Transcriptomic Analysis of Non-Specific Immune Responses in the Rice Field Eel (Monopterus albus) Infected with Pallisentis (Neosentis) celatus

Abstract

Parasitic infestations present significant threats to the physiological health and ecological stability of aquatic species, frequently compromising immune defenses and elevating mortality rates. This study was conducted to elucidate the non-specific immune responses induced by Pallisentis (Neosentis) celatus infection in Monopterus albus, with a focus on intestinal histopathology and transcriptome gene expression. A histopathological examination revealed minor alterations in intestinal villi under low-level infection. A transcriptome analysis, performed using Illumina sequencing technology, identified 347 upregulated and 298 downregulated genes involved in critical biological pathways, such as lipid metabolism, immune responses, and the regulation of inflammatory processes. GO and KEGG analyses indicated the upregulation of immune-related pathways, including the RIG-I-like and IL-17 signaling pathways, highlighting a robust intestinal immune response. Conversely, the complement pathway was found to be downregulated, with significant suppression of C9, suggesting that the parasite may engage in immune evasion. Fluorescein-labeled C9 antibody assays confirmed reduced complement C9 levels in the infected tissues. A real-time PCR analysis identified the differential expression of eight genes, including C5, maats1, CFI, and gmnc, which were consistent with the sequencing results. These findings suggest that Pallisentis (Neosentis) celatus infection compromises intestinal health, induces inflammation, and activates non-specific immune responses in Monopterus albus. However, Pallisentis (Neosentis) celatus appears to evade the host immune response by suppressing the activation of complement components, thereby facilitating its reproductive parasitism.

1. Introduction

The rice field eel, Monopterus albus Zuiew, 1793, is an omnivorous, facultatively carnivorous fish species. It holds considerable commercial significance as one of China’s key primary aquatic species, being extensively distributed across diverse aquatic habitats, including ponds, paddy fields, and ditches throughout the country [1]. Nonetheless, parasitic pathogens pose a significant threat to the health of aquatic organisms, hindering the growth of aquaculture and resulting in substantial economic losses for the industry. Intestinal infections in fish caused by parasites such as nematodes and tapeworms typically do not result in severe or overt mucosal damage, primarily attributable to their relatively superficial interaction with host tissues [2]. In contrast, parasitism by the majority of echinoderm parasites is recognized to induce considerable tissue damage [3]. Helminths that parasitize M. albus are classified within the phylum Acanthocephala, specifically in the order Gyracanthocephala, with Pallisentis (Neosentis) celatus [4,5,6] and Pallisentis unbellatus [7] recognized as the sole known species inhabiting the intestinal tract. Among these, P. (Neosentis) celatus stands out as the most prevalent parasite in M. albus. This parasite adheres to and invades the host’s intestinal wall through a specialized head structure equipped with hooked anterior spines, thereby facilitating nutrient extraction and instigating a cascade of pathological responses [6].

The intestine serves as a vital organ in maintaining homeostasis, executing diverse functions including digestion, nutrient absorption, and transportation [8,9,10]. Similarly to other vertebrates, the digestive tract in fish functions as a principal pathway for pathogen infection, including parasitic agents, while simultaneously serving as a crucial barrier to restrict or prevent pathogen entry [11,12]. Following helminth infestation, mucosal surfaces, including the gastrointestinal tract, experience a rapid proliferation of mucus-secreting cells, excessive mucus production, fluid exudation, and epithelial transformation [13]. The intestinal mucosa possesses the ability to recognize parasitic antigens, thereby eliciting a defensive immune response. In contrast, microbial or dietary antigens are frequently tolerated asymptomatically, facilitating the maintenance of homeostasis within the organism [14]. The intestine is currently recognized as a pivotal organ for investigating mucosal immune responses in fish [15,16,17]. Nevertheless, research examining the immunological functions of the intestine in M. albus remains markedly insufficient.

