All Cockroaches were provided by American cockroach breeding farm in Chizhou city, Anhui province, China. The colony was maintained at 27°C with a relative humidity of 70-80% in breathable plastic box, and the animals received lab mice diet and water. To obtain pools of synchronized animals, the oothecae were hatched uniformly and molt numbers were recorded. Newly 6^th instar nymphs were selected from the colony and placed in separate plastic boxes, supplied with mice diet and water. The experimental cockroaches were anesthetized with ice for sample collection.

The full-length transcript sequence of PaSCLec was obtained from cockroach leg by transcriptomic sequencing. Transcript sequence was used for primer design to obtain full-length cDNAs by Rapid Amplification of cDNA Ends (RACE) using the HiScript-TS 5’/3’ RACE Kit (Vazyme, China). A cDNA copy of PaSCLec was obtained using the following primers: 3′RACE (5′- ATGGGCGCTGGAAGCTGTATACCGG-3′) and 5′RACE (5′ -AGGGGTTCATGCGGCTTATGA-3′). The sequence of PaSCLec was subjected to nanopore sequencing by Beijing Tsingke Biotech Co., Ltd. The full-length cDNA sequence of PaSCL-Reg and PaSCL-Ad were amplified by RT-PCR using corresponding primers and re-sequenced for confirmation (Supplementary Table S1). Homology analysis of PaSCL-Reg and PaSCL-AD amino acid sequence were performed using BLAST (http://www.ncbi.nlm.nih.gov/). Multiple sequences alignment analysis was performed using the DNAMAN. The characterizations of protein were predicted on the ExPASY server (http://www.expasy.org/). SignalP 5.0 server was used to predict the signal peptide (https://services.healthtech.dtu.dk/service.php?SignalP-5.0). Domain architecture prediction of the proteins was performed using SMART (http://smart.embl-heidelberg.de/). MEGA 5 was used for phylogenetic analysis. The modeling structures were generated by predicted by using Alphafold3 server (https://alphafoldserver.com/).

The codon-optimized sequences of rPaSCL-Reg and rPaSCL-Ad were synthesized de novo by Beijing Tsingke Biotech Co., Ltd and subsequently cloned into the plasmid (pET28a) before transformation into BL21 (DE3) cells to generate recombinant plasmids. Positive clones underwent PCR screening with primers T7-F and T7-R, followed by sequence confirmation. A single colony of transformed BL21 (DE3) cells was induced to express the recombinant protein with 0.1 mM IPTG at 28°C for 6 h. The resulting rPaSCL-Reg and rPaSCL-Ad were purified using a Ni column (Qiagen, Germany) and underwent desalination via Amicon^® Ultra (Merck, Germany), followed by analysis using 10% SDS-PAGE and Coomassie brilliant blue R-250 staining. Recombinant protein concentration was determined using BCA protein Assay Kit (Solarbio, China).

Following SDS-PAGE separation, the purified proteins were transferred to a nitrocellulose membrane (Pall, America). The membranes underwent blocking for 2 h with 5% non-fat milk in 1×TBST (10 mM Tris·HCl, 150 mM NaCl, 0.1% Tween, pH 7.2-7.6 in 25°C). After washing, the membrane was incubated with 1/5000 primary antibody (Rabbit anti-His-tag antibody, Abclonal, China) in TBST containing 5% non-fat milk at 4°C for 12 h. The washed membrane was then incubated with a secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG, BBI, China) at 37°C for 2 h. The Western blot analysis was performed on UVP ChemStudio815 using an enhanced Chemiluminescence Substrate Kit (Yuanye, China).

