Functional Analysis of a Multiple-Domain CTL15 in the Innate Immunity, Eclosion, and Reproduction of Tribolium castaneum

C-type lectin X (CTL-X) plays critical roles in immune defense, cell adhesion, and developmental regulation. Here, a transmembrane CTL-X of Tribolium castaneum, TcCTL15, with multiple domains was characterized. It was highly expressed in the early and late pupae and early adults and was distributed in all examined tissues. In addition, its expression levels were significantly induced after being challenged with pathogen-associated molecular patterns (PAMPs) and bacteria. In vitro, the recombinant TcCTL15 could recognize bacteria through binding PAMPs and exhibit agglutinating activity against a narrow range of bacteria in the presence of Ca2+. RNAi-mediated TcCTL15-knockdown-larvae infected with Escherichia coli and Staphylococcus aureus showed less survival, had activated immune signaling pathways, and induced the expression of antimicrobial peptide genes. Moreover, silencing TcCTL15 caused eclosion defects by impairing ecdysone and crustacean cardioactive peptide receptors (CCAPRs). Suppression of TcCTL15 in female adults led to defects in ovary development and fecundity, accompanied by concomitant reductions in the mRNA levels of vitellogenin (TcVg) and farnesol dehydrogenase (TcFDH). These findings imply that TcCTL15 has extensive functions in developmental regulation and antibacterial immunity. Uncovering the function of TcCTL15 will enrich the understanding of CTL-X in invertebrates. Its multiple biological functions endow the potential to be an attractive target for pest control.


Introduction
Insects lack an adaptive immune response and rely solely on a powerful innate immunity to resist invading pathogens [1]. When pathogen invasion occurs, an innate immune response is activated by a variety of pattern recognition receptors (PRRs), which can specifically recognize non-self and bind to the evolutionarily conserved pathogen-associated molecular patterns (PAMPs) on the surfaces of pathogens, triggering a series of extracellular and intracellular signal cascade reactions, such as Toll-like receptors, immune deficiency (IMD), and prophenoloxidase pathways, ultimately killing and eliminating invasive pathogens [2]. To date, several types of PRRs corresponding to different PAMPs have been investigated in various insect orders and found to participate in innate immunity, including C-type lectins (CTLs), scavenger receptors, Toll-like receptors, and peptidoglycanrecognition proteins [3,4].
CTLs belong to the lectin superfamily possessing one or more carbohydrate-recognition domains (CRDs), which exist widely in many organisms, ranging from animals and plants to viruses [3,5]. Based on their domain structure and phylogenetic relationships, CTLs can be sorted into three different groups: CTL-S, which possesses only one CRD; IML, with two CRDs; and CTL-X, with one CRD and additional conserved domains. In a short period of evolutionary time, gene duplication and merging produced a diversity of members in the IML group, whereas there were fewer changes in the CTL-S and CTL-X groups, implying that they might have conserved functions [3]. During the past few years, many CTL-Ss in (http://smart.embl-heidelberg.de/ accessed on 27 January 2023) server was applied to predict the motif features of TcCTL15. The isoelectric point (PI) and molecular weight (MW) of TcCTL15 were determined using the ExPASy online tool Compute pI/Mw (https: //web.expasy.org/compute_pi/ accessed on 27 January 2023).
Total RNAs from whole T. castaneum bodies or different tissues were extracted using RNAiso™ Plus (Takara, Dalian, China). The concentration and integrity of RNA were determined by measuring the absorbance at 260 nm/280 nm using a spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and 1% agarose gel electrophoresis, respectively. HiScript II Reverse Transcriptase (Vazyme, Nanjing, China) was used to reverse-transcribe first-strand cDNA using 500 ng RNA samples.
qRT-PCR was used to detect the expression levels of target genes using SYBR Green Master Mix (Vazyme, Nanjing, China). The primer sequences used for qRT-PCR are provided in Table S1. qRT-PCR was performed in triplicate for each biological sample using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) based on our previous method [21]. The transcript levels of target genes were normalized against T. castaneum ribosomal protein S3 (RPS3) and quantified using the 2 −∆∆CT method [24]. All experiments were performed with at least three biological replications.

Immune Challenges
To investigate the changes in TcCTL15 transcript levels upon immune challenge, each larva was injected with 200 nL of injection buffer (IB, 373 g/L KCl and 0.038 g/L Na 3 PO 4 12H 2 O, pH 7.0) containing bacteria (Staphylococcus aureus Rosenbach (approximately 7.0 × 10 4 cells) or Escherichia coli Migula (approximately 7.8 × 10 4 cells)) or PAMPs (lipopolysaccharides (LPS) or peptidoglycan (PGN), 0.08 mg/mL), whereas the larvae were injected individually with 200 nL of IB as the control group. After injection, all larvae were cultured in the same conditions mentioned above. At 12,24,36,48,60, and 72 h post-challenge, three surviving individuals were randomly selected for RNA extraction and cDNA synthesis. Subsequently, qRT-PCR was employed to determine TcCTL15 expression profiles. Each sample included three biological replicates.

Recombinant Expression, Purification, and Western Blot Analysis
A 444 bp DNA fragment coding the TcCTL15-CRD domain was amplified using the primers listed in Table S1. After that, the amplicons were digested with EcoR I and Xho I and then ligated to the pET-28a expression vector (Novagen, Darmstadt, Germany). The pET-28a-TcCTL15 construct was transformed into E. coli strain BL21 (DE3) competent cells (TransGen, Beijing, China) via induction with a final concentration of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37 • C for 4 h. The recombinant TcCTL15 (rTcCTL15) was expressed as inclusion bodies and subjected to denaturation, refolding, and purification according to a method described earlier [7]. The purified rTcCTL15 fused with a His-tag was checked using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with anti-His mouse polyclonal antibodies (TransGen, Beijing, China). The successfully purified rTcCTL15 was quantified using the total protein quantitative assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with bovine serum albumin (BSA) as a standard and was then ready for the binding and agglutination assays.