Endoparasitic helminths commonly provoke inflammatory responses, leading to significant alterations in tissue structure and function [18]. Notably, intestinal helminths frequently induce localized inflammation and stimulate a host immune response [19,20,21]. Inflammation encompasses a multifaceted network of homeostatic processes that coordinate interactions among the nervous, circulatory, and immune systems, acting as a crucial response to tissue damage or pathogenic invasion while promoting tissue repair and pathogen clearance [19,22]. Additionally, parasites display a broad spectrum of immune evasion strategies. Through co-evolution with their hosts, they have developed a heightened ability to adapt to specific host environments. Such adaptations can manifest as the evasion of the host immune system or the development of host-specific behavioral and physiological traits [23,24,25,26,27].

Prior research has detailed the histopathological alterations in M. albus resulting from P. (Neosentis) celatus infection [28,29], alongside the biological traits of P. (Neosentis) celatus within the gastrointestinal environment of the M. albus [5,30]. Nevertheless, studies exploring the complex interactions between P. (Neosentis) celatus and its eel hosts, as well as the host specificity of this parasite, remain scarce. Furthermore, while the expression patterns of immune-related genes in M. albus have been thoroughly investigated [31,32,33,34,35,36,37], a significant gap persists in transcriptomic data at the molecular level that elucidates the mechanisms underlying their immune response to P. (Neosentis) celatus following infection. The transcriptomic analysis performed in this study effectively bridges this knowledge gap, offering essential insights for forthcoming controlled investigations into the physiological adaptations of M. albus to pathogenic influences or various environmental factors. Furthermore, it lays robust scientific groundwork for the development of targeted parasite vaccines and innovative disease management.

2. Materials and Methods

2.1. Ethics Statement

All experimental procedures involving animals were conducted in strict accordance with operational guidelines, fully complying with the ethical principles and regulatory standards outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978). All procedures were performed under anesthesia using ethyl m-aminobenzoate methane sulfonate (MS-222, Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 50 mg/L, with every effort made to minimize animal suffering. All experimental protocols were approved by the Academic Committee of the Hunan Fishery Sciences institute, Changsha, China (Approval Code: No. HFSI2023-01).

2.2. Sampling and Collection of M. albus

Samples for transcriptome analysis were obtained from wild M. albus collected from rice farms and ponds within their natural habitat in the Dongting Lake region. The captured M. albus were housed in open fiberglass tanks with a capacity of 200 L, where the water temperature was carefully maintained at 25 ± 1 °C, under a 12:12 light–dark cycle to replicate natural environmental conditions. The fish were fed commercial feed at 2% of their body weight once daily. Key water quality parameters, including pH, ammonia, and nitrate levels, were regularly monitored and maintained within optimal ranges throughout the acclimation period. Following a one-week acclimatization in rearing ponds, the fish were euthanized by immersion in MS-222 (50 mg/L), followed by a blow to the head prior to dissection. Intestinal samples, including those infected with P. (Neosentis) celatus (PI) and those uninfected (NI), were promptly harvested and preserved in tubes containing 1.5 mL of RNAlater (Sigma-Aldrich, St. Louis, MO, USA).

Three parallel samples were collected for each condition (PI and NI). The samples were initially stored at 4 °C for 24 h to stabilize their condition, then they were transferred to −20 °C for long-term preservation prior to RNA purification and transcriptome library preparation. Following transcriptome sampling, the intestines were collected, and the number of P. (Neosentis) celatus infections per M. albus was quantified. Additionally, the M. albus specimens were carefully examined to identify the presence of any other parasitic species that could potentially influence the results [38].

2.3. Histological Observations of the M. albus Intestine

The experimental M. albus were anesthetized with 50 mg/L MS-222 during the sampling process. Fresh intestinal specimens were then rapidly excised and immediately fixed in 10% neutral formalin for tissue sectioning. The tissue samples underwent standard processing, including gradient alcohol dehydration, paraffin embedding, sectioning at approximately 5 μm thickness, deparaffinization with xylene, hematoxylin and eosin (HE) staining, clearing with xylene, and sealing with neutral gum. The sections were subsequently observed and photographed using a Nikon Eclipse E200MVR light microscope (Nikon, Tokyo, Japan).

