Identification and Functional Insights of Knickkopf Genes in the Larval Cuticle of Leptinotarsa decemlineata

Simple Summary

The Colorado potato beetle, Leptinotarsa decemlineata, poses a significant threat to potato crops worldwide, causing substantial agricultural damage. To better understand how these pests develop and potentially find ways to control them, we studied a group of genes called Knickkopf (Knk) genes in the larvae of the beetle. These genes are crucial for forming and maintaining the insect’s outer shell, known as the cuticle, which protects them from the environment. We identified four LdKnk-family genes in the beetle and analyzed when and where these genes are expressed during the insect’s growth stages. By using RNA interference (RNAi) to silence these genes, we observed that larvae became weaker, with damaged cuticles and higher death rates. These findings indicate that LdKnk-family genes are vital for the beetle’s survival and development. Targeting LdKnk-family genes could provide a new strategy for pest control, potentially weakening the beetles’ defenses and making them more susceptible to environmental factors and insecticides, ultimately helping to protect potato crops.

Abstract

The Colorado potato beetle (Leptinotarsa decemlineata) is a major pest of potato crops. While Knickkopf (Knk) genes are essential for insect cuticle formation, their roles in pests like L. decemlineata remain unclear. This study aims to identify and characterize Knk genes in L. decemlineata and explore their functions in larval development and cuticle integrity. We used genomic and transcriptomic databases to identify LdKnk-family genes, validated through RT-PCR and RACE. Gene expression was analyzed at various developmental stages and tissues using qRT-PCR. RNA interference (RNAi) and Transmission electron microscopy (TEM) were applied to determine the functional roles of these genes. Four LdKnk-family genes were identified. Spatio-temporal expression analysis indicated significant gene expression during larval molting and pupal stages, especially in the epidermis. RNAi experiments showed that silencing LdKnk and LdKnk3-5′ led to reduced larval weight, cuticle thinning, and increased mortality, while LdKnk3-FL knockdown caused abnormal cuticle thickening and molting disruptions. LdKnk2 knockdown increased epicuticle and endocuticle thickness without visible phenotypic changes. The study highlights the essential roles of LdKnk-family genes in maintaining cuticle structure and integrity, suggesting their potential as targets for RNAi-based pest control.

1. Introduction

The insect cuticle is essential for maintaining an insect’s shape and providing protection against dehydration, pathogens, and environmental damage [1,2]. To accommodate growth, insects periodically shed their cuticle through molting, replacing the old cuticle with a new one [3,4,5,6]. TEM has been crucial for studying the ultrastructure of the insect cuticle, revealing its multi-layered architecture and the organization of chitin microfibrils. TEM studies have shown that the cuticle consists of three layers forming an extracellular matrix. The outer waxy envelope (env) acts as the primary barrier against water loss and foreign substances. Beneath it, the protein-rich epicuticle (epi) contributes to the insect’s overall body shape. The innermost procuticle (pro), composed of a protein-chitin matrix, provides stability [7,8,9].

Chitin, a homopolymer of β-1,4-linked N-acetylglucosamine units, is the main structural component of the insect cuticle [10,11,12,13,14,15]. When associated with proteins, chitin arranges into microfibrils that align parallel to each other, forming stacked helicoidal laminae [16,17,18,19]. Cuticle-associated proteins significantly influence chitin organization [1,20,21]. Among these, the chitin-binding protein Knickkopf (Knk) has been extensively studied in Drosophila melanogaster, Tribolium castaneum, and Locusta migratoria [22]. In D. melanogaster, Knk is crucial for chitin organization within the tracheal system, the larval body, and the adult wing cuticle [23,24]. Knk proteins comprise a signal peptide, two DM13 domains of unknown function, and a dopamine-monooxygenase (DOMON) domain essential for its activity [25]. It interacts with the chitin-binding protein Obstructor-A (Obst-A) and the chitin deacetylase Serpentine (Serp) to form a complex at the apical membrane, facilitating the deposition and organization of chitin fibers [26,27].