Microorganism Binding and Agglutination Assays
Gram-positive bacteria (G + ) (S. aureus, Bacillus subtilis Ehrenberg, and Bacillus thuringiensis Berliner) and Gram-negative bacteria (G − ) (E. coli and Pseudomonas aeruginosa Schroeter) were used for the binding assay. Microbes in the mid-logarithmic phase were collected via centrifugation at 12,000× g for 1 min, washed three times with sterilized Tris-buffered saline (TBS), and then resuspended in TBS. The purified rTc-CTL15 (600 µg/mL) was mixed with a microbe suspension in the presence or absence of 10 mM CaCl 2 with gentle rotation at 28 • C for 2 h. Then, the bacteria were pelleted via centrifugation and washed with TBS five times. The microorganism pellets were eluted with a 7% SDS solution for Western blot analysis.
As mentioned above, five microorganisms were also used to evaluate the agglutination activity of rTcCTL15. An agglutination assay was performed as previously reported [25]. Bacteria cultured in an LB medium overnight were harvested via centrifugation, washed three times with TBS, resuspended, and diluted in TBS at 2.0 × 10 8 cells/mL. Subsequently, the purified rTcCTL15 (200 µg/mL) and recombinant methyl-CpG-binding domain (rTcMBD) in T. castaneum (as a negative control) were incubated with a microorganism suspension in the presence or absence of 10 mM CaCl 2 for 1 h at 28 • C. The agglutination reactions were observed and photographed under a fluorescent microscope (Nikon, Shanghai, China).

Polysaccharide-Binding Assay
The binding activity of rTcCTL15 to PAMPs was examined using an enzyme-linked immunosorbent assay (ELISA) according to previous reports [8].
Briefly, 50 µL of LPS or PGN (80 µg/mL) was coated on the wells of a 96-well microplate at 37 • C overnight and then evaporated at 60 • C for 30 min. After blocking with BSA (100 µL/well of 1 mg/mL) in TBS for 2 h at 37 • C, 100 µL serial concentrations of rTcCTL15 (0-70 µg/mL) were added to each well and incubated at room temperature for 3 h. The plate was washed with TBS, and 100 µL/well of an anti-His mouse polyclonal antibody (TransGen, Beijing, China) diluted to 1:2000 with TBS was added and incubated at 37 • C for 1 h. The plate was rewashed with TBS, and 100 µL/well of a goat anti-mouse IgG horseradish peroxidase (HRP) conjugate (TransGen, Beijing, China) diluted to 1:5000 with TBS was added and incubated at 37 • C for 1 h. After washing with TBS, 100 µL/well of a soluble 3,3 ,5,5 -tetramethylbenzidine (TMB) solution (Solarbio, Beijing, China) was added and incubated at 28 • C for 10 min under darkness and then stopped with 100 µL/well of 2 mol L −1 H 2 SO 4 . After the termination of the reaction, an automatic enzyme-linked immunosorbent assay reader (BioTek, Winosky, VT, USA) was utilized to measure the absorbance at 450 nm. Each binding assay was repeated three times.

Double-Stranded RNA Synthesis and RNAi Assay
To synthesize double-stranded RNA (dsRNA), DNA templates of TcCTL15 and TcVer were amplified using a T7 promoter sequence at the 5 -end-linked primers. The PCR products (TcCTL15 (490 bp) and TcVer (609 bp)) were purified and were utilized to synthesize dsRNA in vitro using the TranscriptAid TM T7 High Yield Transcription Kit (Thermo Fisher Scientific, Shanghai, China). After synthesis, agarose gel electrophoresis was used to verify the quality and size of the dsRNA. Each dsRNA was quantified with spectrophotometry and diluted with diethylpyrocarbonate-treated water at an equal concentration of 2 µg/µL. The same amount of dsTcVer for the specific visible phenotype in the eyes was used as a control [26].
For the dsRNA-dependent knockdown experiment, a total of 200 ng of the dsTcCTL15 or dsTcVer was injected into the hemocoels of each larva using an Inject- Man 4 (Eppendorf, Hamburg, Germany). On day 3 post-injection, three individuals were randomly collected from the different experimental groups for RNA extraction and cDNA synthesis. The efficiency of dsRNA-mediated gene silencing was further confirmed using qRT-PCR. To study the effect of TcCTL15 on reproduction, dsRNA was injected into 5-day-old female and male pupae. Each microinjected pupa that developed to the adult stage was individually matched with a pupa with a different sex and then transferred into a glass bottle. The total number of eggs was counted and calculated for each adult pair for three consecutive days after mating. Meanwhile, to explore the roles of TcCTL15 in growth and development, dsTcCTL15 or dsTcVer were injected into the 12-day-old larvae and 1-day-old pupae. In addition, to evaluate the larval survival rate after TcCTL15 knockdown followed by bacterial infection, the TcCTL15-deficient or TcVer-deficient larvae were injected with E. coli or S. aureus. The number of dead individuals was counted every 24 h for the next seven days.