Bacterial cultures were grown overnight and harvested by centrifugation at 8000 r for 10 min. The bacteria underwent three washing cycles with TBS (10 mM Tris·HCl, 150 mM NaCl, pH 7.2-7.6 in 25°C) and were resuspended in TBS to achieve an OD600 of 1.0. The bacterial suspension (100 μL) in TBS was incubated with purified rPaSCL-Reg and rPaSCL-Ad (50 μg) for 60 min at room temperature under mild rotation. Following incubation, the bacteria were washed four times with TBS. The final bacterial suspension underwent 10% SDS-PAGE and Western blot analysis. Rabbit anti-His-tag antibody (Abclonal, China, 1:5000) served as the primary antibody, while HRP-conjugated Goat Anti-Rabbit IgG (BBI, China, 1:10, 000) functioned as the secondary antibody, respectively.

An Elisa was employed to assess the sugar binding specificity of rPaSCL-Reg and rPaSCL-Ad. LPS from E. coli and PGN from S. aureus were selected for the assay. 96-well ELISA Plates were coated with 10 μg of polysaccharide and incubated at 4°C overnight. After five TBS washes, the microplates underwent blocking with BSA (1 mg/mL, 200 μL) at 37°C for 2 h, followed by TBS washing. Purified rPaSCL-Reg and rPaSCL-Ad (final concentration 0–80 μg/mL in TBS with 0.1 mg/mL BSA) were added to each coated well and incubated at 37°C for 2 h. The plate underwent five TBS washes. Subsequently, Rabbit anti-His-tag antibody (1:2000) was added (100 μL per well) and incubated at 37˚C for 2 h; 100 μL of HRP-conjugated Goat Anti-Rabbit IgG (1:5000) was introduced to each well at 37°C for 1 h. Following five TBS washes, color development occurred with TMB (BBI, China) at room temperature for 30 min. The OD value was measured at 450 nm. Each binding assay was performed in triplicate.

Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and Gram-negative bacteria (Escherichia coli and Salmonella Typhimurium) were selected to evaluate the bacterial agglutination properties of rPaSCL-Reg and rPaSCL-Ad. A volume of 25 μL rPaSCL-Reg and rPaSCL-Ad (100 μg/mL) was incubated with an equal volume of bacterial suspension (1×10⁸ CFU/mL) at room temperature (~25°C) for 1 h, both with and without 10 mmol/L CaCl₂. rBSA in TBS and TBS+Ca²⁺ (10 mM Tris·HCl, 150 mM NaCl, 10 mM CaCl₂, pH 7.2-7.6 in 25°C) served as negative controls. Following incubation, agglutination was observed and documented using a fluorescence microscope (Nikon, Japan).

A total of 100 μg of each protein was incubated with a bacterial suspension (1 × 10⁵ CFU/mL) in fresh LB (Tryptone 10 g/L, Yeast Extract 5g/L, NaCl 10g/L) in a 96-well culture plate. CaCl₂ was added to a final concentration of 10 mM, with each well containing a total volume of 200 μL. The plate was incubated at 37 °C for 24 h. Bacterial growth was assessed by measuring the absorbance at 600 nm using a microplate reader. Each assay was performed in triplicate wells per protein and repeated in three independent experiments. rBSA served as a negative control. Data are presented as mean ± standard deviation (SD), and statistical significance was evaluated using student t-test.

Hemolymph extracted from P. americana was fixed using 200 μL of anticoagulant mixture (62 mM NaCl, 100 mM glucose, 10 mM EDTA, 30 mM Sodium citrate, 26 mM citric acid) and 4% paraformaldehyde, followed by centrifugation at 600 g for 10 min at 4°C. The isolated hemocytes were placed on poly-L-lysine-Prep slides (BBI, China) to facilitate cell adhesion during microscopic analysis, washed with TBS, and blocked with 5% BSA at 37°C for 30 min. Following TBS washing, the hemocytes were incubated with 10 μg rPaSCL-Reg or rPaSCL-Ad at 4°C for 12 h. The samples were then washed with TBS and incubated with 5% anti-His-tag antibody (1:1000 in 5% BSA) at room temperature for 2 h, followed by TBS washing and incubation with Alexa Fluor 488-conjugated Goat anti-rabbit IgG (Abclonal, China, 1:1000 in 5% BSA) for 1 h at 37°C in darkness. After six washes, the hemocytes were treated with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime, China) for 10 min at room temperature and washed six times. Fluorescence was examined using a laser scanning confocal microscope (LSCM) (Nikon, Japan). Nis-ElemeViewer software was utilized to assess the binding capability of rPaSCL-Reg and rPaSCL-Ad to hemolymph membranes.