2.4. Transcriptome Analysis

2.4.1. RNA Extraction and Sequencing

Intestinal samples (infected n = 3; uninfected n = 3) from M. albus were homogenized in a lysis buffer containing 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) using a TissueLyser II (Qiagen, Hilden, Germany) to ensure complete cell disruption and lysis. Total RNA was extracted from each sample using the RTN350 kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions, followed by treatment with DNase I (Thermo Scientific, Waltham, MA, USA) to eliminate any residual genomic DNA.

The quality and integrity of the extracted RNA were evaluated using an Agilent Bioanalyzer 2100 Total RNA Nano Series II Microarray (Agilent, Amstelveen, The Netherlands), with the results further validated by 2% agarose gel electrophoresis and ethidium bromide staining. RNA sequencing libraries were prepared from 500 ng of total RNA using the Illumina TruSeq™ Stranded mRNA LT Sample Prep Kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol. All libraries, featuring insert sizes ranging from 300 to 500 bp, were sequenced on an Illumina HiSeq2500 platform using single-end reads of 50 nucleotides in length, following the manufacturer’s guidelines. Image analysis and base calling were conducted using the Illumina data processing pipeline.

The raw sequencing data were processed using fastp (v0.23.4) to remove low-quality reads, including spliced and undetermined nucleotide (N base) reads, resulting in clean reads. The quality metrics, including Q20, Q30, and GC content, were calculated for the clean reads. These clean reads were subsequently aligned to the M. albus genome (https://www.ncbi.nlm.nih.gov/genome/?term=Monopterus+albus, accessed on 7 July 2024) using Hisat2 (v2.0.5) [39]. Aligned reads for each sample were assembled into reference transcripts using StringTie (v1.3.3b). FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values were calculated for each gene based on transcript length and the number of aligned reads. A differential expression analysis was subsequently performed between the control and P. (Neosentis) celatus-infected groups using the DESeq2R package (v1.20.0) [40]. Genes exhibiting differential expression were identified based on statistical significance, with an adjusted p-value (padj) ≤ 0.05 and an absolute log2 fold change of ≥ 1. To further investigate the relationships among the six samples, a principal component analysis (PCA) was employed, alongside Pearson’s correlation coefficient, to assess the overall correlation and variance structure within the dataset.

2.4.2. GO and KEGG Enrichment Analyses of Differentially Expressed Genes (DEGs)

A Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was conducted using the clusterProfiler R package (v3.8.1), with adjustments made for gene length bias to ensure robust and accurate enrichment results. GO terms with a corrected p-value of <0.05 were considered significantly enriched, emphasizing key biological processes, molecular functions, and cellular components linked to the observed gene expression changes. Additionally, The KEGG (Kyoto Encyclopedia of Genes and Genomes) database provides a comprehensive resource for understanding the high-level functional roles and utilities of biological systems, including cells, organisms, and ecosystems, particularly through the integration of large-scale molecular datasets derived from genome sequencing and other high-throughput experimental methodologies (http://www.genome.jp/kegg/, accessed on 7 July 2024). A statistical enrichment analysis of DEGs within KEGG pathways was also performed using the clusterProfiler R package (v3.8.1), facilitating the identification of broader metabolic and signaling networks associated with these genes.