Expression Profiling of Transcription Factors and AMP Genes
To investigate the potential function of TcCTL15 in regulating the expression of downstream immune-related genes, the dsTcCTL15-injected larvae were challenged with E. coli or S. aureus. In the RNAi following bacterial injection, qRT-PCR was used to determine the mRNA levels of nine AMP genes and four transcription factor genes. The primers used for the qRT-PCR analysis of these immune genes are shown in Table S1.

Statistical Analysis
All analyses were carried out using GraphPad Prism v.8.3 (GraphPad Software, Inc., San Diego, CA, USA), and data are expressed as means ± SDs of triplicate experiments. The statistical significance between treatments was assessed using a one-way ANOVA followed by Tukey's honestly significant difference (HSD) test. Differences between the means of treatment and control groups were compared using Student's t-test for unpaired samples. Statistical significance was set at p ≤ 0.05.

Molecular Characteristics and Bioinformatics Analysis of TcCTL15
In the T. castaneum genome database, the gene encoding TcCTL15 was annotated as TC030754 and mapped to chromosome 3 (LG3). The 3336-base-pair open reading frame (ORF) of TcCTL15 encodes a single transmembrane protein with 1111 amino acid residues, a predicted molecular mass of 122 kDa, and an isoelectric point (pI) of 5.36. The protein domain analysis indicated that TcCTL15 had one coagulation factor 5/8 C-terminal domain, eleven CCP domains, one canonical CRD with a theoretical pI of 7.77, and a transmembrane region, whereas the signal peptide was absent. In addition, a WND (Trp-Asn-Asp) motif existed in the CRD of TcCTL15 (Supplementary Figure S1).

Spatiotemporal Profiles and Induced Expression of TcCTL15
To better investigate the characteristics of TcCTL15, its expression patterns were analyzed in different stages and larval tissues using qRT-PCR. The results revealed that TcCTL15 was expressed at all examined developmental stages and was especially highly expressed in the early pupae, late pupae, and early adults ( Figure 1A). Moreover, TcCTL15 was expressed in multiple tested tissues of 15-day-old larvae, with the greatest abundance being localized to the integument, followed by the CNS, fat bodies, and hemocytes ( Figure 1B). Figure 2 shows the time-course expression profiles of TcCTL15 after injections of PAMPs or bacteria. The TcCTL15 mRNA level was significantly up-regulated at 12 and 24 h and at 72 h post-infection with LPS and PGN, respectively (Figure 2A,B). The mRNA abundance of TcCTL15 was significantly increased at 60 and 24 h after infection with E. coil and S. aureus, respectively ( Figure 2C,D). To analyze the immune role of TcCTL15 in vivo, we knocked down the TcCTL15 expression using RNAi following bacterial infection and assessed the survival rate relative to the control. TcCTL15-deficient larvae infected with E. coli or S. aureus showed less survival than TcVer-deficient larvae infected with E. coli or S. aureus ( Figure 2E). These results indicate that TcCTL15 modulates immune activation in T. castaneum. alyzed in different stages and larval tissues using qRT-PCR. The results revealed that TcCTL15 was expressed at all examined developmental stages and was especially highly expressed in the early pupae, late pupae, and early adults ( Figure 1A). Moreover, TcCTL15 was expressed in multiple tested tissues of 15-day-old larvae, with the greatest abundance being localized to the integument, followed by the CNS, fat bodies, and hemocytes ( Figure  1B). Figure 2 shows the time-course expression profiles of TcCTL15 after injections of PAMPs or bacteria. The TcCTL15 mRNA level was significantly up-regulated at 12 and 24 h and at 72 h post-infection with LPS and PGN, respectively (Figure 2A,B). The mRNA abundance of TcCTL15 was significantly increased at 60 and 24 h after infection with E. coil and S. aureus, respectively ( Figure 2C,D). To analyze the immune role of TcCTL15 in vivo, we knocked down the TcCTL15 expression using RNAi following bacterial infection and assessed the survival rate relative to the control. TcCTL15-deficient larvae infected with E. coli or S. aureus showed less survival than TcVer-deficient larvae infected with E. coli or S. aureus ( Figure 2E). These results indicate that TcCTL15 modulates immune activation in T. castaneum. The tissue distribution of TcCTL15 in CNS, fat bodies, gut, integument, and hemocytes from 15-day-old larvae. Transcript levels were normalized using RPS3 and are shown as means ± SDs from three biological replicates. The different lowercase letters above each bar represent statistically significant differences at the 0.05 level and were analyzed using a one-way ANOVA followed by Tukey's test. The tissue distribution of TcCTL15 in CNS, fat bodies, gut, integument, and hemocytes from 15-day-old larvae. Transcript levels were normalized using RPS3 and are shown as means ± SDs from three biological replicates. The different lowercase letters above each bar represent statistically significant differences at the 0.05 level and were analyzed using a one-way ANOVA followed by Tukey's test. The survival rates of TcCTL15-deficient larvae infected with E. coil and S. aureus. TcVer-deficient larvae infected with E. coil and S. aureus were used as a control. Data were normalized against RPS3 and are shown as means ± SDs. The asterisks above the bars indicate significant differences. Differences were calculated using Student's t-test for unpaired samples (* p < 0.05; ** p < 0.01; *** p < 0.001).

rTcCTL15 Binds to and Agglutinates Bacteria
To further elucidate the role of TcCTL15 in antibacterial immunity, the recombinant plasmid pET-28a-TcCTL15 was expressed using an E. coli expression system. After IPTG induction, rTcCTL15 was successfully expressed as inclusion bodies ( Figure 3A). Then, The survival rates of TcCTL15-deficient larvae infected with E. coil and S. aureus. TcVer-deficient larvae infected with E. coil and S. aureus were used as a control. Data were normalized against RPS3 and are shown as means ± SDs. The asterisks above the bars indicate significant differences. Differences were calculated using Student's t-test for unpaired samples (* p < 0.05; ** p < 0.01; *** p < 0.001).

