A Pseudotumorous Syndrome Associated with an As-Yet-Unidentified Eukaryotic Parasite Causing Functional Gonadal Arrest in Largefin Longbarbel Catfish (Hemibagrus macropterus)

TL;DR

A new disease in catfish causes tumor-like growths and reproductive issues, possibly due to an unknown eukaryotic parasite.

Contribution

First documented case of a eukaryotic parasite-induced pseudotumorous syndrome in fish.

Findings

Pseudotumors are composed of hyperplastic host cells and invasive Type III cells.

Metagenomic analysis suggests infection by an unidentified eukaryotic parasite.

Pseudotumors act as a 'nutrient sink,' causing cachexia and reproductive arrest.

Abstract

This study presents the first documented case of a disease syndrome in cultured largefin longbarbel catfish (Hemibagrus macropterus). The condition is characterized by massive abdominal pseudotumor formation, severe cachexia, and functional gonadal arrest. Comprehensive pathological investigation revealed that the pseudotumor was encapsulated by fibroblasts and primarily composed of host-derived, poorly differentiated hyperplastic cells, interspersed with invasive, basophilic Type III cells. These cells and associated inflammatory–fibrotic lesions were also disseminated in the gill, kidney and spleen. Systematic diagnostic approaches, including microbiology and transmission electron microscopy, found no evidence of conventional bacterial or viral pathogens. Metagenomic analysis further supported these findings and suggested a link to infection by an as-yet-unidentified eukaryotic parasite, with Microsporidia or Ichthyosporea being the primary candidates. Functional (KEGG) profiling of the pseudotumor tissue further revealed a molecular signature consistent with active cellular proliferation and metabolism. We propose that the pseudotumor acts as a metabolically active “nutrient sink,” driving the systemic catabolism that underlies the severe cachexia and reproductive arrest. This work provides the first case of a eukaryotic parasite-induced pseudotumorous syndrome in fish, which represents an emerging threat to conservation aquaculture and offering novel insights into parasite-mediated host metabolic hijacking and tumor-mimicry.

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1. Introduction

The largefin longbarbel catfish (Hemibagrus macropterus), an endemic species of the family Bagridae that inhabits the upper reaches of the Yangtze River and Zhujiang River [1], functions dually as an ecological indicator and a species with substantial economic potential [2]. Wild populations of this species have undergone a continuous decline, primarily driven by dam construction, overfishing, and habitat fragmentation [3]. Although classified as “Least Concern (LC)” by the International Union for Conservation of Nature (IUCN) [4], severe reductions in population size have led to its inclusion in provincial-level conservation initiatives across Sichuan, Hunan, and other regions [5].

The sustainable utilization of H. macropterus plays a key role in the conservation of regional aquatic biodiversity: its benthic habit makes it a key indicator of river ecosystem health, while its nutritional profile—featuring 62.09% crude protein, 40.76% essential amino acids, and 29.7% umami amino acids—coupled with minimal intermuscular bones, renders it an ideal candidate for the development of high-quality food fish [6]. However, the ongoing Yangtze fishing moratorium has limited access to wild broodstock [7], and artificial reproduction efforts continue to encounter challenges such as asynchronous gonadal maturation and inadequate sperm cryopreservation protocols for this genus [8]. As a result, conservation aquaculture has emerged as a pivotal strategy for population recovery. In this context, effective health management of cultured stocks is essential to sustainable expansion, and any emerging disease may pose a risk to the conservation of this vulnerable species, particularly if it affects reproductive fitness.

