The fry of grass carp (11 ± 1 cm, 11.4 ± 0.5 g) was purchased from an aquaculture pond in Jiangmen, Guangdong Province, where the prevalence and intensity of S. acheilognathi in grass carp were 40% and 3.2, respectively in previous survey. Grass carp were kept temporarily for 3 days prior to the formal experiment.

Grass carp were randomly divided into four buckets, fed with a normal diet (ND) with 5% fat content or a high-fat diet (HFD) with 10% fat content (Huaian Tongwei Feed Co., Ltd., China; Table 1) to apparent satiation twice daily (10: 00, 17: 00 oor ersity of se mationertebradomesticated in 100 L plastic buckets under a natural photoperiod (12 h light: 12 h dark), and kept at a water depth of 60 cm and a water temperature of 29°C. Following a 4-week feeding trial, the grass carp (n = 63) were anesthetized with eugenol (0.2 mL/L) before sampling.

The growth indices of the grass carp were measured, and blood samples were drawn from the caudal vein. Liver samples for gene expression analysis and oil red O staining were stored at −80°C and fixed in 4% paraformaldehyde (PFA) solution, respectively. The intestine was aseptically excised from visceral organs. The foregut and midgut were used for parasites checking under a stereomicroscope (Leica, Germany) to determine whether the host was infected with S. acheilognathi. Hindgut contents, intended for 16S rDNA high-throughput sequencing, were stored at −80°C for DNA extraction, and hindgut samples for hematoxylin and eosin (HE) staining were fixed in 4% PFA.

Based on the helminth infection status and the fat content in the feed, the samples were divided into four groups: ND (5% normal fat diet with no S. acheilognathi infection), ND + SA (5% normal fat diet with S. acheilognathi infection), HFD (10% high fat diet with no S. acheilognathi infection) and HFD + SA (10% high fat diet with S. acheilognathi infection). After dissection, it was recorded that the mean intensity of S. acheilognathi in grass carp was 3.9 (2–7).

The standard length (L) and body weight (W) of the grass carp were measured to calculate the condition factor (CF). The liver weight (Wl), viscera weight (Wv), and mesenteric fat weight (Wm) were measured individually to determine the hepatosomatic index (HSI), mesenteric fat index (MFI), and visceral index (VSI), respectively. The formulas for calculation of these indicators were shown as follows: CF = W/(L³) × 100%; HSI = Wl/W × 100%; VSI = Wv/W × 100%; MFI = Wm/W × 100%.

Blood samples were centrifuged at 3,000 rpm for 10 min at 4°C, after which the supernatant serum was carefully collected and stored at −80°C until used. The activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), as well as the concentration of high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), total triglycerides (TG), and total cholesterol (TC) in serum were measured through a biochemical analyzer, using commercially available reagent kits (Seville Biotech Co., Ltd., Wuhan, China).

Fresh hindgut tissues were immersed in 4% PFA solution for 48 h, and then processed routinely. These tissues were sectioned into 6 μm slices and stained with hematoxylin and eosin for examination under a light-microscopic (Zeiss, Germany). Photographs were analyzed using software ImageJ (National Institutes of Health, Bethesda, MD, United States). The thickness of muscularis mucosa (MM), the height of microvilli (MV) and perimeter ratio (PR) were measured, respectively. The PR was calculated using the following formulas: PR = the internal perimeter (IP) of the intestine lumen (villi and mucosal folding length)/the external perimeter (EP) of the intestine.

Liver samples from grass carp were excised and preserved in 4% PFA for 24 h. Subsequently, the tissues were trimmed to ensure smoothness and then dehydrated in 30% sucrose solution at 4°C for 24 h. Once the surface liquid of the liver tissue was evaporated, it was embedded in OCT embedding agent (SAKURA, United States). After freezing at −80°C, the tissue was then sectioned into 8 μm thick slices using a cryostat microtome (Leica, Germany) and stained with oil red O. Photomicrographs were captured using a light microscope (Zeiss, Germany). A total of six random fields of view were chosen from each sample, and the areas of lipid droplets were calculated using software ImageJ.