rTcCTL15 Binds to and Agglutinates Bacteria
To further elucidate the role of TcCTL15 in antibacterial immunity, the recombinant plasmid pET-28a-TcCTL15 was expressed using an E. coli expression system. After IPTG induction, rTcCTL15 was successfully expressed as inclusion bodies ( Figure 3A). Then, Cells 2023, 12, 608 8 of 18 rTcCTL15 was purified using denaturation and renaturation and Ni 2+ affinity chromatography. The SDS-PAGE and Western blotting results confirmed that purified rTcCTL15 migrated as a single distinct band ( Figure 3A,B), showing that rTcCTL15 was successfully purified. Then, the potential binding to various bacteria of rTcCTL15 was analyzed. The Western blotting results revealed that rTcCTL15 could directly bind to all the tested bacteria in the presence of CaCl 2 , while no binding activity was detected in the absence of CaCl 2 ( Figure 4A). To clarify whether the microbial binding ability of rTcCTL15 was mediated by cell surface PAMPs, we then examined the potential binding of rTcCTL15 to two typical PAMPs (LPS and PGN) using an ELISA assay. The results showed that rTcCTL15 could bind to these ligands in a dose-dependent manner ( Figure 4B). These results suggest that TcCTL15 is capable of binding to various bacteria by recognizing and interacting with their cell surface PAMPs.
The agglutination activity of rTcCTL15 was detected via incubation with five microorganisms. In the presence of Ca 2+ , rTcCTL15 agglutinated the G + S. aureus and B. thuringiensis and the G -E. coli and P. aeruginosa but not the G -B. subtilis ( Figure 4C). Notably, the agglutinating activity was completely lost in the absence of Ca 2+ and in the rTcMBD group ( Figure 4C), which demonstrated that TcCTL15 is a Ca 2+ -dependent C-type lectin. Based on these results, we concluded that TcCTL15 protects T. castaneum against pathogens via agglutinating pathogens.
Cells 2023, 12, x FOR PEER REVIEW rTcCTL15 could bind to these ligands in a dose-dependent manner ( Figure 4B sults suggest that TcCTL15 is capable of binding to various bacteria by recog interacting with their cell surface PAMPs.

Involvement of TcCTL15 in Regulating the Expression of Transcription Factors and AMP Genes
To address whether TcCTL15 regulates the expression of transcription factors and AMP genes during bacterial infection, S. aureus or E. coli infections were performed at 3 d after dsTcCTL15 or dsTcVer injections. The 12-day-old larvae were, respectively, injected with equal volumes of dsTcCTL15 or dsTcVer. After 3 d, 15-day-old larvae were infected with E. coli or S. aureus, respectively. The mRNA abundances of transcription factors and AMP genes were assessed using qRT-PCR at 24 h after the injection of E. coli or S. aureus. The dsTcVer + E. coli and dsTcVer + S. aureus groups were used as control groups. The results revealed that TcCTL15 was dramatically down-regulated post-dsRNA-treatment following an E. coli or S. aureus challenge, and the RNAi efficiencies (4 d after dsRNA injection) were 71.4% and 89.5%, respectively ( Figure 5A,D), compared with those of dsTcVer injections. Moreover, compared with the control beetles (dsTcVer + E. coli), transcription factors (Rel and Stat) and AMPs, including attacin2 (att2), attacin3 (att3), defensin3 (def3), cecropin2 (cecr2), and cecropin3 (cecr3), were markedly down-regulated in the TcCTL15-silenced larvae challenged with E. coli ( Figure 5B,C). Meanwhile, the expression levels of transcription factors (Dif1, Rel, and Stat) and AMPs, including attacin1 (att1), att2, defensin1 (def1), defensin2 (def2), and def3, were significantly decreased in the TcCTL15-silenced larvae challenged with S. aureus ( Figure 5E,F). All the above results indicate that TcCTL15 could be involved in the beetle's antibacterial immune responses by coordinating the gene expression of transcription factors and AMPs.

Involvement of TcCTL15 in Regulating the Expression of Transcription Factors and AMP Genes
To address whether TcCTL15 regulates the expression of transcription factors and AMP genes during bacterial infection, S. aureus or E. coli infections were performed at 3 d after dsTcCTL15 or dsTcVer injections. The 12-day-old larvae were, respectively, injected with equal volumes of dsTcCTL15 or dsTcVer. After 3 d, 15-day-old larvae were infected with E. coli or S. aureus, respectively. The mRNA abundances of transcription factors and AMP genes were assessed using qRT-PCR at 24 h after the injection of E. coli or S. aureus. The dsTcVer + E. coli and dsTcVer + S. aureus groups were used as control groups. The results revealed that TcCTL15 was dramatically down-regulated post-dsRNA-treatment following an E. coli or S. aureus challenge, and the RNAi efficiencies (4 d after dsRNA injection) were 71.4% and 89.5%, respectively ( Figure 5A,D), compared with those of dsTcVer injections. Moreover, compared with the control beetles (dsTcVer + E. coli), transcription factors (Rel and Stat) and AMPs, including attacin2 (att2), attacin3 (att3), defensin3 (def3), cecropin2 (cecr2), and cecropin3 (cecr3), were markedly down-regulated in the TcCTL15-silenced larvae challenged with E. coli ( Figure 5B,C). Meanwhile, the expression levels of transcription factors (Dif1, Rel, and Stat) and AMPs, including attacin1 (att1), att2, defensin1 (def1), defensin2 (def2), and def3, were significantly decreased in the TcCTL15-silenced larvae challenged with S. aureus ( Figure 5E,F). All the above results indicate that TcCTL15 could be involved in the beetle's antibacterial immune responses by coordinating the gene expression of transcription factors and AMPs.  profiling of AMP genes post-dsRNA-treatment followed by E. coli (C) and S. aureus (F) challenge. The bars represent means ± SDs. Each experiment was repeated at least in triplicate. The asterisks denote significant differences from the control groups based on t-tests for unpaired samples (* p < 0.05 and ** p < 0.01).