This study documents the first report of a novel pathological phenotype in captive H. macropterus, characterized by extensive abdominal adipoid hyperplasia in the absence of ascites, accompanied by severe muscle wasting. These proliferative masses impede gonadal development through mechanical compression and nutrient sequestration, leading to a functional arrest of gonadal development and significantly compromising the reproductive viability of cultured populations. This syndrome constitutes a cryptic yet highly detrimental condition: although not associated with acute mortality, its insidious progression results in reproductive failure, thereby posing a potential risk to both economic sustainability and conservation objectives. Parasites are well known to impair host reproductive capacity and, in some cases, to cause severe functional reproductive failure by destroying gonadal tissue or diverting reproductive resources. Nematodes of the family Philometridae have been documented to infect fish gonads and, under heavy infestation, to feed on ovarian fluid or physically damage ovarian tissue, resulting in reduced fecundity or loss of reproductive function [9]. Parasite-induced reproductive dysfunction (e.g., parasitic castration) is a widely recognized strategy that can alter host life-history traits and population dynamics across diverse host–parasite systems [10]. These examples support the plausibility that chronic eukaryotic parasitism could contribute to the gonadal degeneration and reproductive arrest observed in our study. Consequently, this study aims to describe and characterize this urgent pathological anomaly, with dual implications for the conservation of endangered fishery resources and exploring its potential association with eukaryotic parasitic infection.

2. Materials and Methods

2.1. Necropsy

Four specimens of H. macropterus displaying the most severe characteristic asymmetric abdominal swelling and cachexia were selected for this study. These individuals were identified through visual screening from a cohort of approximately 30 symptomatic fish within a conservation aquaculture stock of about 1000 two-year-old individuals. The estimated prevalence of the syndrome in this stock was ~3%. Notably, the condition was not observed in one-year-old fish within the same system. The disease presented as a chronic condition, with no associated acute mortality reported at the time of sampling. Due to the absence of macroscopically identifiable gonads, the sex of the affected individuals was not determined. The selected diseased individuals had a mean body weight of 545 ± 91 g and a total length of 36.4 ± 2.1 cm. Following euthanasia with MS-222 in accordance with humane protocols, a comprehensive postmortem examination was conducted to assess both external and internal pathological changes. Tissues and organs including liver, spleen, kidney, intestine, gill, heart, and the abnormal abdominal mass were systematically examined for gross lesions. All animal procedures were approved by the Animal Care and Use Committee of the Sichuan Academy of Agricultural Sciences (Permit No. 20250703001A; approval date: 3 July 2025).

2.2. Microbiological Examination

Standard microbiological assays were performed to screen for common bacterial and viral pathogens. For bacterial isolation, samples (~0.1 g each) of liver, spleen, and the abnormal abdominal mass were aseptically collected and homogenized in sterile physiological saline. Homogenates were inoculated onto Brain Heart Infusion (BHI) (Hopebio, Qingdao, China) agar and incubated at 28 °C for 48–72 h. Observed colonies were further subcultured for purity and examined microscopically after Gram staining. For virological examination, tissue homogenates (prepared as above) were filtered through 0.22-μm membrane filters to remove bacteria and large debris. Filtrates were inoculated onto monolayers of epithelioma papulosum cyprini (EPC) cells, which are commonly used for isolating a broad range of fish viruses. Cultures were maintained at 20 °C and examined daily for cytopathic effects (CPE) over 7 days. Blind passages were performed if no CPE was observed initially.

2.3. Histopathology

Tissue samples, including the adipoid mass, liver, spleen, kidney, intestine, gill, heart, brain, eye, swim bladder, skin, and muscle, were collected from four diseased fish and four healthy control fish (n = 4 per group) for histopathological examination. For structural preservation, tissues were immediately fixed in 10% neutral buffered formalin. Following fixation and decalcification where necessary, samples were dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections of 4 μm thickness were obtained using a microtome and stained with hematoxylin and eosin (H&E) for routine histological evaluation [11]. To further characterize abnormal cellular components, additional staining procedures were performed, including Alcian blue (AB), periodic acid–Schiff (PAS), and combined Alcian blue–PAS (AB-PAS) staining. For lipid visualization, portions of the adipoid pseudotumor tissue (~0.5 cm^3^) from diseased fish were mounted on tissue supports, frozen, sectioned at 5 μm using a cryostat, and stained with Oil Red O (Sigma-Aldrich, Beijing, China) at low temperature prior to microscopic examination.