To assess the phagocytic activity of hemocytes, Hemolymph was collected with anticoagulant and centrifuged at 600 g for 10 min at 4 °C to isolate hemocytes. Hemocytes were gently washed twice with TBS and resuspended. Escherichia coli was cultured overnight, harvested by centrifugation at 5000 rpm for 10 min, washed three times with TBS, and resuspended. Bacteria were labeled with 0.1 mg/mL FITC which dissolved in DMSO for 2 h in the dark, washed three times with TBS to remove excess dye, and finally resuspended in TBS at an OD600 = 1. For the phagocytosis assay, 100 µL of hemocyte suspension (1×10⁶ cells/mL) was added to each well of a confocal culture dish containing culture medium. The treatment group received 10 µg of rPaSCL-Reg and rPaSCL-Ad, whereas the control group received rBSA, with three replicates per group. Cells were incubated at 28 °C for 1 h. Subsequently, FITC-labeled bacteria were added at a 1:10 ratio of hemocytes to bacteria. Hemocytes were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, and 10 µL of the cell-bacteria mixture was placed onto a microscope slide for 10 min sedimentation in a humid chamber. Phagocytosis was observed under a LSCM (Nikon, Japan), and images were captured. For each treatment, at least 150 hemocytes from randomly selected fields were counted to calculate the phagocytic rate.

Morphological changes in recombinant protein-bound bacteria were examined using field-emission scanning electron microscopy (Verios G4 UC, Thermo Fisher Scientific, America). Bacterial suspensions (1×10⁸ CFU/mL) were incubated with rPaSCL-Reg and rPaSCL-Ad at 2 mg/mL in TBS and TBS+Ca²⁺ (TBS, 10 mM CaCl₂) at 37°C for 2 h, using rBSA protein (2 mg/mL) as the negative control. After incubation, cells underwent fixation with 2.5% (v/v) glutaraldehyde in 0.1 M Phosphate buffer at 4°C for 12 h, followed by gradual dehydration using increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) for 20 min at each step. The samples were subsequently dried, gold coated, and analyzed using field-emission scanning electron microscopy.

For gene expression analyses, 10-fold diluted cDNA served as templates for qRT-PCR. Reactions were performed in triplicate using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711). The relative expression levels were calculated using the 2^-ΔΔCt method and there were three biological replicates and three technical replicates for each sample. Actin was chosen as a reference gene for qRT-PCR analysis (20). The primers used for qRT-PCR and the amplification efficiency (29) of each primer are shown in Supplementary Table S1.

Double-stranded RNA (dsRNA) was synthesized using the T7 RiboMAX Express RNA interference (RNAi) System (Promega, P1700). Following purification, dsRNA was prepared at a concentration of 2 μg/μL, and 2μg was microinjected into the coxa of each nymph. To sustain RNAi efficiency throughout the regeneration process, injections were repeated every 7 days (15 nymphs per replicate, three biological replicates). The dsRNA-synthesizing primer sequences are detailed in Supplementary Table S1. A dsMock targeting a clone vector sequence was used as the negative control. For the analysis of differentially expressed genes following RNAi of target genes, dsRNA was performed together with amputation, and the coxa and trochanter at 7 days post-amputation (dpa) were harvested.