TcCTL15 Modulates Eclosion
To determine the potential functions of TcCTL15, we injected dsTcCTL15 or dsTcVer into the hemocoel of 1-day-old pupae. The RNAi efficiency of TcCTL15 was confirmed using qRT-PCR, and TcCTL15 transcripts were down-regulated to approximately 28% after 72 h of dsTcCTL15 relative to the expression after dsTcVer injection ( Figure 6B), showing that the knockdown of TcCTL15 was successful. Visible eclosion defects were observed after TcCTL15 knockdown ( Figure 6A). TcCTL15-deficient pupae had reduced viability, with 46% pupa eclosing deficiency and death in comparison to the dsTcVer group ( Figure 6C). Cells 2023, 12, x FOR PEER REVIEW 11 of 18 expression profiling of AMP genes post-dsRNA-treatment followed by E. coli (C) and S. aureus (F) challenge. The bars represent means ± SDs. Each experiment was repeated at least in triplicate. The asterisks denote significant differences from the control groups based on t-tests for unpaired samples (* p < 0.05 and ** p < 0.01).

TcCTL15 Modulates Eclosion
To determine the potential functions of TcCTL15, we injected dsTcCTL15 or dsTcVer into the hemocoel of 1-day-old pupae. The RNAi efficiency of TcCTL15 was confirmed using qRT-PCR, and TcCTL15 transcripts were down-regulated to approximately 28% after 72 h of dsTcCTL15 relative to the expression after dsTcVer injection ( Figure 6B), showing that the knockdown of TcCTL15 was successful. Visible eclosion defects were observed after TcCTL15 knockdown ( Figure 6A). TcCTL15-deficient pupae had reduced viability, with 46% pupa eclosing deficiency and death in comparison to the dsTcVer group ( Figure  6C). The percent of individuals with abnormal phenotypes and mortality after injecting dsRNA. At least 36 individuals were observed and counted in each group, with three biological replicates. The bars are expressed as means ± SDs. The asterisks denote a significant difference from the control. Differences were calculated using Student's t-test for unpaired samples (*** p < 0.001).
It is well-known that insect metamorphosis and development are mainly controlled by neuropeptides, ecdysone (20E), and juvenile hormone (JH) [27]. To investigate the mechanisms of suppressed metamorphosis, we detected the mRNA levels of genes involved in the synthesis and metabolism of 20E and JH and the regulation of eclosion after TcCTL15 silencing. The results showed that the expression of spook, shadow, and shade, which are involved in the 20E synthesis pathway, was down-regulated in TcCTL15-deficient pupae, while there was no significant effect on the expression levels of E74, Broad complex (Br-C), and the fushi tarazu factor-1 (Ftz-F1) ( Figure 7A). The expression level of the key enzyme gene in the JH synthesis pathway, JH acid methyltransferase (JHAMT), exhibited no obvious changes between TcCTL15-deficient pupae and TcVer-deficient pupae ( Figure 7B). The receptors of eclosion-related neuropeptides, crustacean cardioactive The percent of individuals with abnormal phenotypes and mortality after injecting dsRNA. At least 36 individuals were observed and counted in each group, with three biological replicates. The bars are expressed as means ± SDs. The asterisks denote a significant difference from the control. Differences were calculated using Student's t-test for unpaired samples (*** p < 0.001).
It is well-known that insect metamorphosis and development are mainly controlled by neuropeptides, ecdysone (20E), and juvenile hormone (JH) [27]. To investigate the mechanisms of suppressed metamorphosis, we detected the mRNA levels of genes involved in the synthesis and metabolism of 20E and JH and the regulation of eclosion after TcCTL15 silencing. The results showed that the expression of spook, shadow, and shade, which are involved in the 20E synthesis pathway, was down-regulated in TcCTL15-deficient pupae, while there was no significant effect on the expression levels of E74, Broad complex (Br-C), and the fushi tarazu factor-1 (Ftz-F1) ( Figure 7A). The expression level of the key enzyme gene in the JH synthesis pathway, JH acid methyltransferase (JHAMT), exhibited no obvious changes between TcCTL15-deficient pupae and TcVer-deficient pupae ( Figure 7B). The receptors of eclosion-related neuropeptides, crustacean cardioactive peptide receptors (CCAPR1 and CCAPR2), were significantly decreased after TcCTL15 knockdown, whereas Rickets, encoding the bursicon receptor, did not change ( Figure 7B). These results indicate that the eclosion failure caused by TcCTL15 knockdown is mediated by ecdysone synthesis and eclosion-related regulatory genes. Altogether, these results indicate that TcCTL15 is necessary for the viability of T. castaneum and is involved in eclosion. knockdown, whereas Rickets, encoding the bursicon receptor, did not change ( Figure 7B). These results indicate that the eclosion failure caused by TcCTL15 knockdown is mediated by ecdysone synthesis and eclosion-related regulatory genes. Altogether, these results indicate that TcCTL15 is necessary for the viability of T. castaneum and is involved in eclosion. The transcript levels of the key genes of JH biosynthesis and eclosion regulation after injecting dsTcCTL15 in 1-day-old pupae. The bars are expressed as means ± SDs. The asterisks denote significant differences from the controls. Differences were calculated using Student's t-test for unpaired samples (* p < 0.05 and ** p < 0.01).

