Abstract

   Bacillus thuringiensis (Bt) has been extensively applied in
   agricultural pest management, posing a notable ecological risk to
   beneficial insects like Bombyx mori (silkworms). However, the
   toxicological mechanisms of Bt at low concentrations on silkworms
   remain largely unexplored. In this study, we determined the LC[50] (96
   h) of Bt for fifth-instar silkworm larvae to be 0.08 × 10^−3 mg/L.
   Exposure to a sub-lethal concentration of Bt (1/2 LC[50]) led to
   significant reductions in body weight, pupal size, and the weights of
   both the whole cocoon and cocoon shell. Histopathological and
   ultrastructural examinations revealed that Bt exposure caused severe
   damage to the microvilli and epidermal cells of the midgut.
   Transcriptome sequencing of the midgut identified 290 differentially
   expressed genes (DEGs), with these genes predominantly involved in
   metabolic processes and apoptotic pathways. Notably, apoptosis-related
   genes such as Apaf-1 and Caspase-3 were upregulated by 5.08-fold and
   1.27-fold, respectively. Further validation through TUNEL assays and
   Western blotting analysis confirmed a significant activation of
   apoptotic signaling. These findings suggested that low concentrations
   of Bt could trigger apoptotic pathways in the midgut of silkworm
   larvae, providing valuable insights into the toxicological evaluation
   of Bt at sub-lethal doses in insect species.

1. Introduction

   Bacuillus thuringiensis (Bt) is a Gram-positive microorganism that
   produces a range of proteins during both sporulation and vegetative
   growth phases [[36]1,[37]2]. Once solubilized and proteolytically
   activated in the insect midgut, these transform into potent toxins that
   compromise the midgut’s integrity, ultimately causing the insect’s
   death [[38]3,[39]4,[40]5]. Bacuillus thuringiensis is not only easy to
   cultivate but also demonstrates low persistence and significant
   insecticidal effects [[41]6,[42]7]. As a result, Bt has been applied as
   a biological insecticide against lepidopteran, coleopteran, dipteran,
   and other pests for over five decades [[43]8,[44]9]. However, the
   extensive use of Bt has raised concerns regarding its chronic toxicity
   in non-target organisms due to environmental residues. Studies have
   shown that exposure to low concentrations of Bt can adversely affect
   the larval development of various insect species, including Aedes
   aegypti (Linnaeus, 1762), Leptinotarsa decemlineata (Say, 1824),
   Helicoverpa armigera (Hübner, 1808), and Sesamia nonagrioides
   (Lefèbvre, 1827) [[45]10,[46]11,[47]12,[48]13,[49]14]. Despite these
   findings, the molecular mechanisms underlying midgut injury caused by
   low-dose Bt exposure in insects remain poorly understood.

   Bombyx mori Linnaeus, 1758 (Lepidoptera: Bombycidae), a lepidopteran
   insect, holds significant economic and scientific value
   [[50]15,[51]16]. It has become highly sensitive to external
   environmental factors after more than 5000 years of selective breeding
   [[52]17,[53]18]. Many Bt crops transformed with genes encoding various
   Bacillus thuringiensis (Bt), such as Bt rice, which have shown
   resistance to one or more lepidopteran pests of rice in the field, such
   as yellow stem borer (Scirpophaga incertulas) [[54]19]. The widespread
   cultivation of Bt crops, such as Bt rice and maize, has resulted in the
   dispersion of Bt pollen onto mulberry leaves through wind, impacting
   silkworms that rely on these leaves for sustenance, and this exposure
   has led to a marked reduction in silk production
   [[55]20,[56]21,[57]22].

   Apoptosis is one of the signaling pathways of programmed cell death
   [[58]23], which plays a vital role in biological growth and
   development, as well as cell proliferation and cell differentiation
   [[59]24]. Apoptosis is characterized by morphological changes and
   biochemical events such as cell contraction, vacuolization, chromatin
   condensation, DNA fragmentation, exteriorization of phosphatidylserine,
   cell phagocytosis, and activation of caspases such as caspase 3, 8, and
   9. Caspase activation can occur by ligating membrane-binding receptors
   or by mitochondrial depolarization and release of cytochrome c and
   APAF-1 [[60]25].