Through transcriptome analysis of leg regeneration in Periplaneta americana, a unique secreted C-type lectin (PaSCLec) with high expression was identified. Notably, an alternate donor site event in the transcript of PaSCLec generating a frameshift mutation revealed two distinct isoforms (designated PaSCL-Ad and PaSCL-Reg) (Supplementary Figure S1). Using 5′/3′ RACE and Nanopore sequencing, we found that transcripts corresponding to PaSCL-Ad constituted approximately 70–80% of the total isoform population (Primer 3′RACE: 79.06%; Primer 5′RACE: 75.46%). Therefore, the transcript of PaSCL-Ad was considered the main transcript. Sequence analysis showed the ORF of PaSCL-Ad (accession no. XP_069685827.1) encodes 214 amino acids with a molecular weight (MW) of 23.9 kDa and an isoelectric point of 4.97, while the ORF of PaSCL-Reg (accession no. XP_069685825.1) encodes 377 amino acids with a molecular weight (MW) of 41.3 kDa and an isoelectric point of 4.65. Both PaSCL-Ad and PaSCL-Reg proteins contain a 22-amino acid signal peptide and a 173-amino acid C-type lectin-like domain (Figure 1A). The amino acid sequence analysis indicates PaSCL-Reg contains 163 additional amino acids compared to PaSCL-Ad at its C-terminal (Figure 1B). A comparison of protein structure reveled that the overall architecture of PaSCL-Ad is relatively compact, with a well-defined core domain (Figure 1B1). In contrast, although PaSCL-Reg also possesses a recognizable core structure, it appears more loosely organized, suggesting that its overall structural stability may be lower (Figure 1B2). Phylogenetic analysis of PaSCL-Ad, PaSCL-Reg and other CTLs from invertebrate and vertebrate species revealed that, unlike Regenectin, PaSCL-Ad and PaSCL-Reg cluster with CTLs from Gryllus bimaculatus, which also possesses leg regeneration capability (Figure 1C).

Tissue expression profiling revealed that total PaSCLec exhibited highest expression in the hemolymph, followed by fat body, coxa and gut (Figure 2A). To investigate the potential role of PaSCLec in innate immunity, sixth instar P. americana were subjected to bacterial injection to induce a systemic immune challenge. Compared with the control group, the mRNA levels of PaSCLec in hemolymph were significantly upregulated at 24 h post-injection with either E. coli (Figure 2B) or S. aureus (Figure 2C). These results suggest PaSCLec is involved in the immunoregulatory network of P. americana.

The alternative splicing variants rPaSCL-Ad and rPaSCL-Reg were expressed using a prokaryotic expression system to assess their biological activities and purified using the Ni-NTA Agarose (QIAGEN, Germany). The purification of rPaSCL-Ad and rPaSCL-Reg was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 3A). To assess the immune functions of rPaSCL-Ad and rPaSCL-Reg, another P. americana C-type lectin, Regenectin, which is expressed during leg regeneration but has no reported immune activity, was also purified as a comparative control (Figure 3B).The purified rPaSCL-Ad, rPaSCL-Reg and Regenectin were analyzed using Western blot (Figure 3C, D, E), revealing apparent molecular weights of approximately ~26 kDa and ~40 kDa for rPaSCL-Ad and rPaSCL-Reg respectively, which correspond precisely to their predicted sizes.

C-type lectin exhibits pathogen recognition functions through binding to the Pathogen-Associated Molecular Patterns (PAMPs). Direct binding analysis using enzyme-linked immunosorbent assay (ELISA) demonstrated that rBSA had no binding ability (Figure 4A) and rPaSCL-Ad and rPaSCL-Reg bound to lipopolysaccharide (LPS) from E. coli and peptidoglycan (PGN) from S. aureus, with binding ability showing dose dependence (Figures 4B, C). Additionally, rRegenectin displayed lower binding capacity compared to rPaSCL-Ad and rPaSCL-Reg at equivalent concentrations (Figure 4D). Furthermore, rPaSCL-Ad demonstrated binding capability to Mannan and β-Glucan at low doses (PAMPs predominantly found in fungal cell walls). Western blotting analysis confirmed the binding affinity of rPaSCL-Ad and rPaSCL-Reg to bacteria. The results indicated that rPaSCL-Ad exhibited superior binding capacity for microorganisms including GP bacteria S. aureus, Bacillus subtilis, and GN bacteria Salmonella Typhimurium and E. coli (Figure 4E).