Knocking down TcCTL15 Affects Female Egg-Laying and Ovary and Testis Development
To determine the function of TcCTL15 in T. castaneum reproduction, RNAi was used to knock down the expression of TcCTL15 in 5-day-old pupae. When compared with the wildtype (WT), the silencing of TcCTL15 resulted in a dramatic reduction in egg numbers ( Figure 8B). dsTcCTL15 beetles laid an average of about 4.76 ± 0.57 eggs/female during 3 d of mating, while about 37.17 ± 2.18 eggs were produced by the WT females. When the mating experiments were performed on WT females and dsTcCTL15 males, the females laid a normal number of eggs. In contrast, dsTcCTL15 females crossed with WT males laid fewer eggs ( Figure 8B). Further observation of the ovary and testis development status revealed that ovaries were much smaller in the dsTcCTL15-injected females than in dsTcVer-injected females of the same age ( Figure 8D), whereas the testis morphology group exhibited no obvious differences between the dsTcCL15 and dsTcVer-injected groups ( Figure 8E).
To investigate the mechanisms of TcCTL15 in reproduction, we examined the expression of genes involved in the reproduction-related gene TcVg, JH-biosynthesis-related genes such as TcJHAMT and TcFDH, and the transcription factor TcFOXO, which has been reported to act in the regulation of Vg expression and JH signaling [28]. As shown in Figure 8C, the expression of TcVg and TcFDH was significantly down-regulated in dsTcCTL15-injected females, whereas the levels of TcJHAMT and TcFOXO transcripts exhibited no obvious changes between the dsTcCTL15 and dsTcVer groups. Taken together, these results indicate that the knockdown of TcCTL15 resulted in a reduction in TcVg and TcFDH at the mRNA level, caused female reproductive capacity and ovary development defects, and finally led to impaired fecundity. The transcript levels of the key genes of JH biosynthesis and eclosion regulation after injecting dsTcCTL15 in 1-day-old pupae. The bars are expressed as means ± SDs. The asterisks denote significant differences from the controls. Differences were calculated using Student's t-test for unpaired samples (* p < 0.05 and ** p < 0.01).

Knocking down TcCTL15 Affects Female Egg-Laying and Ovary and Testis Development
To determine the function of TcCTL15 in T. castaneum reproduction, RNAi was used to knock down the expression of TcCTL15 in 5-day-old pupae. When compared with the wildtype (WT), the silencing of TcCTL15 resulted in a dramatic reduction in egg numbers ( Figure 8B). dsTcCTL15 beetles laid an average of about 4.76 ± 0.57 eggs/female during 3 d of mating, while about 37.17 ± 2.18 eggs were produced by the WT females. When the mating experiments were performed on WT females and dsTcCTL15 males, the females laid a normal number of eggs. In contrast, dsTcCTL15 females crossed with WT males laid fewer eggs ( Figure 8B). Further observation of the ovary and testis development status revealed that ovaries were much smaller in the dsTcCTL15-injected females than in dsTcVer-injected females of the same age ( Figure 8D), whereas the testis morphology group exhibited no obvious differences between the dsTcCL15 and dsTcVer-injected groups ( Figure 8E).
To investigate the mechanisms of TcCTL15 in reproduction, we examined the expression of genes involved in the reproduction-related gene TcVg, JH-biosynthesis-related genes such as TcJHAMT and TcFDH, and the transcription factor TcFOXO, which has been reported to act in the regulation of Vg expression and JH signaling [28]. As shown in Figure 8C, the expression of TcVg and TcFDH was significantly down-regulated in dsTcCTL15-injected females, whereas the levels of TcJHAMT and TcFOXO transcripts exhibited no obvious changes between the dsTcCTL15 and dsTcVer groups. Taken together, these results indicate that the knockdown of TcCTL15 resulted in a reduction in TcVg and TcFDH at the mRNA level, caused female reproductive capacity and ovary development defects, and finally led to impaired fecundity.

Discussion
In this study, TcCTL15 was demonstrated to be a member of the CTL-X group and the sole selectin in the T. castaneum genome, which was homologous to that of D. melanogaster furrowed. TcCTL15 contained multiple domains, such as CCP, CRD, and a single transmembrane region, which were common to the structure of selectin family members [12,15]. TcCTL15, as a PRR, could recognize, bind to, and agglutinate bacteria using its CRD and activate immune signaling pathways to induce the production of AMPs, thus modulating insect innate immunity, which was consistent with the functions of TcCTL12 [21], indicating that TcCTL15 could also participate in various immune responses. In addition, CTL-Xs were reported to be involved in developmental regulation in insects [5]. A recent study showed that TcCTL12 was required for the metamorphosis, reproduction, and muscle remodeling of T. castaneum [21]. Here, TcCTL15 could regulate essential physiological processes, including metamorphosis and reproduction, implying that TcCTL15 could also be involved in numerous developmental processes. Above all, the major functions of TcCTL15 in innate immunity and development in T. castaneum were in accordance with vertebrate selectins involved in immunity and furrowed (required for growth and development in D. melanogaster), which might be a consequence of analogous functional evolution.
It is known that CTLs bind to carbohydrates on the surfaces of microbes in a Ca 2+dependent manner through characteristic motifs located in the CRD, such as EPN (Glu-Pro-Asn), QPD (Gln-Pro-Asp), and WND [29,30]. EPN and QPD are specific for mannose and galactose, respectively, and WND can increase the affinity and specificity of CTLs binding to different carbohydrates [29,30]. The carbohydrate-binding region of the