Studies on T. castaneum have revealed that Knk proteins bind to chitin, protecting it from enzymatic degradation during molting and ensuring proper laminar organization, playing roles in cuticle formation at different developmental stages and in different parts of the insect anatomy [28,29,30]. Additionally, in L. migratoria, LmKnk contributes to correct chitin content, pore canal formation, and lipid deposition during molting, with LmKnk3-5′ being essential for cuticle formation [31,32]. These findings suggest that Knk-family proteins may exhibit functional diversity across different insect species.

The Colorado potato beetle, L. decemlineata, is a significant agricultural pest that poses a severe threat to potato crops and farmers’ livelihoods [33,34]. Although insecticides have historically been effective in suppressing L. decemlineata populations, the emergence of insecticide-resistant populations has created significant management challenges [35,36,37]. Understanding the genetic and molecular mechanisms of cuticle formation in this species could offer valuable insights for pest management.

In this study, we aim to identify and characterize Knk genes in L. decemlineata and investigate their roles in larval development and cuticle integrity. We identified four Knk-family genes: LdKnk, LdKnk2, LdKnk3-full length (LdKnk3-FL), and LdKnk3-5′. Using qRT-PCR, we analyzed their expression across various developmental stages and tissues. RNAi experiments assessed their impact on larval development and cuticle structure. Our results demonstrate that Knk-family genes exhibit developmentally regulated and tissue-specific expression patterns. Specifically, LdKnk, LdKnk3-FL, and LdKnk3-5′ are essential for larval development in L. decemlineata, as confirmed by TEM analysis. These findings highlight potential targets for pest management and contribute to understanding the function of Knk genes in insects.

2. Materials and Methods

2.1. Experimental Insect

The rearing of L. decemlineata followed standardized protocols as previously described [38]. Adults were collected from potato fields in Urumqi, Xinjiang (43.82° N, 87.61° E) during their spring emergence. The beetles were kept at 28 ± 1 °C with a 14:10 light-dark cycle and 50–60% humidity and were fed fresh potato leaves. Eggs were laid on the underside of potato leaves and hatched into larvae within seven days. The larvae underwent four instars, with the first three instars, each lasting two days and the fourth instar lasting 3.5 days. After the larval period, the fourth-instar larvae ceased feeding, burrowed into the soil for pupation, and subsequently emerged as adults.

2.2. Identification of LdKnk-Family Genes

Using the known amino acid sequences of Knk proteins from T. castaneum, D. melanogaster, and L. migratoria as queries, a genome-wide TBLASTN search was conducted on the L. decemlineata genome data (https://www.hgsc.bcm.edu/arthropods/colorado-potato-beetle-genome-project (accessed on 1 January 2024), PRJNA854273). This search resulted in the identification of four putative Knk-like genes in L. decemlineata. These sequences were further used to identify additional Knk-like genes from the nonredundant (nr) NCBI nucleotide database and transcriptome databases (PRJNA464380 and our unpublished database) of L. decemlineata.

The sequences were verified via polymerase chain reaction (PCR) using primers listed in Table S1. They were then completed by 5′- and 3′-RACE using the SMARTer RACE cDNA amplification kit (Takara Bio., Dalian, China). Table S1 provides the gene-specific and nested primers for the 5′ and 3′ ends. The full-length Knk-family gene sequences have been submitted to GenBank, with Accession numbers listed in

Table 1. Characteristics of four LdKnk-family genes in the L. decemlineata.

Gene Name	Accession Number	cDNA (bp)	Coding Region	5′-UTR (bp)	3′-UTR (bp)	aa	pl	Mw(kDa)
LdKnk	PP962378	2118	61–2088	60	30	675	6.35	76.03
LdKnk2	PP962379	2142	1–2109	0	33	702	5.09	78.90
LdKnk3-FL	PP962380	5100	49–4236	48	864	1395	7.41	156.48
LdKnk3-5′	PP962381	1042	61–975	60	67	304	7.65	33.81

UTR–untranslated region; aa–amino acid; pl–isoelectric point; Mw–molecular weight.