The agglutination activity of all recombinant proteins was evaluated using FITC method across various bacteria. All recombinant proteins demonstrated agglutination activity toward bacteria in the presence of Ca²⁺ (Figure 5), except for the control rBSA (Figure 5A). rPaSCL-Ad demonstrated the strongest agglutination activity (Figure 5B). Both rPaSCL-Reg and rRegenectin exhibited comparatively weaker agglutination, with rRegenectin showing almost no detectable activity toward E. coli (Figures 5C, D); however, no agglutinating activity was observed toward any bacteria without Ca²⁺ (Supplementary Figure S2), indicating Ca²⁺-dependent agglutinating activity.

Following the observation that rPaSCL-Reg and rPaSCL-Ad bind to bacterial surfaces, their potential bacteriostatic or bactericidal activity was examined. Bacterial cultures exposed to intact rPaSCL-Reg, rPaSCL-Ad and rRegenectin at concentrations of 100 μg/ml showed significant variations in bacterial growth compared to control cultures with rBSA control group. The results revealed that only rPaSCL-Ad demonstrated direct bacteriostatic activity against all bacteria (Figure 6A), while rPaSCL-Reg and rRegenectin specifically inhibited B. subtilis growth (Figure 6B and C). Notably, scanning electron microscopy (SEM) analysis revealed that both GN E. coli and GP S. aureus bacteria exhibited membrane pore formation following rPaSCL-Ad treatment (Figure 6B). In contrast, rPaSCL-Reg did not compromise cell membrane integrity of GN or GP bacteria (Supplementary Figure S3).

To determine whether rPaSCL-Ad, rPaSCL-Reg and rRegenectin are involved in hemocyte-mediated phagocytosis, their ability to bind to the hemocyte surface of Periplaneta americana was examined by immunocytochemical analysis. The results showed that all three recombinant proteins bound to the hemocyte surface (Figure 7A).

Subsequently, collected hemocytes were placed in cell culture plates and pre-incubated separately with each recombinant proteins, using rBSA as the control group. FITC-labeled E. coli was then added for co-incubation. After fixation, hemocytes phagocytosis of E. coli was visualized by microscopy and quantitatively analyzed. The results demonstrated that rPaSCL-Ad and rPaSCL-Reg significantly enhanced hemocyte phagocytosis of E. coli compared with the control, whereas rRegenectin had no detectable effect (Figure 7B and B’). These findings suggest that rPaSCL-Ad and rPaSCL-Reg promote hemocyte phagocytic activity by facilitating interactions between bacterial and hemocytes.

To investigate whether PaSCLec indirectly participates in immune responses through AMP expression regulation, dsPaSCLec was synthesized in vitro and injected into 6th P. americana. Following stimulation with E. coli and S. aureus, decreased relative mRNA levels of PaSCLec were observed (Figures 8A, D). Six AMPs were evaluated: Attacin-1 (Att-1), Termicin-1 (Term-1), Termicin-2 (Term-2), Defensin-1 (Def-1), Defensin-2 (Def-2) and Defensin-3 (Def-3). Bacterial challenges significantly downregulated the expression of antimicrobial peptides Att-1, Term-2, Def-1 and Def-3 (Figures 8B, E). Conversely, Term-1 and Def-2 expression were equally upregulated in the dsPaSCLec transfected cockroaches. To examine the potential role of PaSCLec in AMP regulation in vivo, its involvement in Toll, IMD, and JAK/STAT pathways was investigated via transcription factors Dorsal, Relish, and STAT. The results indicated significant reduction in Dorsal, Relish and STAT expression (Figures 8C, F).