2.4. Transmission Electron Microscopy

For ultrastructural analysis, pseudotumor tissue samples from four diseased fish (n = 4) were processed for transmission electron microscopy. Healthy control fish were not included in TEM analysis because no corresponding tissue was present. Tissue fragments were first fixed rapidly in 2.5% glutaraldehyde and then post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4). After dehydration through a graded ethanol series, the tissues were embedded in epoxy resin. Ultrathin sections (65–75 nm thick) were cut from the embedded blocks using a microtome equipped with a glass knife, collected on uncoated copper grids, and stained sequentially with uranyl acetate and 0.2% lead citrate. The prepared grids were examined using a Hitachi H-600 transmission electron microscope (Hitachi, Tokyo, Japan) for observation and imaging of ultrastructural morphology.

2.5. Metagenomic Library Construction and Sequencing

To gain molecular insights complementary to the histopathological findings and to explore potential microbial etiologies, metagenomic analysis was performed. The adipoid tissues (~0.1 g per sample) were aseptically excised from the abdominal proliferative masses of four individual diseased fish, yielding four independent biological samples. Healthy controls were not subjected to metagenomic sequencing because the adipoid masses is a disease-specific tissue absent in healthy individuals. Genomic DNA was extracted using the FastPure Stool DNA Isolation Kit (MJYH, Shanghai, China). The concentration of the extracted DNA was quantified with a Quantus Fluorometer (using Picogreen dye; Promega, Madison, WI, USA), and its integrity was verified by 1% agarose gel electrophoresis. The DNA was then fragmented to an average size of 350 bp using a Covaris M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). Sequencing libraries were prepared from the fragmented DNA using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, USA) and subsequently sequenced on an Illumina NovaSeq™ X Plus platform (Illumina, San Diego, CA, USA).

Raw metagenomic reads were quality-filtered using fastp (v0.23.4) to remove adapter sequences and low-quality reads (read length < 50 bp or average quality score < 20). Host-derived sequences were removed by aligning the filtered reads against the reference genome of the closely related species Tachysurus fulvidraco (GenBank: GCF_022655615.1). This species was selected as the closest available genomic relative to H. macropterus in public databases. A well-annotated and readily downloadable reference genome for H. macropterus (GenBank: PRJNA182666) was not available for bioinformatic filtering at the time of this analysis. The alignment was performed using Bowtie2 (v2.5.1) in end-to-end mode to require full-length matches, with the sensitive preset to balance alignment accuracy and sensitivity. Additional parameters included -I 200 -X 400 to define the valid paired-end insert size range, and threads 8 for parallel computation. The remaining non-host reads were assembled de novo using MEGAHIT (v1.2.9) with default parameters, retaining contigs ≥ 300 bp. Open reading frames (ORFs) were predicted from assembled contigs using Prodigal (v2.6.3), and ORFs shorter than 100 bp were discarded.

To reduce redundancy, predicted genes were clustered using CD-HIT (v4.8.1) at 90% sequence identity and 90% coverage, and the longest sequence from each cluster was retained as the representative gene. Clean reads were mapped back to the non-redundant gene set using SOAPaligner (soap2, v2.21), and gene abundance was calculated as counts per million (CPM). CPM-normalized abundances were used to estimate the relative abundance of genes, functional pathways, and taxonomic groups within pseudotumor samples.

Taxonomic annotation of predicted protein sequences was performed by alignment against the NCBI non-redundant (NR) database using DIAMOND (v2.1.9). Functional annotation was primarily based on KEGG and eggNOG databases. KEGG pathway profiles and gene composition were generated based on CPM-normalized abundances. Analyses focused on within-sample functional and taxonomic composition rather than statistical comparisons between diseased and healthy groups, as the pseudotumor represents a disease-specific tissue lacking a healthy counterpart.