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2.3. Bioinformatics and Phylogenetic Analysis

Knk proteins open reading frames (ORFs) and amino acid sequences were identified, and their theoretical isoelectric points and molecular weights were calculated using ExPASy’s online server (https://web.expasy.org/peptide_mass/, accessed on 1 January 2024). The results are detailed in Table 1. The exon-intron organization was predicted by aligning ORFs with their corresponding genomic sequences, and these organizations were illustrated using Adobe Illustrator CS6. Domain analysis was performed using SMART software (http://smart.embl-heidelberg.de/ (accessed on 1 January 2024)).

To investigate evolutionary relationships, a phylogenetic tree based on Knk amino acid sequences from ten insect species was constructed using MEGA6 software. The unrooted phylogenetic tree was generated using the neighbor-joining method and the Jones-Taylor-Thornton (JTT) substitution model, based on full-length protein sequence alignments. Bootstrap analysis with 1000 replications was performed, and bootstrap values greater than 50% are indicated on the tree. Sequence accession numbers for Knk genes from various insects are provided in Table S2.

2.4. Expression Analysis of LdKnk-Family Genes

Quantitative real-time PCR (qRT-PCR) was employed to evaluate the temporal and spatial expression patterns of LdKnk-family genes. Gene-specific primers (Table S1) were designed using the PRIMER PREMIER 5 program. cDNA templates were generated from different developmental stages and tissues.

For developmental stage expression analysis, cDNA templates were obtained from eggs, first- to third-instar larvae, wandering larvae, prepupae, and pupae at one-day intervals, and from fourth-instar larvae at twelve-hour intervals. For tissue-specific expression analysis, RNA samples were extracted from the epidermis, foregut, midgut, hindgut, Malpighian tubules, fat body, hemocytes, and trachea of day-3 fourth-instar larvae. Each sample consisted of RNA from 5–30 individuals and was processed in triplicate.

RNA extraction was performed using the SV Total RNA Isolation System (Promega), followed by DNase I treatment to remove residual DNA. mRNA expression levels were quantified using the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the TaKaRa SYBR Premix Ex Taq kit, following a standard PCR protocol. Internal control genes (Table S1) were used for normalization as per our published results [39]. RT negative controls (without reverse transcriptase) and non-template negative controls were included to ensure the absence of genomic DNA and contamination. Each sample was tested in triplicate. Data analysis was performed using the 2^−ΔΔCT method, normalized to the geometric mean of the internal control genes, and validated according to the MIQE guidelines [40].

2.5. Preparation of dsRNA Using Bacterial Expression

To enhance the reliability of RNAi experiments, two different dsRNA fragments were designed for each LdKnk-family gene. The dsRNAs were prepared as described previously [41]. Unique regions of each gene were amplified using gene-specific primers (Table S1) and introduced into a T7 dual promoter expression system in Escherichia coli HT115 (DE3) cells, which lack RNase III, to produce dsRNA. As a control, dsRNA targeting the enhanced green fluorescent protein (EGFP) gene from Aequorea victoria was produced to monitor the nonspecific effects of dsRNA.

Bacterial cultures were inoculated and grown to an OD₆₀₀ of 1.0. dsRNA expression and were then induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. The dsRNA was extracted and confirmed by electrophoresis on a 1% agarose gel. Post-verification, the bacteria were harvested by centrifugation at 5000× g for 10 min and resuspended in 0.05 M phosphate-buffered saline (PBS, pH 7.4) at a 1:1 ratio for bioassays. The final concentration of dsRNA in the bacterial suspension was approximately 0.5 g/mL.