   The early study found that Bt could induce the vacuolation of midgut
   cells in Bombyx mori [[61]26]. Vacuolation and nuclear condensation of
   midgut cells in other insects were subsequently observed
   [[62]27,[63]28]. Swelling and vacuolation of mitochondria have also
   been found in other tissues such as the malpighian tubule [[64]29].
   However, it has not been suggested that these phenomena are closely
   related to apoptosis signals, and morphological changes have only been
   observed through light and electron microscopy, without involving
   specific biochemical experiments. Our study firstly identified
   apoptosis as a primary mechanism underlying midgut damage caused by low
   concentrations of Bt. Then, we further verified the apoptotic signal by
   morphology and molecular chemistry. These findings opened up a new
   avenue of research into the effects of low-dose insecticides on
   non-target organisms.

2. Materials and Methods

2.1. Insects and Chemicals

   The silkworm larvae (Huakang 3 strain) were purchased from Rugao
   Sericulture Station (Nantong Jiangsu, China), fed with fresh mulberry
   leaves (Yu-711 strain) three times in our laboratory, the Sericulture
   Institute of Soochow University, under standard conditions (25 ± 1 °C,
   12:12 h light/dark photoperiod, 75 ± 5% relative humidity). Bt (8000
   IU/μL) was purchased from Beijing Green Agricultural Science and
   Technology Group Co., Ltd. (Beijing, China).

2.2. Toxicity Evaluation and Low Concentration of Bt Treatment

   Bacuillus thuringiensis (8000 IU/μL) is dissolved in sterilized water
   and diluted to different working solutions (0.01 × 10^−3, 0.04 × 10^−3,
   0.05 × 10^−3, 0.1 × 10^−3, and 0.2 × 10^−3 mg/L). Mulberry leaves were
   soaked in different solutions for 1 min and air-dried naturally for
   about 30 min. Three liters of working solution were used to soak the
   mulberry leaves. Silkworm larvae at the third day of the fifth instar
   were randomly selected for Bt treatment until cocooning. The treatment
   group was fed Bt-treated mulberry leaves, and the control group was fed
   sterilized-water-treated leaves. Thirty insects per replicate were used
   for survival statistics. Three insects per replicate were chosen for
   dissection. Thirty insects per replicate were collected to measure the
   body weight and cocooning rate. The pupa and cocoon were photographed
   and observed. Mortality was defined as the index of poisonous
   silkworms, which was recorded at 96 h. The LC[50] (lethal concentration
   50) value and 95% confidence intervals were calculated using standard
   probit analysis [[65]30].

2.3. Sample Collection and Extraction of Total RNA

   The midgut (MG) of silkworms was dissected on ice at 96 h following Bt
   (0.04 × 10^−3 mg/L) exposure, immediately cleaned in pre-cooled 1× PBS
   buffer (Sangon Biotechnology Co., Ltd., Shanghai, China), and dried
   with sterilized filter paper. Three midgut tissues were randomly
   selected and placed into 1.5 mL RNase-free tubes, frozen instantly in
   liquid nitrogen, and finally stored at −80 °C for the next experiments.

   Frozen tissues were thawed, ground into powder with a sterilized
   mortar, and 50 mg of each sample was weighed out. Total RNA from the
   midgut tissues was extracted using RNAiso Plus (Takara, Dalian, China),
   and impurities were removed with trichloromethane, isopropanol, and 75%
   ethanol. The quality of RNA was detected with 1% agarose gel
   electrophoresis and NanoDrop-2000 (Thermo Fisher Scientific, Boston,
   MA, USA).

2.4. Transcriptome Sequencing and Identification of Differentially Expressed
Genes (DEGs)

   High High-quality RNA was used as material for the construction of cDNA
   libraries, and 150-base paired-end reads were generated on the DNBSEQ™
   platform (BGI). Sequencing data were filtered with SOAPnuke, and
   paired-end clean reads were mapped and spliced to the silkworm
   reference genome using HISAT2 [[66]31,[67]32]. Then, the
   transcriptional location information was identified and revised by
   STRINGTIE [[68]33,[69]34]. Finally, HTSEQ was adopted to calculate the
   number of reads and the level of gene expression [[70]35]. DESeq2 was
   used to screen DEGs between two groups with the criteria of adjusted
   q-value < 0.05 and | Log[2]FoldChange | > 0.5 [[71]35]. Gene Ontology
   (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and
   enrichment pathway analysis of DEGs were performed to assign putative
   functional annotations.