3. Results

3.1. Gross Pathology and Cachectic Phenotype

Diseased H. macropterus exhibited distinct gross pathological features, presenting a typical cachectic phenotype. A dorsal overview (Figure 1A) revealed extreme abdominal distension accompanied by severe caudal emaciation, with the morphological discrepancy between the anterior and posterior body segments constituting a hallmark of cachexia. Magnified observations of the dorsal and ventral regions (Figure 1B,C) demonstrated asymmetric bilateral swelling, with prominent hemorrhagic and congestive lesions. Higher magnification of the abdominal protrusion (Figure 1D) showed mechanical abrasion on the epidermis, albeit without overt hemorrhage or ulceration. The adipoid mass displayed evident vascular infiltration (Figure 1E) and exhibited significant morphological divergence from normal adipose tissue. Upon dissection of the adipoid mass (Figure 1F), a well-defined serosal layer was observed, enclosing adipose-like tissue internally. Systematic gross inspection of major internal organs (liver, spleen, kidney, and intestine) in diseased individuals failed to detect classic pathological hallmarks indicative of acute bacterial or viral infections, including marked organomegaly, focal hemorrhage, tissue necrosis, or ascites. This finding, combined with the negative outcomes of comprehensive bacteriological culture and virological assays, provided limited evidence for a primary bacterial or viral etiology. Furthermore, during systematic anatomical examination of the diseased fish, no distinct gonadal structures were macroscopically identifiable at the expected anatomical location, which appeared largely occupied by the expansive pseudotumor. This is presumably due to either compressive effects from the abdominal adipoid mass, physical displacement, or severe gonadal developmental arrest/atrophy induced by prolonged nutritional deprivation, culminating in a functional gonadal arrest.

3.2. Pseudotumor Histoarchitecture

Systematic histopathological investigations were consistent with that the adipose-like masses in diseased H. macropterus were pseudotumor tissues, accompanied by mild to moderate-severe lesions in several organs including the gills, mesonephros, spleen, and head kidney. The pseudotumor exhibited a specific histological structure, consisting primarily of a fibroblast-derived capsule enclosing a proliferative tissue mass (Figure 2A). High-magnification observation revealed an infiltrated vascular network and a heterogeneous population of lesion-associated cells, which were categorized into four distinct morphological types (Figure 2B). Type I cells formed the majority and appeared as irregularly rounded cells with faint pink cytoplasm, poorly defined cell membranes, and small peripherally located nuclei measuring approximately 8.5 ± 1.5 μm in diameter. Type II cells, also a principal component, exhibited a signet-ring morphology with deeply stained cell membranes and large internal vacuoles that occasionally contained pale material, measuring about 10 ± 3 μm in diameter. The primary distinction between Type I and II cells lay in their degree of cytoplasmic vacuolation, potentially representing different growth stages. Type III cells were elliptical with intensely basophilic cytoplasm and were often separated from other cells by significant interstitial spaces; most lacked visible nuclei and measured around 4 ± 0.7 μm, while a minority displayed multiple nuclei with a diameter of approximately 9 ± 2 μm. Type IV cells were sporadically distributed as irregular, rhomboid satellite cells with blue-stained cytoplasm and a visible nucleus, averaging 7 ± 2 μm in diameter. Oil Red O staining indicated that adipocytes were sparse within the pseudotumor, accounting for a relatively low proportion of its overall structure (Figure 2C). Alcian blue staining showed that Type I, II, and IV cells were negative, while Type III cells stained red, albeit less intensely than erythrocytes (Figure 2D). In contrast, all four cell types were negative for AB-PAS staining, which instead highlighted extensive fibrous scaffolds throughout the pseudotumor tissue (Figure 2E). Despite the presence of a vascular network and diverse cell populations, the pseudotumor tissue did not exhibit features of acute suppurative inflammation, such as significant neutrophil infiltration, or the presence of bacterial microcolonies.

3.3. Parasitic Cysts in Gill Tissue

The gills represented the primary target organ among native tissues, displaying numerous cystic structures (Figure 3A)—a pathological feature suggestive of a parasitic infection, for instance myxosporidiosis. However, non-cystic gill lamellae remained structurally intact without other overt lesions. Magnified views of gill lamella cyst regions (Figure 3B) revealed cyst walls composed of epithelial tissue, with intracystic contents consisting of basophilic Type III cells and cellular debris. AB-PAS staining of gill tissues (Figure 3C) demonstrated abundant fibrous tissue within cysts, while Alcian blue staining (Figure 3D) confirmed numerous type III cells within the cystic structures.