2.6. Dietary dsRNA Bioassays

Bioassays were conducted as previously described [14,15]. Newly ecdysed second-instar larvae were selected to assess the impact of dsRNA ingestion. Ten larvae per treatment group were fed leaves immersed in specific dsRNA targeting LdKnk-family genes for three days, following a four-hour starvation period. Control groups included a blank control (PBS buffer) and a negative control (dsGFP). Each treatment was replicated 15 times.

On the third day, total RNA was extracted from three replicates of each treatment group (10 larvae per replicate) to assess target gene transcript levels using qRT-PCR. The remaining 12 replicates per treatment were provided with untreated leaves under standard conditions for further observation. This included phenotypic analysis, weight measurements on days 3 and 6, and larval survival rates on day 6. Additionally, cuticle structure analysis was performed using TEM, and quantifications of overall procuticle, epicuticle, envcuticle, and layered procuticle thickness were measured using ImageJ software based on larval cuticle TEM images. Each bioassay was performed with three biological replicates to ensure accuracy and reproducibility.

2.7. Transmission Electron Microscopy (TEM) Analysis

For TEM analysis, larvae were dissected on the third day of the fourth instar following the LdKnk-family genes knockdown to obtain integument samples. The dorsal integument was excised and fixed overnight in 3% glutaraldehyde at 4 °C. Samples were then rinsed three times with PBS buffer (pH 7.2) for 15 min each and postfixed with 1% osmium tetroxide (OsO₄) solution for 1–2 h. Following postfixation, the samples were rinsed in 0.1 M PBS (pH 7.2) and dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 95%, and 100%).

The dehydrated samples were embedded in Epon 812 at room temperature for an appropriate duration. The resin blocks were then trimmed to 60–70 nm thickness using an ultramicrotome (Leica UCT, Wetzlar, Germany) and transferred onto copper grids. Sections were stained sequentially with uranyl acetate and lead citrate. After drying, images were captured using a Transmission Electron Microscope (Hitachi HT-7800, Tokyo, Japan) at 80 kV.

2.8. Data Analysis

Data from three biological replicates were pooled and presented as means ± SE. Statistical analyses were conducted using One-way ANOVA with the Tukey-Kramer test to determine significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001), utilizing Prism 10 software. No significant differences were found between dsRNAs targeting two distinct regions of each of the four genes: dsKnk-1 and dsKnk-2, dsKnk2-1 and dsKnk2-2, dsKnk3-FL-1 and dsKnk3-FL-2, dsKnk3-5′-1 and dsKnk3-5′-2. Consequently, data from the RNAi treatments for each gene were combined for further analysis.

3. Results

3.1. Identification and Characterization of LdKnk-Family Genes in L.decemlineata

To identify Knk genes in L. decemlineata, comprehensive searches were conducted using genomes [42,43] and transcriptome databases [44]. Amino acid sequences of Knk genes from T. castaneum, D. melanogaster, and L. migratoria were used as queries in BLAST searches. which resulted in the identification of four candidate gene fragments encoding LdKnk-like proteins.

These gene fragments were validated through reverse-transcription PCR (RT-PCR) followed by sequencing. The application of rapid amplification of cDNA ends (RACE) subsequently identified four full-length LdKnk gene sequences in L. decemlineata, named LdKnk, LdKnk2, LdKnk3-FL, and LdKnk3-5′. The accession numbers are listed in Table 1.

The open reading frames (ORFs) and protein characteristics of these genes are as follows. LdKnk comprises 2028 nucleotides encoding a 76.03 kDa protein with 675 amino acids and an isoelectric point (pI) of 6.35. LdKnk2 comprises 2109 nucleotides encoding a 78.90 kDa protein with 702 amino acids and a pI of 5.09. LdKnk3-FL comprises 4188 nucleotides encoding a 156.48 kDa protein with 1395 amino acids and a pI of 7.41. LdKnk3-5′ comprises 915 nucleotides encoding a 33.81 kDa protein with 304 amino acids and a pI of 7.65. Additionally, two alternative splicing transcripts of LdKnk3 were identified. LdKnk3-FL encodes a full-length protein, while LdKnk3-5′ encodes a C-terminally truncated protein (Table 1).