2.5. Quantitative Real-Time PCR (qRT-PCR) Analysis

   The Reverse Transcriptase M-MLV reagent kit was purchased from Sangon
   Biotechnology Co., Ltd. (Shanghai, China) for synthesizing cDNA. The
   primers ([72]Table S1) for qRT-PCR were designed on NCBI (Primer
   designing tool (nih.gov)) and synthesized by Sangon Biotechnology Co.,
   Ltd. (Shanghai, China). Each 10 μL qRT-PCR reaction mixture contained
   25 ng cDNA, 0.4 μL of each primer (10 μM), 5 μL TB Green Premix Ex Taq
   II (2×) (Takara, Dalian, China), and 4.1 μL sterilized water.
   Quantitative real-time PCR was performed on a CFX Opus Real-time PCR
   System (Bio-Rad, USA). The tubes were incubated at 95 °C for 30 s,
   followed by 40 cycles of the thermal program (5 s at 95 °C, 30 s at 60
   °C). Actin3 was selected as an internal reference gene [[73]36]. Three
   biological and technical replicates were performed for each gene. The
   relative transcription levels of target genes were measured using the
   2^−ΔΔCt method [[74]37] and visualized using GraphPad Prism 8.0.1.

2.6. Immunohistochemistry (IHC) and Transmission Electron Microscopy (TEM)

   The fresh midgut tissues were fixed in 4% paraformaldehyde, embedded in
   paraffin, and then sliced into 5 μm sections and fixed on glass slides.
   The histopathological sections were examined blindly with the Nikon
   DS-U3 (Nikon, Tokyo, Japan) imaging system after hematoxylin-eosin
   staining.

   The fresh midgut tissues were fixed in 4% glutaraldehyde, then dark
   fixed at 4 °C for 2 h, washed with 1 × PBS buffer three times, and
   fixed with 1% osmium tetroxide for 1 h, dehydrated with a graded series
   of ethanol (75%, 85%, 95%, and 100%), stained with 0.5% uranyl acetate
   in water overnight, and after that, coated with Epon 812 and stained
   with lead citrate and uranium dioxide acetate. Finally, 100 nm sections
   were obtained with the Leica UC6 (Leica, Berlin, Germany) and examined
   under HT7700 transmission electron microscopy (Hitachi, Tokyo, Japan).

2.7. Apoptosis Detection in Frozen Sections of Midgut

   The fresh midgut tissues were cleaned with 1 × PBS buffer, then frozen
   sections were prepared according to the manufacturer’s instructions
   using a previously described protocol [[75]38]; subsequently, they were
   stained with TUNEL Kit (chromogenic method) (Beyotime, Shanghai,
   China), and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride
   (DAPI) (Solarbio, Beijing, China) was used for staining cell nuclei,
   and the Nikon DS-U3 (Nikon, Japan) imaging system was used for
   detecting apoptotic signals.

2.8. Western Blot Assay

   The midgut samples were lysed in lysis buffer (Beyotime, Shanghai,
   China) with 1mM phenylmethylsulfonylfluoride (Beyotime, Shanghai,
   China). The lysed tissues were centrifuged at 12,000 rpm at 4 °C for 10
   min, then the supernatant was collected and the protein concentration
   was determined by the BCA method [[76]39]; finally, 60 μg of total
   protein was used for the Western blot assay. The prepared samples were
   separated using 10% SDS-PAGE and transferred to PVDF membranes. The
   membrane was incubated with primary antibodies at 4 °C overnight after
   being blocked with 5% BSA in TBST at 25 °C for 2 h. The primary
   antibodies used were β-tubulin at a dilution of 1:1000 (CST, Boston,
   MA, USA) and Caspase-3 at a dilution of 1:500 (GenScript, Nanjing,
   China). The primary antibody-binding was visualized with a horseradish
   peroxidase (HRP)-conjugated goat anti-rabbit IgG (CST, Boston, MA, USA)
   at a dilution of 1:1000 at 25 °C for 1 h. All results of the Western
   blot were revealed by using the Chemiluminescent HRP Substrate Kit
   (Bio-Rad, Hercule, CA, USA) and image analysis software (ImageJ
   2.1.4.7, Bethesda, MD, USA).