3.4. Renal Fibrosis and Degeneration

The mesonephros was also identified as a target organ for pathological changes, characterized by fibrotic infiltration (Figure 4A). A unique pathological pattern—predominantly glomerular lesions with secondary tubular and interstitial involvement—was observed, which is relatively rare in fish histopathology. Glomeruli exhibited degeneration and dilation, while renal tubules showed degenerative changes; melano-macrophage centers were also noted (Figure 4B). Overall glomerular changes included extreme dilation of intraglomerular capillaries, swelling of glomerular endothelium, and presence of type III cells (Figure 4C). Interstitial regions contained Type III cells and mild cellular necrosis (Figure 4D). Additionally, calcium deposits were detected in renal tubules of some regions (Figure 4E).

3.5. Immune and Other Organs Involvement

The spleen and head kidney showed predominantly mild pathological changes, with activation of the immunophagocytic reticuloendothelial system and mild necrosis as key features. The spleen maintained relatively intact overall architecture, containing numerous basophilic Type III cells, mild granulomatous hyperplasia, and slight reticuloendothelial cell necrosis (Figure 5A). In the head kidney, prominent reticuloendothelial system activation was observed, characterized by increased reticuloendothelial cells with rounded nuclei, accompanied by scattered reticuloendothelial cell necrosis with pyknotic nuclei and cytoplasmic margination (Figure 5B); the structure of interstitial hematopoietic cells in the kidney remained relatively intact without significant lesions (Figure 5B).

Mild pathological changes were observed in the skin and liver. Compared with normal skin and muscle tissues (Figure S1A), diseased fish exhibited epidermal loss and mild peritoneal inflammation, with no significant changes in muscle tissue (Figure S1B). Relative to normal hepatic tissues (Figure S2A), hepatocytes showed mild steatosis and edema; the proportion of leukocytes in hepatic vein blood cells was increased, with enhanced eosinophilic staining in plasma (Figure S2B). High-magnification views revealed marked edema between hepatic cords and scattered Type III cells (Figure S2C). In contrast to normal tissues (Figures S3–S5), the intestine, heart, brain, swim bladder, and eyes of diseased fish showed no characteristic pathological changes, maintaining normal histological architecture.

3.6. Ultrastructural Pathology of Pseudotumor

Transmission electron microscopy analysis revealed that the pseudotumor tissue was encapsulated by a monolayer of epithelial cells and contained a monomorphic population of poorly differentiated, hyperplastic cells with marked atypia of diverse morphology (Figure 6A). The primary ultrastructural characteristics included marked cellular pleomorphism, with cells exhibiting highly irregular sizes and shapes, prominent nucleoli, increased nuclear-to-cytoplasmic ratios, indistinct intercellular boundaries, and an abundance of lysosomes (Figure 6A,A′). Further examination detailed the morphology of the different cell types. Type I and Type II cells were primarily distinguished by their nuclear morphology (Figure 6B,C), with Type II cells displaying lighter chromatin that often aggregated into a single prominent nucleolus (Figure 6C), which likely contributed to their vacuolated appearance in H&E-stained sections. Type III cells were characterized by a disproportionately large nucleus and electron-dense cytoplasm (Figure 6D). Type IV cells contained numerous lysosomes (Figure 6E). Additionally, a minority of cells exhibited other distinct morphologies, including those with mitochondrial proliferation (Figure 6F), cells undergoing nuclear division (Figure 6G), spindle-shaped cells (Figure 6H), cells with indistinguishable plasma membranes (Figure 6I), cells containing large autophagolysosomes (Figure 6J), and residual autophagolysosomal structures (Figure 6K). Mitochondria constituted the most prominently altered organelles within these tumor-like cells, predominantly exhibiting swelling, cristae fragmentation or complete disappearance, internal vacuolization, or the presence of autophagosomes (Figure 6L,M). These ultrastructural observations were consistent with the histopathological findings of a highly atypical and poorly differentiated proliferative lesion. Critically, thorough examination of multiple grid squares revealed no viral particles, bacterial organisms, or distinct parasitic spore structures within the neoplasm-like cells or the interstitial spaces. This ultrastructural evidence corroborated the histological and microbiological findings, collectively arguing against a conventional viral or bacterial pathogenesis for the pseudotumor formation.