RNAiso Plus Reagent (Takara Bio Inc., Beijing, China) was used to extract total RNA. After being treated with RNase free DNase I (Promega, Wisconsin, United States), single-strand cDNA was synthesized using One−Step gDNA Remover kit (Servicebio, Wuhan, China). qPCR was carried out in a CFX96™ Real Time Detection System (BIO-RAD Bio Inc., Shanghai, China) using TB Green^® Premix Ex Taq™ (Beijing, China). Gene-specific primers (Table 2) were used to amplify the target gene fragments. The β-actin gene of grass carp (Accession No. M25013.1) served as the reference gene. The qPCR cycle conditions were as follows: an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 34 s, and a final Melt Curve analysis. The Ct method (2^–ΔΔCT) (Livak and Schmittgen, 2001) was used to determine the relative expression levels of immune and lipid metabolic related genes in liver of grass carp.

The total bacterial DNA was extracted using the TGuide S96 magnetic bead method for soil/fecal genome DNA (Tiangen Biotech, Beijing, China), following the protocol provided by the manufacturer. The purity and concentration of genomic DNA were determined using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, United States). The extracted DNA was preserved at −80°C. The V3-V4 hypervariable region of the bacterial 16S rDNA gene was amplified using the primers 338F (5′−ACT CCT ACG GGA GGC AGC A−3′) and 806R (5′−GGA CTA CHV GGG TWT CTA AT−3′) (Mori et al., 2013). The PCR amplification program was the same as previously reported (Fu et al., 2019). PCR products were purified with Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN) and quantified with the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer. Following individual quantification, equimolar amounts of amplicons were combined into a single pool. Sequencing was conducted by Biomarker technologies (Qingdao, China) on the Illumina NovaSeq 6000 sequencing platform. The raw 16S rRNA sequence data can be obtained in the NCBI SRA database (Bioproject: PRJNA1045724).

The raw sequencing data were analyzed using the QIIME2 Pipeline, version 2021.4.¹ The raw data were initially processed using Trimmomatic 0.35 (Bolger et al., 2014) for quality filtering, followed by the identification and removal of primer sequences with Cutadapt version 1.9.1 (Martin, 2011). DADA2 was subsequently employed to correct errors in the merged reads and to identify amplicon sequence variants (ASVs) (Callahan et al., 2016). The ASVs were then classified taxonomically using a native Bayes classifier (Wang et al., 2007), pre-trained on the SILVA 138 (Balvočiūtė and Huson, 2017).

Alpha diversity (ACE, Chao1, Simpson and Shannon index) of gut microbiota was calculated by QIIME2 and visualized in software R version 3.5. Beta diversity was used to evaluate the similarity between microbial communities across different samples in QIIME2. PERMANOVA analysis, non-metric multidimensional scaling (NMDS), Principal coordinate analysis (PCoA) and unweighted pair group mean algorithm (UPGMA) were used to visualize the beta diversity based on weighted Unifrac or Bray-curtis distance. The Pearson correlation coefficient was conducted using software PAST version 4.13 to examine the linear correlation between helminth infection status and the abundance of bacteria. Differences in taxa among groups were tested with Venn diagram and linear discriminant analysis coupled with effect size (Lefse). The metagenomic information of the samples was predicted from the 16S rDNA gene sequence data by employing PICRUST 2.0 in conjunction with the KEGG database (Langille et al., 2013). STAMP version 2.1.3 was used to perform all statistical analyses on the functional profiles (Parks et al., 2014).

Data analysis among the four groups utilized the One-Way ANOVA, supplemented by LSD post hoc testing. Students’ t-test was used to perform statistically analysis between two groups. All the statistical tests were performed in software SPSS 20 at the 0.05 significance threshold.

The growth indices of grass carp were presented in Figures 1A–D. Among the four groups, HFD + SA had the highest CF, MFI and VSI (Figures 1A,C,D). The CF of HFD + SA group was significantly higher than that of the HFD group (P = 0.015 < 0.05) and ND group (P = 0.004 < 0.05) (Figure 1A); the MFI of HFD group was merely higher than that of ND group (P = 0.039 < 0.05, Figure 1C); and the VSI was only showed significant difference between the HFD + SA group and the ND group (P = 0.025 < 0.05, Figure 1D). The HSI did not exhibit any significant differences across the four groups (P > 0.05, in all cases, Figure 1B).