2.9. Statistical Analysis

   Statistical analysis was performed with SPSS 19.0 (SPSS, USA) using
   Student’s T-test and one-way analysis of variance (ANOVA). The level of
   significance with p < 0.05 (*) and p < 0.01 (**) was considered with
   statistical significance. All quantitative data were presented as mean
   ± standard deviation (SD). All figures were drawn with GraphPad Prism 8
   (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Toxicity of Bt Product Against Silkworms

   Silkworm larvae were fed mulberry leaves treated with varying
   concentrations of Bt, and mortality was recorded after 96 h ([77]Table
   S2). The mortality curve indicated that Bt exerted cumulative damage on
   silkworms ([78]Figure 1A). Based on a toxic regression equation (y =
   0.425x + 0.9383), the LC[50] (96 h) for fifth-instar silkworm larvae
   was determined to be 0.08 × 10^−3 mg/L. Following Bt exposure for 96 h,
   larval body weight decreased by 24.9% ([79]Figure 1B). Additionally, Bt
   significantly impacted the size of both pupae and cocoons, leading to a
   marked reduction in whole cocoon and cocoon shell weight ([80]Figure
   1C,D). However, the proportion of the cocoon shell to the total cocoon
   weight remained unaffected by Bt treatment ([81]Table 1).

Figure 1.

   [82]Figure 1
   [83]Open in a new tab

   The toxicity and effect of Bt treatment on the growth of silkworms. (A)
   The toxicity curve of different concentrations of Bt treatment to
   silkworms at 96 h. (B) The effect of Bt treatment on larval body weight
   of silkworm. (C) The effect of Bt treatment on pupae. (D) The effect of
   Bt treatment on cocoons. Scale bar of 1 cm.

Table 1.

   Effect of low concentration of Bt on cocoon quality.
   Goup Name Cocoon Weight (g) Cocoon Shell Weight (g) Ratio of Cocoon
   Shell
   Control 1.75 ± 0.03 0.38 ± 0.02 0.22 ± 0.01
   Bt 1.12 ± 0.03 *** 0.20 ± 0.03 *** 0.18 ± 0.02
   [84]Open in a new tab

   The results were shown as mean ± SEM. SPSS 19.0 was used for one-way
   ANOVA (*** p ≤ 0.001).

3.2. Histopathological and Ultrastructural Analysis of the Midgut

   Histopathological analysis was performed to assess Bt-induced damage to
   the midgut. In the Bt-treated group, the midgut cell layer was thinner,
   with numerous nuclei displaced into the lumen, and the microvilli were
   detached from the underlying structure, and chromatin was contracted
   ([85]Figure 2A–C). In contrast, the midgut of the control group
   displayed well-preserved epithelial cells, with tightly organized
   goblet and columnar cells and an intact, clearly defined basement
   membrane ([86]Figure 2D–F).

Figure 2.

   [87]Figure 2
   [88]Open in a new tab

   Histopathological changes in the midgut of silkworm larvae at 96 h
   after Bt exposure. (A–C) Represent the Bt treatment group. (D–F)
   represent the control group. Columnar cells (green arrow), goblet cells
   (black arrow), basement membrane (blue arrow), epithelium (EP), lumen
   (L), cells fell off (red arrow), and chromatin condensation (yellow
   arrow). Scale bar of 50 μm.

   Transmission electron microscopy (TEM) further revealed ultrastructural
   changes in midgut cells. In the Bt-treated group, numerous vacuoles
   were observed ([89]Figure 3A). The microvilli had sloughed off from
   goblet cells, leading to significant loss ([90]Figure 3B,C). In the
   control group, there were few vacuoles ([91]Figure 3D), while the
   microvilli appeared dense and uniformly distributed ([92]Figure 3E,F).
   These ultrastructural findings aligned with the histopathological
   observations.