Comparing the full-length cDNA sequences with the genomic DNA sequences of L. decemlineata allowed us to construct gene structures. Both LdKnk and LdKnk2 contain nine exons, LdKnk3-FL contains eleven exons, and LdKnk3-5′ contains six exons. Notably, the first 281 nucleotides from the 5′-end of the LdKnk2 cDNA sequence could not be located in the genome database, preventing the determination of the first intron location (indicated by a grey line). Exons 1–5 of LdKnk3-5′ are identical to the 5′ end sequence of LdKnk3-FL (Figure 1A).

The domain architectures of these four Knk proteins were predicted using their amino acid sequences and SMART software. All four predicted proteins contain a signal peptide and two N-terminal DM13 domains. Additionally, LdKnk, LdKnk2, and LdKnk3-FL contain a dopamine monnoxygenase (DOMON) domain in the middle, which is absent in LdKnk3-5′ (Figure 1B).

A phylogenetic analysis was conducted to identify and classify the putative Knk-like proteins in L. decemlineata. This analysis involved aligning the amino acid sequences of Knk-like proteins from ten representative insect species. As a result, four Knk proteins were identified in L. decemlineata and were organized into three clades (Figure 2). LdKnk and LdKnk2 belong to the Knk and Knk2 protein clades, respectively, while both LdKnk3-FL and LdKnk3-5′ belong to the Knk3 protein clade (Figure 2). This suggests the functional diversification of Knk proteins within L. decemlineata.

3.2. Spatio-Temporal Expression Patterns of LdKnk-Family Genes

The expression patterns of LdKnk-family gene transcripts were analyzed at various developmental stages, including egg, larvae (first to fourth instar), wandering larvae, prepupae, and pupae, using qRT-PCR. The results indicate significant differences in gene expression (Figure 3). LdKnk exhibited relatively stable expression levels throughout the larval stages, with high expression at the end of the first to third instars and prepupal stages. A noticeable increase was observed during the prepupal and pupal stages, peaking in the mid-pupal stage (day 3). LdKnk2 was expressed in all developmental stages, with its highest expression observed during the pupal stage. LdKnk3-FL displayed distinct expression patterns, with high transcript levels sharply increasing during the end stages of the first to third instars, prepupal, and mid-to-late pupal stages. Its expression was low or undetectable during other periods. LdKnk3-5′ exhibited elevated levels during the late stages of the first to third instars and the prepupal stage, with the highest expression observed from days 1 to 3 of the pupal stage. Unlike LdKnk3-FL, LdKnk3-5′ also exhibited high expression at the early third instar and 24 h into the fourth instar (Figure 3).

The tissue-specific expression patterns of the LdKnk-family genes were analyzed in fourth-instar larvae. Relative transcript levels were measured in various tissues, including the epidermis (EP), foregut (FG), midgut (MG), hindgut (HG), Malpighian tubules (MT), fat body (FB), hemocytes (HE), and trachea (TR) (Figure 4). LdKnk, LdKnk3-FL, and LdKnk3-5′ showed predominant expression in the epidermis. However, LdKnk2 was highly expressed in the trachea. Additionally, LdKnk and LdKnk3-5′ exhibited moderate expression in the trachea, and all four LdKnk-family genes were expressed in the digestive system, particularly in the foregut and/or hindgut. These findings suggest that the LdKnk-family genes play potential roles in cuticle formation and molting processes.

3.3. Effects of Silencing LdKnk Genes on Larval Development and Epidermis Structure

RNAi experiments were conducted to investigate the biological functions of four LdKnk-family genes in L. decemlineata larvae. dsRNA specific to each LdKnk-family gene was produced using Escherichia coli HT115 and administered to newly molted second-instar larvae through ingestion of treated potato leaves. The larvae were fed dsRNA-soaked leaves for three days, followed by fresh potato leaves until pupation. dsGFP was used as the control. RNA was extracted from larvae three days after ingestion of dsRNA, and qRT-PCR was used to measure the transcript levels of the targeted genes. Results indicated significant gene silencing for the four LdKnk-family genes, evidenced by reduced transcript levels in treated larvae compared to controls (Figure 5A–D).