2.5. Real-Time Quantitative PCR

The validation of RNA-seq data were performed using quantitative real-time PCR (qRT-PCR) on 8 selected genes (n = 3), including complement component 5 (C5), Proline-rich 7 (prr7), complement factor I (cfi), Carboxypeptidase A1 (CPA1), Mitochondrial ATPase Associated Transmembrane Protein 1 (maats1), Geminin Coiled-coil Domain Containing Protein (gmnc), Cyclin-Dependent Kinase 14 (cdk14), and complement component 9(C9). Real-time PCR assays were conducted on an AriaMx real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) using cDNA as the template. Gene-specific primers and probes (Table S1) were employed in qPCR reactions set up with TB Green^® QPCR Master Mix (Takara, Dalian, China), containing a MgCl₂ concentration of 5.5 μM. To ensure the specificity of amplification, each qPCR assay was evaluated with SYBR Green qPCR assays, followed by a melting curve analysis to confirm the absence of primer dimers and the production of a single specific amplicon. The qRT-PCR reactions were conducted in a total volume of 20.0 μL, comprising 10.0 μL of TB Green^® Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Dalian, China), 0.8 μM of each forward and reverse primer, 6.4 μL of DNase/RNase-free H₂O, and 2.0 μL of cDNA template. Thermal cycling conditions included an initial step at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. Data obtained from the qRT-PCR were analyzed using the 2^−ΔΔCt method [41], with expression levels normalized against the mean of three reference genes: 18S-1, 18S-2, and β-actin.

3. Results

3.1. Histopathological Findings

The prevalence P. (Neosentis) celatus infection in M. albus from Dongting Lake was observed at 30.15%, with parasite loads ranging from 2 to 11 individual per host. An analysis of P. (Neosentis) celatus infection across different body length groups of M. albus revealed that the infection rate was consistent with the trend in infection intensity. As illustrated in Figure 1, when the body length of M. albus was below 40 cm, both the prevalence and intensity of P. (Neosentis) celatus infection increased significantly with rising body length. Conversely, when the body length surpassed 50 cm, both metrics infection prevalence and intensity exhibited a declining trend. Furthermore, a notable positive correlation was observed between the infection rate and host body length.

For a detailed histopathological analysis, three intestinal samples from M. albus with an infection intensity of five parasites, along with three uninfected control samples, were selected for a detailed histopathological analysis. Following HE staining, the results are presented in Figure 2. In the control group, the intestinal tissue of M. albus exhibited a well-defined structure across all layers. The mucosal layer showed abundant, uniformly shaped intestinal villi with continuous epithelial cells, orderly arranged epithelial cells and numerous goblet cells. The lamina propria exhibited no discernible abnormalities, and the submucosal layer displayed fine connective tissue without noticeable defects. The muscle fibers in the muscularis propria were consistently stained and arranged in an orderly manner, exhibiting no signs of pathological changes. Post-infection, the intestinal tissues of M. albus demonstrated villous atrophy and shortening with an irregular morphology. A significant number of epithelial cells were observed to have detached from the lamina propria (as indicated by black arrows), and a modest reduction in goblet cell numbers was noted (as indicated by yellow arrows). No apparent abnormalities were detected in the submucosal layer or the muscularis propria, nor was there any noticeable infiltration of inflammatory cells. These findings indicate that low-level infections induce only subtle alterations in the structure of intestinal villi.

3.2. Transcriptome Sequencing and Classification of DEGs

Six cDNA libraries were constructed from total intestinal RNA extracted from three control group (NI) and three P. (Neosentis) celatus-infected group (PI). Following the filtering of raw data, clean reads of 41,283,578 (6.19 G), 39,236,422 (5.89 G), 40,798,166 (6.12 G), 40,194,048 (6.03 G), 40,184,514 (6.03 G), and 40,395,150 (6.06 G) were obtained from the respective libraries, with Q20 values exceeding 97.7% and Q30 values surpassing 93.3% (Table S2). The paired-end clean reads were subsequently aligned to the M. albus reference genome using Hisat2 (v2.0.5), achieving unique alignment rates between 84.16% and 85.0% (Table S3). Gene expression quantification, including novel gene detection, was performed using the feature counts function in subread, based on gene location annotations from the reference genome. FPKM values were calculated for all genes across the six samples, and Pearson’s correlation coefficients were employed to evaluate the consistency and reliability of the differential expression analyses, with R² values exceeding 0.8 for all samples (Table S4). The clean reads from the six transcriptomes have been submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1163312, accessed on 22 September 2024).