The serum biochemical indices of grass carp were shown in Figures 1E–J. HFD significantly increased the activity of ALT (Figure 1E, P = 0.012) and AST (Figure 1F, P = 0.004), elevated serum levels of TG (Figure 1G, P = 0.005) and CHO (Figure 1H, P = 0.045), and reduced serum concentration of HDL (Figure 1I, P = 0.001) in comparison with normal diet. but the concentration of LDL between ND and HFD showed no significance (Figure 1J, P > 0.05). Comparing to HFD, helminth infection significantly reduced the activity of AST (Figure 1F, P = 0.044), decreased the concentration of LDL (Figure 1J, P = 0.008), and elevated the serum HDL content (Figure 1I, P = 0.006) in grass carp fed with HFD. The activity of ALT and the levels of TG and CHO in the serum showed a slight decrease in the HFD + SA group compared to the HFD group, but the differences were not significant (Figures 1E,G,H, P > 0.05 in all cases).

The results of the hindgut sections stained with H&E were presented in Figures 2A–D. Helminth infection significantly decreased the MM (Figure 2F, P = 0.015) and PR (Figure 2G, P = 0.022) in grass carp fed on HFD, with no effect on MV (Figure 2E, P > 0.05). In the normal diet, helminth infection had no affection on the MM, MV, and PR (Figures 2E–G, P > 0.05 in all cases).

The liver lipid content was determined using oil red O staining. The results indicated that the lipid droplets in the HFD group were larger and denser comparing with the other three groups (Figures 2H–K). Helminth infection significantly decreased the fat ratio value in liver of grass carp fed with HFD (P = 0.000, Figure 2L). However, helminth infection significantly increased the fat ratio value in grass carp fed normal diet (P = 0.025, Figure 2L).

The mRNA expression levels of immune and lipid metabolism related genes were showed in Figure 3. Helminth infection significantly promoted the expression of transforming growth factor-beta 1 (TGF-β1) (Figure 3C, P = 0.004), interleukin-10 (IL-10) (Figure 3D, P = 0.000) and apolipoprotein E (ApoE) (Figure 3G, P = 0.001), and significantly down-regulated the expression of interleukin-1beta (IL-1β) (Figure 3A, P = 0.009), tumor necrosis factor-alpha (TNF-α) (Figure 3B, P = 0.027), peroxisome proliferator-activated receptor-gamma (PPAR-γ) (Figure 3E, P = 0.003) and lipoprotein lipase (LPL) (Figure 3F, P = 0.001) in grass carp fed on HFD, but no affection on the expression of carnitine palmitoyltransferase 1(CPT1) (Figure 3H, P > 0.05). Meanwhile, helminth infection significantly elevated the relative expression levels of PPAR-γ (Figure 3E, P = 0.000), and significantly reduced the expression levels of ApoE (Figure 3G, P = 0.011) and CPT1 (Figure 3H, P = 0.007), but no impact on the expression levels of IL-1β, TNF-α, TGF-β1, IL-10, and LPL (Figures 3A–D,F; P > 0.05 in all cases) in grass carp fed with normal diet.

In growth performance, helminth infection with grass carp fed on HFD pointedly increased the CF, and slightly increased VSI and MFI. Numerous investigations on fish species, including Micropterus salmoides (Guo et al., 2019; Yin et al., 2021), Nibea japonica (Han et al., 2014), and Rachycentron canadum (Wang et al., 2005), have revealed a positive correlation between the VSI and MFI and the level of dietary lipids. The rise in CF observed in this study was associated with a concurrent upward trend in both VSI and MFI. The level of dietary fat usually causes no change of CF in fish (Guo et al., 2022; Han et al., 2014; Yin et al., 2021). Thus, the significant increasing of CF in HFD + SA group indicated that helminth infection boosted the growth of grass carp fed with HFD.