Discussion
In this study, TcCTL15 was demonstrated to be a member of the CTL-X group and the sole selectin in the T. castaneum genome, which was homologous to that of D. melanogaster furrowed. TcCTL15 contained multiple domains, such as CCP, CRD, and a single transmembrane region, which were common to the structure of selectin family members [12,15]. TcCTL15, as a PRR, could recognize, bind to, and agglutinate bacteria using its CRD and activate immune signaling pathways to induce the production of AMPs, thus modulating insect innate immunity, which was consistent with the functions of TcCTL12 [21], indicating that TcCTL15 could also participate in various immune responses. In addition, CTL-Xs were reported to be involved in developmental regulation in insects [5]. A recent study showed that TcCTL12 was required for the metamorphosis, reproduction, and muscle remodeling of T. castaneum [21]. Here, TcCTL15 could regulate essential physiological processes, including metamorphosis and reproduction, implying that TcCTL15 could also be involved in numerous developmental processes. Above all, the major functions of TcCTL15 in innate immunity and development in T. castaneum were in accordance with vertebrate selectins involved in immunity and furrowed (required for growth and development in D. melanogaster), which might be a consequence of analogous functional evolution.
It is known that CTLs bind to carbohydrates on the surfaces of microbes in a Ca 2+dependent manner through characteristic motifs located in the CRD, such as EPN (Glu-Pro-Asn), QPD (Gln-Pro-Asp), and WND [29,30]. EPN and QPD are specific for mannose and galactose, respectively, and WND can increase the affinity and specificity of CTLs binding to different carbohydrates [29,30]. The carbohydrate-binding region of the TcCTL15 lectin domain (QPN) was a hybrid between QPD and EPN, suggesting that TcCTL15 might recognize both galactose and mannose and bind to more potential carbohydrate ligands.
Moreover, the TcCTL15 lectin domain also contained the WND motif, indicating that the multi-motif of TcCTL15 might possess more sites involved in PAMP recognition and binding against various pathogens in T. castaneum. In this study, rTcCTL15 was able to bind microbial cell wall components (LPS and PGN) and exhibited strong binding activities to all the tested microbes in the presence of Ca 2+ , suggesting that TcCTL15 could serve as a PRR for the recognition and binding of various foreign pathogens.
In invertebrates, agglutination activity is a principal feature of lectin and a crucial innate immune method of attacking various invading pathogens [31,32]. In the case of rTcCTL15, it displayed agglutinating activity against G + S. aureus and B. thuringiensis and G − E. coli and P. aeruginosa but not against G − B. subtilis ( Figure 4C). As stated above, rTcCTL15 could bind to various microbes and different PAMPs, but it did not possess a wide range of agglutination against all tested microorganisms, suggesting that TcCTL15 was probably a selective CTL with a relatively narrow range of agglutinating activity. It is highly possible that the mechanisms of binding and agglutinating are different and that the binding was the initial step of immunity, while agglutination might rely on other factors [33,34]. A similar microbial agglutination activity has been reported in CsCTL1 from the tongue sole Cynoglossus semilaevis and CaNTC from the crucian carp Carassius auratus [35,36]. It was suggested that TcCTL15 could perform an antimicrobial activity by agglutinating bacteria.
It has been reported that most CTLs are tissue-specific. Hemocytes are the major tissue of hemolymph lectins and express a variety of immune-related genes to improve immune responses [37][38][39][40][41]. TcCTL15 was expressed in many immune-related tissues and was mainly detectable in the integument. Our results were consistent with those from TcCTL6 of T. castaneum and BmCTL-S2 of Bombyx mori [7,42]. As an important immune tissue of insects, the integument plays a key role in the immune system by supporting the epithelial barrier against the invasion of microorganisms [43]. The tissue expression profile of TcCTL15 indicated that it might participate in various immune responses. Moreover, TcCTL15 transcripts could be significantly up-regulated after LPS, PGN, E. coli, and S. aureus challenges (Figure 2), showing that TcCTL15 could be effectively induced by PAMPs and pathogen stimulation and that it might be involved in defending against pathogens. Similar results were also reported in other CTLs of T. castaneum [7,8,21,25]. In addition, the expression of TcCTL15 could also be down-regulated by PAMPs and bacterial treatments (Figure 2), which might indicate that bacteria might resist host immune attacks via decreasing expression of TcCTL15 [44]. These data suggest that the expression pattern of TcCTL15 was not fixed and was complex against different immune challenges, thus demonstrating that it also exerted distinct immune functions.
Most studies of CTLs have focused on their roles in regulating AMPs, which are a large group of short peptides that directly kill or clear infected bacteria, fungi, and viruses [45,46]. For instance, EsLecH in the crab Eriocheir sinensis could regulate AMP expression via JNK phosphorylation in response to bacterial invasion [46]. Similarly, a novel chimeric CTL of the shrimp Marsupenaeus japonicus could induce the expression of selected AMPs via the JAK/STAT pathway [11]. In the present study, the E. coli challenge could decrease transcription factor (Rel and Stat) and AMP (att2, att3, def3, cecr2, and cecr3) expression in TcCTL15-silenced larvae. Meanwhile, the S. aureus challenge decreased the expression of transcription factors (Dif1, Rel, and Stat) and AMPs (att1, att2, def1, def2, and def3). Knockdown of TcCTL15 followed by bacterial infection resulted in a decline in transcription factors and AMP transcripts, which might explain why injections of E. coli or S. aureus into TcCTL15-silenced larvae caused their mortality to increase significantly ( Figure 2E). Our data demonstrated that TcCTL15 might show antimicrobial activities by positively activating classical immune pathways to selectively induce the expression of AMPs in response to E. coli and S. aureus challenges.