Silencing LdKnk2 in larvae did not result in visible phenotypic changes. There were no significant differences in body weight or mortality between the LdKnk2-silenced group and the control group (Figure 5E,F). The larvae developed normally, molting and pupating successfully, similar to those in the control group (Figure 5G).

In contrast, knockdown of LdKnk caused significant larval weight reduction in larvae on the third and sixth days post-dsRNA feeding (Figure 5E). By the early fourth instar, about 50% of these larvae exhibited localized cuticle damage, and desiccation, leading to death (Figure 5F,H). Silencing LdKnk3-5′ also caused significant changes. on the sixth day post-treatment, these larvae had notable body weight reductions, and some exhibited melanization and death during the early fourth instar (Figure 5E,F,J).

Interestingly, the knockdown of LdKnk3-FL led to a slight increase in body weight on the sixth day, with no significant difference compared to controls (Figure 5E). However, the survival rate was significantly lower (Figure 5F). These larvae stopped developing and died on the soil surface before pupation, with the pupal cuticle trapped within the larval cuticle (Figure 5I). These results highlight the essential roles of LdKnk-family genes in the development and maintenance of the larval epidermis in L. decemlineata.

To investigate the roles of LdKnk-family genes in the structural organization of the larval epidermis, we conducted TEM analysis on the larval cuticle. In the control group treated with dsGFP-dsRNA, TEM revealed that the larval cuticle comprises three distinct layers: the outermost non-conductive transparent waxy envelope layer, the middle epicuticle, and the inner procuticle. The procuticle displayed a clear laminar structure, with a lighter-colored inner endocuticle (endo) and a darker-colored outer exocuticle (exo) (Figure 6(A1–A3)).

RNAi-mediated silencing of LdKnk resulted in significant thinning of the procuticle, including both the endocuticle and exocuticle, and disrupted the laminar organization, causing uneven thickness and localized expansion (Figure 6(B1,B2),F,G vs. Figure S1A,B). Silencing LdKnk3-5′ caused the larval epidermis to become thinner, with both the procuticle and its endocuticle and exocuticle significantly reduced in thickness (Figure 6(E1,E2),F,G vs. Figure S1A,B). The chitin layers were also markedly thinner, with a blurred layer structure (Figure 6(D2)). In contrast, silencing LdKnk3-FL led to a significantly thicker larval procuticle, nearly twice that of the control, with both the endocuticle and exocuticle substantially thicker, while the chitin layer thickness remained similar to the control (Figure 6(D1,D2),F,G vs. Figure S1A,B). Knockdown of LdKnk2 resulted in larvae with procuticle morphology and thickness similar to the control group, with only an increase in procuticle thickness due to a thicker exocuticle (Figure 6(B1,B2),F,G vs. Figure S1A,B).

Measurements of the epicuticle and envcuticle thickness revealed that LdKnk2 knockdown significantly increased the thickness of both layers compared to the control group (Figure 6(B3) vs. Figure S1C,D). In contrast, knockdowns of LdKnk, LdKnk3-FL, and LdKnk3-5′ resulted in significantly thinner epicuticle and envcuticle layers (Figure 6(B3–E3) vs. Figure S1C,D). These findings suggest that LdKnk-family genes play distinct roles in maintaining the structural integrity of the larval cuticle.

4. Discussion

Knickkopf genes are essential for the formation and maintenance of insect cuticles, particularly in organizing and protecting chitin within the exoskeleton [10]. However, research on Knk genes has primarily focused on a few model species, such as D. melanogaster [9,23,26], T. castaneum [28,29,30], and L. migratoria [22,31,32], leaving their broader roles in insects largely unexplored. In this study, we identified and characterized Knk genes in L. decemlineata, a globally destructive pest of potatoes. Our findings reveal the distinct and essential functions of LdKnk-family genes in the larval cuticle, thereby enhancing our understanding of the molecular mechanisms underlying cuticle formation and maintenance and elucidating the developmental roles these genes play.