Helminth infection with grass carp fed with HFD exhibited a notable rise in LDL level, while AST level and HDL concentration significantly declined. AST is primarily situated within hepatocytes, and an increase in its serum level is a response to hepatocellular damage and changes in plasma membrane permeability (Boone et al., 2005). The high-fat diet led to an elevation in serum AST level in grass carp, indicating potential damage to liver cells. However, helminth infection could possibly decrease the liver’s burden imposed by the high-fat diet and facilitate the recovery of liver function. HDL and LDL are typically used as indicators to reflect lipid metabolism in aquatic animals (Guo et al., 2019; Li et al., 2016; Zhao et al., 2016) and mammals (Lu et al., 2022; Teng et al., 2020). High serum LDL is always considered to be risk factors related to fatty livers in fish (Guo et al., 2019; Li et al., 2016; Zhao et al., 2016). HDL has been referred to as good cholesterol, inversely proportional to cardiovascular risk in many studies (Ertek, 2018). In present study, the increase in HDL and decrease in LDL indicated that helminth infection reversed the abnormal lipid metabolism in HFD of grass carp, which could improve health condition of cultured C. idella.

PPAR-γ, LPL, ApoE, and CPT1 are the key regulatory enzymes involved in lipid metabolism within the liver, playing roles in the uptake, transport and oxidation of fatty acids. PPAR-γ is a ligand-activated transcription factor that belongs to the nuclear hormone receptor superfamily and fosters lipogenesis by enhancing the expression of enzymes involved in lipid synthesis (Ahmadian et al., 2013). LPL catalyzes the hydrolysis of intravascular triglycerides contained within lipoproteins, such as chylomicrons and very low-density lipoprotein (VLDL), then the released fatty acids can be taken up by tissues for use in oxidation or for storage purposes (Wu et al., 2021). ApoE is a soluble apolipoprotein primarily synthesized in the liver and brain, and it has an important role in the metabolism of triglyceride rich lipoproteins (TRL) due to its high affinity binding to the LDL receptor (LDLR), the LDL receptor-related protein 1 (LRP1) and the VLDL receptor (VLDLR), which facilitates the hepatic clearance of remnant lipoprotein particles (Huang and Mahley, 2014). LPL-mediated triglyceride hydrolysis would reduce when ApoE concentration was high (Wu et al., 2021). CPT1 is regarded as a pivotal regulatory enzyme in mitochondrial fatty acid oxidation pathway. It catalyzes the conversion of fatty acyl-CoAs into fatty acyl-carnitine molecules, which are then transported into the mitochondrial matrix for further oxidation (Kerner and Hoppel, 2000). In the present study, helminth infection in grass carp fed with HFD resulted in a modest elevation of CPT1 expression, and caused a significantly reduction in the expression levels of PPAR-γ and LPL, while significantly increased the expression level of ApoE. These results indicated that helminth mainly regulate the expression of these lipid metabolism genes to inhibit the absorption of fatty acids, thereby reducing the deposition of lipid in the liver.

Additionally, the lipid content in the liver of the HFD + SA group was significantly lower than that of the HFD group, indicating that helminth infection mitigated the liver lipid deposition induced by the high-fat diet. The protective effect of helminth in grass carp was similar to that in mice (Su et al., 2018). Most of the digested lipids are absorbed by intestinal epithelial cells (Zhang et al., 2021). PR, one of the intestinal morphology parameters, was significantly decreased by helminth infection in this study, reducing the amount of lipid absorbed by intestine, thereby to decrease lipid deposition in the liver.

Similarity to the protective effects of helminth in mammalian metabolic diseases, the primary regulatory pathways through which helminths exert positive effects on metabolic diseases in fish may include the following two aspects: (1) immune regulation, (2) alterations of gut microbiota. The type 2 immune response induced by helminth infection has been proved to ameliorate the side effects of HFD in mice (Su et al., 2020; Su et al., 2018). The intestinal nematode H. polygyrus could protect against obesity by triggering the production of Th2-mediated cytokines, such as IL-4, lL-10 and IL-13, and enhancing the anti-inflammatory M2 macrophages levels (Su et al., 2018). The increased M2 and IL-10 responses resulted in a reduction of the pro-inflammatory cytokine TNF-α expression, which has been shown to modulate lipid metabolism (Khovidhunkit et al., 2004). Similarly, in our study, helminth infection significantly elevated IL-10 levels while concurrently reducing decreased TNF-α expression, indicating that helminth infection protects against the HFD-induced abnormal lipid metabolism via activation of immune responses in teleost.