Differentially expressed genes (DEGs) between the control group (NI) and the P. (Neosentis) celatus-infected group (PI) were identified using the DESeq2 R package (v1.20.0), based on threshold criteria of a ≥2.0-fold change and an adjusted p-value (padj) ≤ 0.05. This analysis resulted in the identification of a total of 645 DEGs in intestinal tissues, comprising 347 upregulated genes and 298 downregulated genes (Figure 3A). The distribution of DEGs was visualized using a volcano plot (Figure 3B). The top 10 significantly upregulated genes (marked in red) included LOC109953876, LOC109968628, LOC109964119, LOC109953068, LOC109958316, LOC109961816, LOC109951655, LOC109967861, and LOC109954698. Conversely, the top 10 significantly downregulated genes (marked in green) included rgn, slc25a48, tbc1d2, hoxc11, LOC109953562, LOC109969192, LOC109965537, LOC109955049, and LOC109974833. A hierarchical clustering analysis was conducted on the identified DEGs to categorize genes exhibiting similar expression patterns. In the heatmap illustrated in Figure 3C, each column denotes an individual sample, while each row corresponds to a distinct gene. The color gradient reflects gene expression levels, with red signifying high expression and green indicating low expression, thereby effectively depicting the differential expression patterns across the samples. The clustering analysis revealed a pronounced separation between the P. (Neosentis) celatus-infected group (PI) and the control group (NI), as anticipated. Furthermore, a three-dimensional PCA (Figure 3D) demonstrated that the six intestinal samples were segregated into two distinct clusters: the three samples from the PI were tightly clustered, while those from the NI positioned themselves at the opposite end, demonstrating a clear separation between the two groups. These results indicate that infection with P. (Neosentis) celatus significantly alters the intestinal transcriptomic profile in M. albus compared to the control group, thereby confirming that the collected samples accurately reflect the organism’s health and pathological status.

3.3. GO and KEGG Enrichment Analyses of DEG_S

Figure 4A,B (Supplementary Table S5) display the top 10 most significantly enriched Gene Ontology (GO) terms for each category, highlighting the key biological processes, molecular functions, and cellular components associated with the 645 differentially expressed genes (PI vs. NI; 347 upregulated and 298 downregulated). Notably, in the biological processes category, the majority of upregulated genes were associated with lipid metabolism, including the methyl-branched fatty acid metabolic process (GO:0097089) and fatty acid alpha-oxidation (GO:0001561). Conversely, the majority of downregulated genes were implicated in inflammation and DNA replication, such as the regulation of acute inflammatory response (GO:0002673) and positive regulation of mitotic cell cycle DNA replication (GO:1903465). For cellular components, upregulated genes were primarily associated with functions in various cellular and extracellular regions, including the extracellular region (GO:0005576) and secretory granule (GO:0030141). In contrast, the downregulated genes were predominantly associated with cell membrane functions, including the membrane attack complex (GO:0005579) and clathrin-coated acetylcholine transport vesicle membrane (GO:0060201). Concerning molecular functions, the majority of upregulated genes were involved in fatty acid and cholesterol metabolism as well as fatty acid enzyme activation, exemplified by cholate-CoA ligase activity (GO:0047747) and phytanate-CoA ligase activity (GO:0050197). Conversely, most downregulated genes played critical roles in immune-related functions, including C-X-C chemokine receptor activity (GO:0016494) and C-X-C chemokine binding (GO:0019958).