In our study, we identified and characterized four LdKnk-family coding gene transcripts in the genome and transcriptome of L. decemlineata. Phylogenetic analysis revealed that the LdKnk-family proteins are divided into three distinct clades: Knk, Knk2, and Knk3. We found single transcripts for LdKnk and LdKnk2 genes, whereas LdKnk3 gave rise to two alternatively spliced transcripts, coding for a full-length protein (LdKnk3-FL) and a C-terminally truncated version (LdKnk3-5′). This pattern in the L. decemlineata is somewhat similar to that in T. castaneum, D. melanogaster, and the hemimetabolous insect L. migratoria. Domain analyses showed that LdKnk, LdKnk2, and LdKnk3-FL each consist of a signal peptide, two tandem DM13 domains, and a DOMON domain, whereas LdKnk3-5′ lacks one DOMON domain, which is essential for Knk function in D. melanogaster [25]. These findings suggest that, despite the presence of three Knk coding genes across different insect species, there may be functional differences among them.

To further investigate the function of LdKnk-family genes in L. decemlineata, we used qRT-PCR to analyze the expression of the four LdKnk-family genes at different developmental stages and in various tissues. The results showed that all four LdKnk-family genes were consistently highly expressed before larval molting, at the late prepupal stage, and throughout the pupal stages, with expression patterns closely mirroring those of 20-hydroxyecdysone (20E). In terms of tissue expression, with the exception of LdKnk2, which was predominantly expressed in the trachea, the other LdKnk-family genes were significantly expressed in the larval epidermis (Figure 3 vs. Figure 4). Similarly, in L. migratoria and T. castaneum, Knk genes (LmKnk, LmKnk3-FL, LmKnk3-5′, TcKnk, TcKnk2, and TcKnk3-FL) showed continuous and high expression in late instar larvae, particularly in the epidermis, with LmKnk-family genes in L. migratoria confirmed as downstream targets of 20E [28,31,32]. These patterns indicate that insect Knk genes are significantly upregulated during cuticle formation and molting processes, highlighting their crucial roles in these stages.

The insect protein Knk is conserved across various insect species and plays a crucial role in cuticle formation and maintenance. Our RNAi experiments demonstrate that silencing LdKnk significantly impacts larval development and cuticle integrity. Knockdown of LdKnk in larvae results in significant weight loss, with more than half dying before the prepupal stage. Most larvae exhibit localized epidermal damage, melanization, dehydration, and death. TEM analysis supports these observations, showing that silencing LdKnk significantly reduces the thickness of the procuticle and its layers, leading to thinner epicuticle and endocuticle layers and compromised cuticle integrity (Figure 5 and Figure 6). Similarly, in L. migratoria, LmKnk localizes to the chitin layer of the cuticle. Injecting dsLmKnk reduces chitin content, disrupts crystalline chitin organization, and lowers lipid content, impairing the cuticle’s barrier function [31]. Previous research in D. melanogaster shows that DmKnk is crucial for cuticle formation and epithelial tube morphogenesis [24,25]. RNAi knockdown results in malformed wings and an amorphous procuticle, while overexpression maintains normal structure [23]. DmKnk, along with Dmkkv and Dmcystic, is essential for organizing chitin in tracheal tubes. Rescue experiments confirm that proper Dmknk expression restores tube integrity [9,45]. In T. castaneum, TcKnk proteins protect newly synthesized chitin from degradation and ensure proper laminar organization. RNAi for TcKnk causes developmental arrest at each molt, highlighting its role in chitin stability [28]. The Retroactive (Rtv) protein is essential for trafficking TcKnk into the procuticle. TcRtv localizes within epidermal cells and promotes TcKnk movement, protecting the new cuticle from chitinases. Loss of TcRtv leads to TcKnk entrapment and molting defects [7,29,30]. The conserved function of Knk protein across insects underscores its essential role in chitin organization and cuticle integrity.