To validate these findings, dsPaSCLec transfected cockroaches were initially injected with rPaSCL-Ad and rPaSCL-Reg protein respectively, followed by E. coli challenge as previously described. The results demonstrated significantly increased Relish and STAT expression in response to E. coli challenge in both rPaSCL-Ad-rescued and rPaSCL-Reg-rescued cockroaches (Figure 9A). Additionally, the expression of Att-1 and Def-3, along with transcription factors Relish and STAT, showed significant increases compared to control cockroaches (Figure 9B). These findings indicate that PaSCLec specifically upregulates the expression of Att-1 and Def-3, suggesting that PaSCLec regulates AMP expression through multiple pathways.

To investigate whether PaSCLec has an effect on leg regeneration, we first evaluated the expression of PaSCLec at 0, 3, 7 and 14 days post-ecdysis (dpe) or dpa. The results showed that the expression of PaSCLec was significantly upregulated during leg regeneration (Figures 10A, B). Then, RNAi of PaSCLec was performed and the interference efficiency of PaSCLec was measured on 7 dpa and 14 dpa, and the mRNA level of PaSCLec was significantly reduced compared with control (Figure 10C). Injection of dsPaSCLec caused the regenerated legs to exhibit morphological abnormalities, while no change was observed when dsMock was injected (Figures 10D, E). The regenerative length (Regenerated leg/Contralateral leg, %) was measured after RNAi treatments of genes (Figure 10F). In the wild-type (WT) group (N = 15), the mean relative regenerative length was 84.046% ± 1.98% (mean ± standard deviation, SD). The dsMock group, which served as a non-targeting control (N = 15), exhibited a similar relative regenerative length of 81.214% ± 2.06%. In contrast, the dsPaSCLec group, in which PaSCLec was knocked down (N = 15), showed a significantly reduced relative regenerative length of 62.473% ± 4.98%. Statistical analysis revealed that both WT and dsMock groups had markedly higher relative regenerative lengths compared to the dsPaSCLec group.

To investigate PaSCLec’s immune function during leg regeneration, we measured mRNA levels of Def-3 and Att-1 regulated by PaSCLec in leg regeneration. Results indicated sustained expression of Def-3 and Att-1 during early regeneration phases (Figures 11A, B), which were downregulated after injection of dsPaSCLec (Figure 11C).

To explore the latent interaction of JAK/STAT signaling pathways with PaSCLec during leg regeneration, RNAi was performed to knockdown the expression of PaSCLec in P. americana. The expression of unpaired was upregulated at 3 dpa and 7 dpa and reduced significant at 7 dpa when the PaSCLec was silenced (Figures 12A, B). Our results showed that PaSCLec knockdown leads to reduced unpaired mRNA levels during leg regeneration, suggesting that PaSCLec may modulate the transcription of JAK/STAT associated cytokines. To confirm the role of the JAK/STAT pathway and specifically the functions of the ligand unpaired, we performed to knockdown the expression of unpaired. Silencing efficiency of RNAi was detected by on 7 dpa (Figure 12C). Knockdown of unpaired in the JAK/STAT signaling disrupted the entire regeneration process (Figure 12D) and the mRNA level of Def-3 was significantly impaired (Figure 12E). In contrast, the mRNA level of Att-1 was upregulated (Figure 12E). Because unpaired is a canonical activator of the JAK/STAT pathway, this correlation implies a possible functional relationship between PaSCLec and JAK/STAT-mediated processes in immunity and regeneration. Unpaired is a canonical activator of the JAK/STAT pathway, this correlation implies a possible functional relationship between PaSCLec and JAK/STAT mediated processes in immunity and regeneration. Nevertheless, the involvement of JAK/STAT signaling remains speculative, as the current study does not provide direct mechanistic evidence. We did not detect physical interactions, receptor activation, or downstream readouts such as STAT phosphorylation or nuclear translocation. Thus, additional experimental work will be required to define whether and how PaSCLec interacts with the JAK/STAT pathway in future.