The KEGG enrichment analysis of the 347 upregulated and 298 downregulated genes in the transcriptome (Figure 5A,B; Supplementary Table S6) identified the 10 most significantly enriched KEGG pathways for each group. Notably, several of the upregulated pathways were associated with non-specific immune responses and inflammation-related processes, including the RIG-I-like receptor (RLRs) signaling pathway and the IL-17 signaling pathway. KEGG annotation of the 298 downregulated genes in the transcriptome revealed that the most significantly affected pathways included the complement and coagulation cascades, followed by pathways involved in valine, leucine, and isoleucine biosynthesis, fatty acid elongation, biosynthesis of unsaturated fatty acids, and glycosylphosphatidylinositol-anchored proteins (GPI-APs).

3.4. Gene Expression of the Complement Pathway

A total of 28 genes associated with the complement pathway were selected for detailed analysis. A hierarchical clustering heatmap, generated based on the expression levels of these genes, revealed that the samples were distinctly grouped into two primary clusters: the PI and NI groups (Figure 6A). The differentially expressed genes identified included several complement components, namely C6, C9, C5, and Cd59, as well as complement factor I (CFI) and the coagulation cascade gene Serpin family C member 1 (SERPINC1). Notably, genes linked to intestinal mucosal immunity, including CFI, C6, and C9, exhibited significant downregulation following infection. An immunofluorescence analysis confirmed the downregulation of C9 in infected tissues (Figure 6B). This downregulation contrasts with the enhanced complement activity typically observed in fish post-bacterial infection, suggesting a unique adaptive strategy by P. (Neosentis) celatus to evade immune detection.

3.5. qRT-PCR Quantification Results

Eight differentially expressed genes (DEGs) were randomly selected for qRT-PCR validation in both the P. (Neosentis) celatus-infected and control groups to confirm the reliability of the RNA-seq results. Significant differences in gene expression were observed between NI and PI groups for all the tested genes (Figure 7). Genes such as C9, CFI, and Prr7 were downregulated in the PI group, whereas C5, gmnc, maats1, CPA1, and cdk14 were upregulated following infection. These results corroborate the RNA-seq findings, reinforcing the observed transcriptomic alterations due to P. (Neosentis) celatus infection.

4. Discussion

4.1. Histopathological Effects of Infection

The prevalence and intensity of intestinal parasite infections largely determine the extent of pathological damage to fish intestinal tissues. High infection rates lead to an increased number of fish infested with parasites, posing a significant threat to population health. Gut damage becomes more severe at high infection intensities, when the number of parasites per fish increases. A large number of parasites can cause extensive mucosal destruction in the intestinal wall, leading to cell degeneration, necrosis, and ulcer formation, and potentially triggering severe hemorrhagic and exudative reactions [42,43,44]. In the intestinal samples of non-infected M. albus, the mucosal layer remained intact, with no significant abnormalities observed in the lamina propria or submucosal layers. However, samples from P. (Neosentis) celatus-infected individuals exhibited marked pathological changes, including villous atrophy, irregular morphology, and detachment of epithelial cells, accompanied by a moderate reduction in goblet cell numbers. Although these changes were primarily concentrated in the superficial structures, no significant abnormalities were observed in the deeper tissue layers, suggesting that low-level parasitic infections predominantly induce subtle structural changes in M. albus. This phenomenon may be due to the fact that acanthocephalans only penetrates the superficial layer of the intestinal wall, with the main damage involving the destruction of the villus-covered mucosal epithelium near the parasite’s attachment site [45].