To some extent, the developmental profiles of the genes reflect their functional importance. For example, TcCTL12 was highly expressed in the late pupae and early adults, implying that it was crucial for the development of epidermis, muscle, and reproduc-tion [21]. Moreover, the high mRNA level of TcCTL15 was detected in the early and late pupae and early adults ( Figure 1A), indicating that it might be involved in the metamorphosis and development of T. castaneum. RNAi of TcCTL15 in 1-and 5-day-old pupae caused suppressed metamorphosis and reproduction, respectively. It is known that insect CTL-Xs are mainly associated with cell adhesion and developmental regulation [5]. However, how CTL-Xs are involved in developmental regulation still needs to be explored.
Insect metamorphosis is mainly controlled by 20E and JH [47]. The ecdysone synthesis genes spook, shadow, and shade were significantly decreased in dsTcCTL15-injected beetles. A reduction in the expression of these genes will hinder the synthesis of ecdysone, which will have a fatal impact on the growth and development of insects [48]. The primary response genes of 20E, such as E74, Br-C, and Ftz-F1, have important regulatory roles in insect metamorphosis and development [49][50][51]. However, their mRNA abundance was not significantly affected between the dsTcCTL15-injected beetles and dsTcVer-injected beetles ( Figure 7A). Hence, we demonstrated that the death from eclosion deficiency in TcCTL15-silenced pupae might have been largely due to the suppressed ecdysone synthesis. Insect metamorphosis processes are not only affected by 20E and JH but are also regulated by crustacean cardioactive peptide, bursicon, and their receptors [52]. A knockdown of CCAPR2 and Rickets showed eclosion arrest in T. castaneum [52]. The phenomenon of eclosion defect was also observed in dsTcCTL15-injected individuals. Additionally, CCAPR1 and CCAPR2 were down-regulated, which indicates that the knockdown of TcCTL15 affected the expression of genes related to eclosion regulation, leading to eclosion failure and then mortality.
The involvement of TcCTL12 (CTL-X) in the reproduction of T. castaneum has been preliminarily studied recently [21]. In this study, a significant reduction in the fertility of offspring and dysplastic ovary were observed in dsTcCTL15-injected female individuals. To our knowledge, this was the first time that it was found that CTLs can affect ovary development in insects, which differs from the previous findings that the knockdown of TcCTL12 resulted in thinner oviducts [21]. It is rare for direct evidence to clarify the relationships between CTLs and the reproduction of insects. In the cotton bollworm Helicoverpa armigera, CTL14 interacted with Vg and participated in innate immune responses under a Beauveria bassiana challenge [53]. The reproductive success of insects mainly depends on Vg biosynthesis and its uptake process during vitellogenesis [54]. The mRNA level of TcVg was significantly decreased in dsTcCTL15-injected females, suggesting that the reduction in Vg levels might be involved in impaired ovary maturation and fecundity. Similarly, the association of the suppression of CTL1 and decreased Vg expression in embryos was also found in T. castaneum [6]. However, the detailed mechanisms remain largely unknown. In adults, JH has well-known roles in regulating insect reproduction, stimulating vitellogenesis, and oocyte maturation, and a knockdown of JH biosynthesis or action-related genes affects Vg biosynthesis or uptake [55,56]. Here, we found that an RNAi-mediated knockdown of TcCTL15 expression significantly down-regulated the mRNA levels of the JH-biosynthesis-related gene TcFDH, which might cause a reduction in Vg mRNA levels and abnormality of reproduction. Taken together, these results indicate that the knockdown of TcCTL15 resulted in reductions in TcVg and TcFDH at the mRNA level, caused female reproductive capacity and ovary development defects, and finally led to impaired fecundity. A recent study found that TcFOXO could bind to the conserved sequence motif of TcFDH and negatively regulate the expression of TcFDH to participate in beetle reproduction [55]. In this study, no difference in TcFOXO at the transcript level was observed between dsTcCL15-injected group and the dsTcVer-injected group. Hence, the underlying mechanisms of TcCTL15 affecting reproduction need further investigation.

Conclusions
TcCTL15, a multiple-domain transmembrane protein of CTL-X was identified in T. castaneum. rTcCTL15 could directly bind to G + and Gbacteria through certain PAMPs and agglutinate various bacteria in vitro. TcCTL15 likely mediated the expression of AMPs by regulating classical immune pathways against pathogens in vivo. Moreover, it was required for metamorphosis and reproduction. These results showed that TcCTL15 plays crucial roles in the innate immunity, metamorphosis, and reproduction of T. castaneum, suggesting it might be a potential target transmembrane protein for pest control. Taken together, the current study deepens our understanding of the functional diversification of CTL-X in T. castaneum and its underlying mechanisms in regulating immunity, growth, and development.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/cells12040608/s1, Figure S1: The nucleotide sequence and deduced amino acid sequence of TcCTL15; Figure S2: Multiple sequence alignment of TcCTL15 with homologues from other model insects; Table S1: Oligonucleotide primers used in the current study.