3.7. Molecular Functional Profiling of the Pseudotumor Tissue

The functional profile of the pseudotumor tissue was investigated to complement its histopathological characterization. Metagenomic analysis was employed to characterize the functional landscape of the pseudotumor tissue. KEGG pathway composition analysis, based on CPM-normalized gene abundances, revealed that a substantial portion of annotated functions fell within broad disease-related categories (Figure 7A). The predominant secondary classifications included Cancer; Cardiovascular disease; Endocrine and metabolic disease; and Immune disease. Among the most represented specific pathways were Viral carcinogenesis (1.18%), Shigellosis (1.16%), Pathways of neurodegeneration-multiple diseases (1.12%), Pathways in cancer (1.11%), and Herpes simplex virus 1 infection (1.11%). It is noteworthy that pathways such as “Pathways in cancer” and “Viral carcinogenesis” encompass genes involved in fundamental cellular processes (e.g., cell cycle, proliferation, survival signaling) that are not exclusive to neoplasia but can also be activated in hyperplastic, inflammatory, or infected tissues. Analysis of the overall KEGG pathway composition indicated that the most abundant pathways were Motor proteins (5.12%), Phototransduction-fly (4.32%), and Metabolic pathways (2.43%) (Figure 7A), suggesting a state of heightened cellular and metabolic activity within the tissue. Gene composition analysis identified several highly represented genes in the pseudotumor (Figure 7B), including myo3, dfnb30 (11.8%); xrn1, sep1, kem1 (1.6%); fam83 (1.37%); c3 (1.25%); obscn, arhgef30 (1.05%); rab36 (1.04%); ptprf, lar (1.02%); krab (0.92%); btn, cd277 (0.89%); lgmn (0.86%); spon1 (0.86%); hvcn1, hv1 (0.86%); v2r (0.82%); tuba (0.69%); and slc25a21, odc (0.66%). Among these, genes such as fam83 (a driver of oncogenic signaling [12]), c3 (complement component involved in inflammatory and tumor microenvironments [13]), and ptprf (a regulator of cell adhesion and migration [14]) have been associated with cell proliferation and stress responses in various contexts [15]. A gene correlation network constructed using genes with high CPM values illustrated a regulatory network centered around myo3, which incorporated several of these proliferation- and metabolism-associated genes (Figure 7C). In summary, the molecular functional profile of the pseudotumor tissue reflects a state of intense transcriptional activity, with enrichment in pathways and genes related to cell proliferation, metabolism, and host response. This molecular signature is consistent with the observed histopathological phenotype of active cellular hyperplasia and provides a complementary perspective to the morphological findings.

3.8. Potential Eukaryotic Pathogens Identification

Given the histopathological evidence suggestive of a parasitic infection, we employed metagenomic sequencing as an exploratory tool to identify associated eukaryotic microorganisms. Taxonomic analysis of the microbial community in the pseudotumor tissue revealed that the microbial community was predominantly composed of bacteria and eukaryotes (Figure 8A). While metagenomic analysis detected bacterial sequences, their taxonomic and functional profiles were inconsistent with those of known primary bacterial pathogens of fish. Given the negative results from standard bacteriological and virological assays, as well as the absence of corresponding histopathological signs (e.g., suppurative inflammation), the detected bacterial sequences were considered more likely to represent background microbiota or secondary colonizers rather than the primary causative agents of the pseudotumor syndrome. Within the kingdom Fungi, the most abundant classes included Glomeromycetes, Agaricomycetes, Sordariomycetes, Chytridiomycetes, and the phylum Microsporidia (Figure 8B). Among the unclassified Eukaryota, the most prevalent taxa were associated with Dinophyceae, Ulvophyceae, and Oomycota (Figure 8C). A literature-based review identified 18 distinct taxonomic groups known to exhibit parasitic lifestyles, including Microsporidia, Chytridiomycetes, Basidiomycota, Plasmodiidae, Amoebae, Ichthyosporea, and Perkinsus (Figure 8D). Morphological and size correlation analysis suggested that Microsporidia, Amoebae (including genera such as Naegleria [primarily N. fowleri, N. gruberi, and N. lovaniensis], Acanthamoeba [primarily A. castellanii], Entamoeba [primarily E. dispar, E. histolytica, and E. invastans], and Amoebophilus [primarily A. protococcus and A. occidentale]), Perkinsus (primarily P. olseni and P. marinus), and Ichthyosporea (primarily Sphaeroforma arctica) were the most probable etiological agents responsible for the disease outbreak in H. macropterus (Figure 8D). Among these candidate parasites, Microsporidia, Ichthyosporea, and Acanthamoeba showed relatively high abundance in the metagenomic profile, whereas Entamoeba was significantly less abundant (Figure 8D).