Figure 3.

   [93]Figure 3
   [94]Open in a new tab

   Ultrastructural of silkworm larvae exposed to Bt treatment for 96 h.
   (A,B) The ultrastructural observation in the Bt treatment group, and
   (C) is the enlarged view of (B) with a red dotted box. (D,E) The
   ultrastructural observation in the control group, and (F) is the
   enlarged view of (E) with a red dotted box. Microvilli (Mv), vacuoles
   (green arrow).

3.3. Transcriptome Sequencing Analysis and Gene Identification in Silkworm
Midguts

   To investigate the molecular mechanisms underlying the silkworm
   response to Bt, transcriptome sequencing was conducted on midgut
   samples from both control and Bt-treated groups. This analysis
   generated over 265 million raw reads, with approximately 44 million
   clean reads obtained from each library, except for the B1 group. The
   Q20 and Q30 scores exceeded 97.5% and 92.1%, respectively, ensuring
   high data quality ([95]Table S3). More than 85.6% of the filtered reads
   were successfully mapped to the reference genome, with 71.0% to 74.4%
   of these reads uniquely aligned ([96]Table S3).

   In total, 21,454 transcripts were detected, including 12,509 novel
   transcripts. Furthermore, 290 DEGs were identified between the control
   and Bt treatment groups, with a |Log[2]FoldChange| greater than 0.5 and
   a q-value below 0.05. Among these DEGs, 107 genes were significantly
   upregulated, while 183 were significantly downregulated ([97]Figure
   4A,B). A detailed breakdown of the DEGs is provided in [98]Table S4.

Figure 4.

   [99]Figure 4
   [100]Open in a new tab

   Transcriptomic analysis in the midgut of silkworm larvae after exposure
   to low concentrations of Bt. (A) Quantities of up- and down-regulated
   DEGs. (B) Volcano map for transcriptomic analysis between Bt-treated
   and control groups. Blue and pink dots represent significant up- and
   down-regulated DEGs, respectively. The red dots are symbolic genes. (C)
   Summary of Gene Ontology (GO) classification of DEGs. (D) Dot plot of
   the GO enrichment analysis.

3.4. Gene Functional Annotation, GO and KEGG Pathway Enrichment Analysis, and
Key Gene Identification

   Functional categorization was performed using the Gene Ontology (GO)
   database to investigate the functional roles of the DEGs. The most
   significantly enriched GO subcategories included “cellular process”
   (77), “binding” (50), and “catalytic activity” (50) ([101]Figure 4C).
   Furthermore, GO enrichment analysis revealed that genes associated with
   “translation”, “ribosome”, and “antibiotic metabolic process” were most
   affected by Bt exposure ([102]Figure 4D).

   To gain deeper insights into the biological changes induced by Bt
   treatment in the silkworm midgut, KEGG pathway analysis was applied to
   all DEGs. The pathways were classified into five main categories, with
   the top three being “cellular process”, “organismal systems”, and
   “metabolism” ([103]Figure 5A). Additionally, KEGG enrichment analysis
   identified 30 significantly enriched pathways (p < 0.05), with the
   majority of DEGs being concentrated in metabolic pathways, followed by
   organismal systems pathways ([104]Figure 5B).

Figure 5.

   [105]Figure 5
   [106]Open in a new tab

   Transcriptomic analysis in the midgut of silkworm larvae after exposure
   to low concentrations of Bt. (A) Summary of KEGG classification of DEGs
   after Bt exposure. (B) Dot plot of the KEGG enrichment analysis. The
   horizontal axis shows the gene proportion, and the vertical axis shows
   the enriched pathway name. Genetic Information Processing (GIP),
   Cellular Processes (CP), Environmental Information Processing (EIP).
   The color scale represents different thresholds of p-value (p < 0.05),
   and the size of the dots represents the quantities of genes
   corresponding to each pathway. (C) The interaction diagram of
   apoptosis-related proteins. Different colored nodes represent different
   MCL clusters (find natural clusters based on the stochastic flow). (D)
   The interaction diagram of proteins related to apoptotic, metabolic
   processes; rRNA processing; collagen trimers and membranes; and
   translation and ribosome pathways. Gray dotted lines indicate GO
   pathways. Network nodes and edges indicate proteins and protein-protein
   associations, respectively.