4.2. Pathways of Non-Immune Response

The KEGG enrichment analysis identified significant pathways, including RIG-I-like receptor (RLR) signaling and IL-17 signaling pathways, which are critical for immune responses to parasitic infections. RIG-I-like receptors (RLRs), which constitute a specialized class of cytoplasmic pattern recognition receptors, are essential for the innate immune response, as their function is to detect viral RNA and initiate antiviral signaling pathways. These cytoplasmic pattern recognition receptors (PRRs) recognize viral RNAs, initiating an antiviral innate immune response and activating additional components of the immune system [46,47,48]. For instance, in fish, PRRs recognize viral nucleic acids, which in turn trigger an antiviral response by activating molecular pathways that culminate in the production of type I interferon (IFN) [49]. The upregulation of the RLR signaling pathway was also observed in the gut of M. albus following P. (Neosentis) celatus infection, indicating the activation of intestinal innate immunity. The inflammatory response constitutes a critical component of the immune defense against parasitic infections [50]. The IL-17 signaling pathway, mediated by IL-17 cytokines, regulates inflammation and defends against pathogen infections [51,52], particularly in mammalian mucosal tissues [53]. This IL-17 signaling pathway promotes the production of pro-inflammatory cytokines and chemokines, which are essential for the recruitment and activation of neutrophils and other immune cells, thereby establishing and sustaining the inflammatory microenvironment [53]. Zhang’s study demonstrated that the expression of certain IL-17 family members was significantly upregulated in mucosal tissues following Edwardsiella piscicida infection, suggesting their critical role in the innate immune response [52]. Similarly, during parasitic invasion in M. albus, the inflammatory response aligns with observations from other fish species, where pro-inflammatory cytokines contribute to the regulation of immune defense mechanisms against infection [54]. Upon infection with P. (Neosentis) celatus, the swift upregulation of the IL-17 signaling pathway signifies a robust inflammatory response, likely serving a protective role by enhancing the activation of immune cells. However, the chronic activation of this pathway poses a risk of significant tissue damage, underscoring the critical need for tightly regulated inflammatory responses. Research on teleost fish has further elucidated that excessive inflammation induces profound alterations in the gut mucosa, impairing both its immune functionality and structural integrity [50]. This upregulation underscores the dual role of the IL-17 signaling pathway: while it is essential for orchestrating an effective immune defense, its dysregulation or hyperactivation can exacerbate inflammatory responses, potentially leading to tissue damage and compromised organ function.

The complement and coagulation cascades are critical biological pathways that regulate inflammation, activate adaptive immunity, and contribute to pathogen eradication in vivo [55]. The downregulation of this may compromise immune defenses, increasing susceptibility to disease, while the downregulation of the coagulation system could lead to bleeding tendencies, thereby impairing wound healing and hemostasis. Furthermore, the downregulation of GPI-APs indicates a reduction in cell surface-associated proteins, potentially impacting biological processes such as cell signaling, adhesion, and immune responses [56,57,58]. These findings suggest that infection by P. (Neosentis) celatus in M. albus damages intestinal epithelial cells, induces local mucosal immune evasion, and may disrupt lipid metabolism.

4.3. Complement System Dynamics: Immune Evasion and Downregulation

The complement system serves as a vital bridge between innate and adaptive immune responses, significantly contributing to the defense against pathogens [59]. It represents one of the fundamental defense mechanisms responsible for the recognition and elimination of invading microorganisms. Initially identified by Jules Bordet in 1896 and subsequently named “complement”, this system comprises a series of small proteins, predominantly synthesized in the liver, that circulate in the blood in an inactive state. Upon the detection of a pathogen, these proteins are sequentially activated in a cascade, tagging foreign particles for destruction and amplifying the immune response [60].