4. Discussion

To our knowledge, this study provides the first systematic description of a novel syndrome in cultured H. macropterus, characterized by massive abdominal pseudotumors, severe progressive emaciation (cachexia), and functional gonadal arrest. Integrated evidence from histopathology, ultrastructural analysis, and metagenomics indicates that this syndrome is unlikely to be primarily caused by traditional bacterial or viral pathogens. This conclusion is supported by the absence of growth in culture, lack of cytopathic effects in cell lines, no corresponding histopathological features, and the dominant eukaryotic signature in metagenomic analysis rather than a pathogen-specific prokaryotic signature. This infection induces a tumor-like tissue response in the host and leads to systemic metabolic and reproductive failure. This finding warrants attention in the context of conservation aquaculture for this species but also offers a new perspective for investigating parasite-induced host tissue remodeling and systemic energy deprivation.

Pathological features suggest a coexistence of parasitic infection and host tumor-like response. Histological observations revealed that the pseudotumor was encapsulated by a fibroblastic capsule and contained four morphologically distinct cell populations. Among these, Type III cells, with intensely basophilic cytoplasm, frequent multinucleation, and significant intercellular spaces, extensively invaded organs such as the gills (forming encapsulated cysts), kidneys, and spleen. Their morphology and distribution strongly suggest a parasitic origin, resembling the proliferative or sporogonic stages of certain parasitic protozoans like myxozoans [32,33]. In contrast, Type I and II cells constituted the bulk of the pseudotumor, appearing as irregularly rounded or signet-ring-shaped with indistinct cell borders, high nuclear-to-cytoplasmic ratios, and varying degrees of cytoplasmic vacuolation. This suggests they may represent different developmental or degenerative stages of the same class of poorly differentiated, proliferative, tumor-like cells. Histological and ultrastructural examinations revealed that the pseudotumor was composed of a monomorphic population of poorly differentiated host cells, characterized by high nuclear-to-cytoplasmic ratios, nuclear atypia, and marked angiogenesis, all enclosed within a fibroblastic capsule. While these features are reminiscent of neoplastic growth, the absence of invasion and the context of infection led us to interpret this lesion primarily as a parasite-induced inflammatory pseudotumor or massive atypical hyperplasia. The molecular functional profile derived from metagenomics provides a complementary, albeit cautious, layer of understanding. The observed enrichment of broad KEGG categories such as “Pathways in cancer” and “Viral carcinogenesis” must be interpreted within the specific context of this study. These pathways encompass fundamental processes like cell cycle progression, survival signaling, and metabolic reprogramming, which are universally upregulated in actively proliferating tissues—whether neoplastic, hyperplastic, or engaged in a sustained inflammatory response [34]. Therefore, in this case, the KEGG profile is best understood not as a diagnostic signature for cancer, but as a molecular correlate of the intense cellular activation and dysregulated proliferation observed histologically within the host tissue. The relatively high representation of genes such as fam83 (involved in proliferation signaling [12]) and ptprf (involved in cell adhesion [14]) further supports the state of active tissue remodeling. This host reaction may be driven by persistent immunomodulatory stimuli from the parasite. Similar to how certain helminth infections can alter local microenvironments to promote host cell proliferation [35], the chronic presence of eukaryotic parasites here appears to have triggered an aberrant and massive proliferative response in susceptible host tissue. This response, morphologically forming a pseudotumor, ultimately functions as a metabolically active “sink”, diverting host resources.