Knk3 exhibits multiple alternative splicing variants that vary across species. LdKnk3 has two transcripts, similar to LmKnk3, while TcKnk3 produces three [28,32]. In L. decemlineata, the roles of LdKnk3-FL and LdKnk3-5′ are particularly notable due to their opposing effects on cuticle thickness. Silencing LdKnk3-FL results in a thicker cuticle, leading to molting disruption before the prepupal stage, while silencing LdKnk3-5′ results in a thinner cuticle, causing melanization and death (Figure 5 vs. Figure 6). This suggests that proper procuticle formation is crucial for insect molting and development. Similarly, in L. migratoria, silencing LmKnk3-5′ causes molting defects and high mortality, significantly decreasing chitin content without affecting cuticle structure and impeding lipid deposition on the cuticle surface [32]. In T. castaneum, dsRNA targeting multiple exons of TcKnk3 had varied effects. Injection of exon 9-specific dsRNA, which depletes TcKnk3-FL transcripts, caused 100% mortality at the pharate adult stage. Silencing TcKnk3-3′ specifically led to molting arrest at the same stage. Knocking down all three TcKnk-family genes significantly reduced total chitin content to varying degrees. TcKnk3 transcripts are crucial for the integrity of body wall denticles and tracheal taenidia but are not essential for the elytral and body wall procuticles [28]. These findings highlight the complex roles of Knk3 isoforms in different insect species and their critical functions in maintaining cuticle structure and integrity.

In this study, silencing LdKnk2 in larvae did not result in visible phenotypic changes. However, measurements revealed that LdKnk2 knockdown significantly increased the thickness of the epicuticle and endocuticle compared to the control group. Similarly, in L. migratoria, no visible phenotypic changes were observed after LmKnk2 was silenced by RNAi [32]. In contrast, silencing TcKnk2 in T. castaneum caused molting arrest at the pharate adult stage, indicating that TcKnk2 is crucial for the integrity of body wall denticles and tracheal taenidia, but not for the elytral and body wall procuticles [28].

Understanding the specific roles of LdKnk-family genes in L. decemlineata provides valuable insights for developing targeted pest management strategies. The essential functions of LdKnk, LdKnk3-FL, and LdKnk3-5′ in maintaining cuticle integrity make them promising targets for RNAi-based pest control. For instance, silencing LmKnk3-5′ in L. migratoria not only reduced procuticle thickness but also increased susceptibility to four insecticides from three different classes. Disrupting these LdKnk-family genes could therefore weaken the protective cuticle barrier, making insects more vulnerable to environmental stressors and enhancing insecticide efficacy. Our findings on the opposite effects of LdKnk3-FL and LdKnk3-5′ on cuticle thickness further underscore the importance of precise regulation of the cuticle in pest control applications. This research enhances our understanding of insect cuticle biology and offers new genetic insights for improving pest management strategies.

5. Conclusions

Our study has successfully identified and analyzed four LdKnk-family genes in the larval epidermis of L. decemlineata. The genes LdKnk, LdKnk2, LdKnk3-FL, and LdKnk3-5′ are integral to cuticle formation and maintenance. RNAi experiments revealed that silencing these genes results in significant changes in cuticle structure, larval development, and survival rates. Specifically, LdKnk and LdKnk3-5′ are essential for maintaining cuticle integrity and preventing damage, while LdKnk3-FL regulates cuticle thickness, ensuring proper molting. These findings suggest that targeting LdKnk-family genes could be an effective strategy for developing RNAi-based pest control methods, potentially reducing the impact of the beetle on potato crops. Future research should further clarify these genes’ roles and optimize RNAi techniques for practical agricultural use.