The complement system enhances phagocytosis, promoting inflammation, and lysing pathogens through the formation of the membrane attack complex (MAC). Previous studies have demonstrated that most pathogens infecting the host attract the serum complement component C3b, which becomes anchored to the pathogen’s surface. This subsequently attracts other complement components such as C5b, C6, C7, C8, and C9, triggering a cascade that culminates in the generation of a MAC. The MAC creates pores in the pathogen’s surface, ultimately resulting in its destruction [61]. The activation of the complement system leads to the formation of complement complexes, which subsequently induce complement-mediated cytolysis [62]. Furthermore, complement activation generates small peptide fragments, such as C3a and C5a, referred to as anaphylatoxins, which augment inflammatory responses and recruit immune cells to the site of infection [63]. These components function synergistically to eliminate pathogens and facilitate the clearance of immune complexes and apoptotic cells [60]. Infection by P. (Neosentis) celatus significantly downregulates the expression of genes linked to intestinal mucosal immunity in M. albus, including CFI, C6, C3, C9, and Cd59, with C9 exhibiting the most pronounced downregulation among the differentially expressed genes. In mammals, these genes are functionally integrated into the complement pathway [64]. Notably, this downregulation stands in stark contrast to the enhanced complement activity commonly observed in numerous fish species following bacterial infection [65,66,67,68,69]. To further validate the downregulation of C9, an immunofluorescence analysis was performed, demonstrating a significant reduction in C9 fluorescence intensity within the intestines of M. albus infected with P. (Neosentis) celatus, thereby validating its downregulation.

Notably, the expression of the SERPINC1 and C5 was not suppressed in the intestinal tissues. Prior research has shown that complement activity in sea bream significantly diminishes three days following repeated stress exposure [70]. Likewise, a decrease in complement protein activity within skin mucus has been documented in both gilthead seabream and sea bream exposed to crowding stress [71]. Both the alternative and classical complement activation pathways were found to be significantly downregulated in darkbarbel catfish following Edwardsiella ictaluri infection, encompassing variably expressed proteins such as C1r, C3, C5, C7, C9, and C1-INH [72]. In goldfish infected with Gyrodactylus kobayashii, the downregulation of complement factor C3 expression, which plays a pivotal role in the microbial response, has also been observed [73]. These findings further corroborate the scientific validity of the complement downregulation mechanism identified in M. albus infected with P. (Neosentis) celatus. Similar expression patterns were observed in the chemokine pathway (Figure 4B). Chemokines serve as key immunomodulators that link adaptive and innate immunity [74], and a reduction in chemokine receptors may impair adaptive immune responses. In Larimichthys crocea, the expression of chemokine receptors CXCR2, LycCXCR3, and LycCXCR4, as well as CXC chemokine ligands 11.3 and 20.3 in channel catfish, were significantly upregulated following bacterial infection [75,76]. However, in the intestines of M. albus, infection by P. (Neosentis) celatus resulted in a significant downregulation of chemokine receptor gene expression, particularly a pronounced decrease in the expression of C-X-C motif chemokine receptor 5 (CXCR5). The success of parasitic worm infections is predominantly contingent upon their capacity to evade or manipulate the host’s immune system [77,78,79]. This phenomenon likely elucidates how P. (Neosentis) celatus reproduces parasitically by downregulating the host’s complement cascade response, thus evading the immune detection.

5. Conclusions

This study represents one of the few efforts to examine the interaction between M. albus and P. (Neosentis) celatus following infection. Although P. (Neosentis) celatus is a well-documented parasite in this host, the immune mechanisms and molecular responses elicited by this interaction remain underexplored in the literature, particularly at the transcriptomic level. This study provides novel insights into the mechanisms underlying the generation of innate immune responses in this host species.

A significant positive correlation was observed between infection rate and host body length, with markedly reduced infection rates in M. albus exceeding 50 cm, suggesting that host size critically impacts parasite establishment. A histopathological analysis revealed pronounced intestinal damage, including villous atrophy and depletion of goblet cells, which implies a marked impairment in digestive function. Transcriptomic analysis indicated a notable upregulation of genes associated with non-specific immune responses, inflammation, and lipid metabolism, especially within the RIG-I-like and IL-17 signaling pathways. The observed downregulation of genes related to the complement system and chemokine signaling (e.g., C9 and CXCR5) suggests that P. (Neosentis) celatus may adopt sophisticated immune evasion strategies.

In conclusion, P. (Neosentis) celatus infection markedly impacts the health and gene expression profiles of M. albus. These findings further inform the development of targeted strategies for effective parasite control in affected fish populations.