   A protein–protein interaction (PPI) network comprising 49 proteins was
   constructed using STRING and Cytoscape, which revealed seven distinct
   clusters ([107]Figure 5C). Notably, these proteins were predominantly
   associated with processes such as apoptosis, metabolism, collagen
   trimer formation, and membrane structure ([108]Figure 5D).

3.5. Effect of Bt on the Apoptosis Levels in Silkworm Midguts

   To assess the activation of apoptotic pathways after Bt treatment
   exposure, the transcription levels of three apoptosis-related genes
   were measured using qRT-PCR. The results revealed significant
   upregulation of Apaf-1, Caspase-3, and Caspase-4, with increases of
   5.08-fold, 1.27-fold, and 1.38-fold, respectively ([109]Figure 6A).
   Additionally, protein levels of Caspase-3 showed a 1.33-fold increase
   (p < 0.01), which aligned with the observed mRNA expression levels
   ([110]Figure 6B). Apoptosis was further evaluated using TUNEL assays,
   which demonstrated widespread apoptotic signals in the midgut tissue of
   Bt-exposed silkworms compared to the control group ([111]Figure 6C).

Figure 6.

   [112]Figure 6
   [113]Open in a new tab

   Low concentrations of Bt exposure promote apoptosis in the midguts of
   silkworms. (A) Transcript levels of apoptosis-related Apaf-1,
   Caspase-3, and Caspase-4. (B) Western blotting analysis of Caspase-3,
   numbers 1 and 2, represents two biological duplications. (C) TUNEL
   assay in frozen sections of the midguts. Cell nuclei are stained with
   DAPI (blue); apoptotic signal is represented by Cy3 (red). The results
   are expressed as mean ± SD, and the significance is determined by
   t-test and p-value, and p-value < 0.05 was statistically different.
   Scale bar of 100 μm.

4. Discussion

   In the evaluation of the toxic side effects of pesticides, most studies
   have focused on assessing the sublethal effects of long-term exposure
   to pesticides through food consumption in insects [[114]40]. Low
   concentrations of insecticides, including biopesticides, can negatively
   influence various aspects of a pest’s life cycle, including growth,
   development, and reproductive capacity [[115]41,[116]42]. In our study,
   we determined the LC[50] (96 h) of Bt for fifth-instar silkworm larvae
   to be 0.08 × 10^−3 mg/L. Prolonged exposure to this low concentration
   of Bt (96 h) significantly reduced larval body weight, as well as pupal
   and cocoon weights, while causing notable damage to the midgut.
   Furthermore, transcriptomic analysis, along with gene and protein
   validation, revealed the activation of apoptosis pathways. These
   findings offered a new perspective on the mechanisms of action of
   low-concentration Bt in non-target organisms.

   LC50 (Lethal concentration 50) represents the dose or inhaled
   concentration of a substance that causes death in 50% of a tested
   population, making it a critical metric for evaluating toxicity
   [[117]43]. Our findings showed that a significantly lower concentration
   of Bt at 0.08 × 10^−3 mg/L, was sufficient to achieve the same lethal
   effect in silkworms, highlighting their heightened sensitivity to Bt
   compared to other pesticides, such as indoxacarb, imidacloprid, and
   thiamethoxam, reported as 1.08 × 10^2, 1.92, 0.66, and 1.66 mg/L,
   respectively [[118]43,[119]44]. This underscored the importance of
   studying Bt’s impact on silkworms. Moreover, our research revealed that
   critical biological traits, including body weight, cocoon weight, and
   cocoon layer ratio, were severely compromised after exposure to low
   concentrations of Bt. In conclusion, even low concentrations of Bt
   could cause substantial harm to silkworms.