Cachexia and arrest of gonadal development are likely outcomes of systemic pathometabolism [36]. The massive abdominal pseudotumor may function as a highly metabolically active “nutrient sink,” continuously consuming host energy and substrates, triggering a systemic catabolic state. This mechanism is similar to cancer-related cachexia, involving accelerated muscle protein breakdown and enhanced fat mobilization [37,38]. The stark contrast between severe caudal emaciation and abnormal abdominal distension observed in this study visually demonstrates the catastrophic consequences of this nutrient redistribution. The enrichment of the “Endocrine and metabolic disease” pathway in the KEGG analysis provides molecular-level support for the existence of systemic metabolic dysregulation. Against this background, the complete stagnation or atrophy of gonad development can be reasonably explained: reproduction is a highly energy-dependent process. When the organism is under severe stress and in a state of energy deficit, the function of the hypothalamic-pituitary-gonadal (HPG) axis is prioritized for suppression [39]. Nutrients and energy are redirected to maintain basic survival and cope with the parasitic infection, leading to functional reproductive arrest [40,41]. Therefore, the potential impact of this syndrome on the sustainability of cultured populations may lie not only in stunted individual growth but, if the condition is transmissible and widespread, in a the functional loss of reproductive capacity it likely causes, as evidenced by the absence of macroscopically identifiable gonads in cachectic individuals. The metabolic reprogramming observed in the pseudotumor, which diverts host resources, parallels the metabolic hijacking strategies employed by various pathogens to support their own replication.

The presence of numerous encapsulated cysts and internal basophilic cells in the gills readily brought to mind common fish parasites—myxozoans (Myxozoa), whose life cycle includes stages that form spore cysts in tissues like gills [42,43]. However, our metagenomic sequencing data did not yield identifiable sequences for myxozoans with sufficient confidence. Conversely, the analysis indicated the presence of other categories of eukaryotic parasites, including microsporidians (Microsporidia), ichthyosporeans (Ichthyosporea), and amoebae (such as Acanthamoeba). Cross-referencing histological morphology with the biological characteristics of these pathogens: The size of Type III cells (diameter ~4 μm, multinuclear form ~9 μm) is closer to the spores of microsporidians (typically 1–10 μm) or the multinucleate trophozoites of ichthyosporeans (5–20 μm) [27,28], and differs significantly from the larger Perkinsus trophozoites (20–100 μm) [28] or many mature myxozoan spores. Microsporidians and some ichthyosporeans are known to form multinucleate syncytial-like trophozoite clusters within the host, also appearing as dark blue cytoplasmic masses in H&E staining [27,28]. Amoebic trophozoites are characterized by their invasiveness and cytoplasmic vacuolization, with a size range (10–50 μm) that could encompass the larger Type III cell units [18,21]. In summary, although myxozoans were initially suspected based on morphology, metagenomic evidence more strongly supports microsporidians, ichthyosporeans, or amoebae as candidate pathogens. Among these, the basophilia, multinucleation tendency, and smaller size of Type III cells best match certain stages of microsporidians or ichthyosporeans, while their invasive behavior aligns with the pathogenic characteristics of amoebae. Future targeted validation using specific molecular detection methods (e.g., in situ hybridization, PCR) with probes for these parasites is required to definitively identify the causative agent.

5. Conclusions

This study reveals a previously unreported disease syndrome, likely associated with a eukaryotic parasitic infection, centered on the formation of a parasitic pseudotumor and leading to systemic host cachexia and loss of reproductive function. It underscores the need to monitor and investigate emerging parasitic diseases in conservation aquaculture and provides a unique perspective for exploring the mechanisms by which parasites mimic tumor characteristics and hijack host metabolism.