An increase of alpha diversity in intestinal microbiota is generally associated with a “healthy” gut homeostasis (Peachey et al., 2017). In this study, a high-fat diet significantly decreased the α diversity of intestinal microbiota in grass carp (Figures 4C–F), indicating that HFD disrupted gut microbial balance. Similar disruptions caused by HFD have also been reported in Monopterus albus (Peng et al., 2019). Additionally, there was no significant difference in the α diversity of gut microbiota between the HFD group and the HFD + SA group (Figures 4C–F), which may be attributed to the regulatory effect of helminth on intestine microbiota.

Microbiota and helminths occupy the same ecological niche within the host’s intestine, where they can interact with each other (Glendinning et al., 2014). Multiple investigations have been carried out to examine the effects of helminth infection on the gut microbiota in fish. Tapeworm and acanthocephalan infection caused alteration in the composition of fish gut microbiota (Fu et al., 2019; Fu et al., 2022; Jiang et al., 2019; Ling et al., 2020). Studies in mammals have shown that helminth infection protects against HFD-induced obesity through modifications to the gut microbiota (Shimokawa et al., 2019; Su et al., 2020).

helminth infection was found to enhance the relative abundance of Cetobacterium in hindgut of grass carp fed with HFD, and the proportion of Cetobacterium in the HFD + SA group was 91.6%. According to Spearman’s correlation analysis, Cetobacterium correlated positively with helminth infection. Cetobacterium, identified as an anaerobic, gram-negative bacterium (Finegold et al., 2003), constitutes a predominant member of the intestinal microbiota in freshwater fish (Di Maiuta et al., 2013; Fu et al., 2022; Larsen et al., 2014; van Kessel et al., 2011). The significant proliferation of Cetobacterium could perform fermentative metabolism of peptides and carbohydrates, resulting in the production of acetate, which in turn modifies glucose homeostasis (Wang et al., 2021). Additionally, this bacterium could produce vitamin B12 (Larsen et al., 2014), thereby enhancing the host couldsicrobioto pathogen infections (Qi et al., 2023). Incorporating Cetobacterium fermentation products into fish feed can effectively enhance intestinal and liver health and decrease liver lipid accumulation (Xie et al., 2022). In zebrafish, cypermethrin (CYP) exposure or CYP and microplastics (MPs) co-exposure increased the abundance of Cetobacterium, which was found to be positively associated with the majority of lipid metabolites (Xu et al., 2024). Consistently, helminth infection may ameliorate the lipid metabolism induced by HFD through increasing the high relative abundance of Cetobacterium to regulate lipid and maintain liver health of grass carp.

Lactobacillus, a group of gram-positive, facultative anaerobic bacteria widely used as probiotics, has been shown to enhance epithelial barrier function and modulate innate immune responses as well as cytokine profiles (Amdekar and Singh, 2016). Strongyloides venezuelensis infection in mice induced the increasing of Lactobacillus spp., which has a positive effect on the glucose metabolism of the host (Pace et al., 2018). In this study, helminth infection showed a significant positive correlation with Lactobacillus. This result indicated that helminth infection can improve host metabolic profile by regulating probiotics.

In study of Caenorhabditis elegans, reactive oxygen species produced by Rhizobium induce DNA damage, which caused abnormal intestinal nuclei divisions (Kniazeva and Ruvkun, 2019). Eubacterium, one of the main SCFAs-producing bacteria in humans (Parada Venegas et al., 2019). Rhodobacter is a gram-negative, purple non-sulfur photosynthetic bacteria. Orally given the Rhodobacter sphaeroides in mice increased the content of SCFAs (Yang et al., 2020). However, SCFA has a significant negative influence on established Oesophagostomum dentatum infection in pigs (Petkevicius et al., 2004). In this study, helminth infection showed significant positive correlation with Rhodobacter, Rhizobium, and Eubacterium, indicating that helminth may improve its survival in the intestine by altering the relative abundance of these three bacteria.