   The midgut epithelium is the primary target of Bt, making it the focus
   for assessing the harmful effects of Bt on silkworms [[120]5]. Previous
   studies have documented midgut damage in other pests exposed to Bt,
   reporting adverse effects such as cellular hypertrophy, microvilli
   damage, vacuolization, mitochondrial swelling, and an increase in
   lysosomes [[121]45,[122]46,[123]47]. Our research similarly revealed
   significant alterations in the midgut epithelium of silkworms following
   exposure to low concentrations of Bt, which is in accordance with these
   earlier findings. These results suggest that midgut damage from Bt
   exposure is a common phenomenon, likely affecting the growth and
   development of silkworms.

   Transcriptomic sequencing analysis identified 290 DEGs, with GO and
   KEGG pathway enrichment analyses revealing that the majority of these
   DEGs were significantly associated with metabolic processes, including
   lipid, amino acid, and carbohydrate metabolism. Our findings were
   consistent with previous studies on silkworms exposed to chlorfenapyr
   [[124]48]. Similarly, recent RNA-seq research on Helicoverpa zea
   (Lepidoptera: Noctuidae) has demonstrated significant alterations in
   lipid and glucose metabolism following Bt exposure [[125]49]. Moreover,
   the PPI network indicated a connection between metabolic processes and
   apoptotic pathways. As with other insecticides, disruption of metabolic
   processes can damage silkworm midguts and initiate apoptosis
   [[126]50,[127]51,[128]52]. Collectively, these results suggest that the
   toxic mechanism of low-concentration Bt may be intricately linked to
   metabolic pathways.

   In this study, the expression of pro-apoptotic genes (Apaf-1 and
   Caspase-4) was elevated, while Caspase-3, a key apoptosis inducer
   [[129]53], showed a significant increase at the protein level. In
   addition, strong apoptotic signals were detected in TUNEL-stained
   frozen midgut sections. These findings clearly demonstrated that
   apoptotic pathways were activated in the silkworm midgut following
   exposure to low concentrations of Bt. Apoptosis can occur via multiple
   pathways, including the extrinsic and intrinsic pathways, the latter
   involving the endoplasmic reticulum, mitochondria, and lysosomes
   [[130]54]. Overall, we hypothesized that low concentrations of Bt
   induced midgut damage by activating the apoptotic pathway. Further
   investigation is needed to determine the specific type of apoptosis
   induced by Bt in the silkworm midgut. Our research showed that low
   concentrations of Bt could lead to damage to the silkworm by inducing
   apoptosis in the midguts. These results not only provided a direct
   basis for the reasonable use of mulberry fields but also provided a
   reference for the safety evaluation of Bt in long-term environmental
   exposure.

Supplementary Materials

   The following supporting information can be downloaded at
   [131]https://www.mdpi.com/article/10.3390/insects15120911/s1, Table S1:
   Primer sequences for quantitative real-time PCR; Table S2: Death number
   of silkworms larvae after exposure to low concentration of Bt for 96 h;
   Table S3: Summary of the transcriptome sequencing data; Table S4: DEGs
   between the control and Bt treatment group in the midguts of silkworms.
   [132]insects-15-00911-s001.zip^ (214.4KB, zip)

Author Contributions

   Methodology and writing, H.Z.; validation, C.T., Q.S. and P.P.;
   investigation, H.Z., H.G. and J.C.; resources, H.Z. and H.G.; data
   curation, B.L. and H.Z.; writing—original draft preparation, B.L. and
   H.Z.; writing—review and editing, B.L., J.C. and C.T.; visualization,
   P.P. and Q.S.; supervision, B.L. and H.Z.; project administration,
   B.L.; funding acquisition, B.L. All authors have read and agreed to the
   published version of the manuscript.

Data Availability Statement

   Data are contained within the article.

Conflicts of Interest

   The authors declare no conflicts of interest.

Funding Statement

   This research was funded by the Earmarked Fund for China Agriculture
   Research System, grant number CARS-18, National Natural Science
   Foundation of China, grant number 32172795, Jiangsu Agriculture Science
   and Technology Innovation Fund, grant number CX (21)2039, and Science
   and Technology support Program of Suzhou, grant number SNG2023016.

Footnotes

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References