Synergistic Impacts of Co‐Exposure to Microplastics and Vibrio harveyi on the Immune and Stress Responses of the Big‐Belly Seahorse Hippocampus abdominalis

ABSTRACT

Microplastics (MPs) originating from synthetic polymers can act as vectors for harmful microorganisms and pollutants, exacerbating ecological risks. Vibrio harveyi, commonly found in seawater, is a major opportunistic pathogen causing vibriosis in marine fish. MPs and pathogenic bacteria such as V. harveyi have increasingly been recognized as co‐contaminants in marine environments. This study investigated the physiological responses of the big‐belly seahorse Hippocampus abdominalis exposed to MPs and V. harveyi, both individually and in combination. Seahorses were exposed to small (0.2 μm) and large (1.0 μm) polystyrene MPs (50 beads/L) and V. harveyi (1 × 10³ CFU/mL) for 4 days under controlled conditions. Biochemical parameters and molecular analyses were conducted to assess hepatic function, immune regulation, oxidative stress, and apoptosis‐related responses. MP exposure promoted V. harveyi proliferation within seahorse tissues, with higher bacterial copy numbers in the kidney than in the liver, indicating the kidney's active immune role. Plasma biochemical indices (alanine aminotransferase, aspartate aminotransferase, and glucose) and immune‐related genes, including lysozyme G, interferon regulatory factor 8, and interleukin 10, were significantly elevated in co‐exposure groups compared with individual exposures. Expression of heat shock protein 75 and caspase 8 was also upregulated, suggesting enhanced oxidative stress and activation of apoptotic pathways. These findings indicate that MPs and V. harveyi exert synergistic physiological stress, disrupting immune homeostasis and promoting apoptosis in seahorses. This study provides mechanistic insights into combined MP‐pathogen toxicity and establishes the big‐belly seahorse as a sensitive bioindicator for complex marine pollution.

Synergistic effects of microplastic and Vibrio harveyi co‐exposure on big‐belly seahorse (Hippocampus abdominalis). Seahorses were exposed to microplastics (50 beads/L of 0.2 μm SMP and 1.0 μm LMP) and injected with V. harveyi (1 × 10³ CFU/mL).

Summary

• Microplastics from synthetic polymers act as vectors for harmful microorganisms.

• Vibrio harveyi, an opportunistic pathogen, is a co‐contaminant with microplastics.

• We investigated responses of big‐belly seahorse exposed to microplastics and V. harveyi.

• Expression of heat shock protein 75 and caspase 8 was also upregulated.

• Microplastics, V. harveyi exert synergistic physiological stress; disrupt immune homeostasis.

Abbreviations

ALT

alanine aminotransferase

AST

aspartate aminotransferase

casp8

caspase‐8

hsp75

heat shock protein 75

IL10

interleukin‐10

IRF8

interferon regulatory factor‐8

lysG

lysozyme G

MPs

microplastics

1. Introduction

Seahorses are highly sensitive to habitat destruction and environmental pollution and are classified as Vulnerable on the International Union for Conservation of Nature (IUCN) Red List (Koning and Hoeksema 2021). These species possess prehensile tails that allow them to anchor onto various substrates; however, they exhibit limited swimming ability, resulting in low mobility, restricted home ranges, and distinctive ecological behaviors (Scales 2010). Owing to their biological vulnerability, seahorses have been recognized as important bioindicators for assessing the impact of marine environmental pollution (Foster and Vincent 2004; Delunardo et al. 2015). Moreover, their feeding habits, which primarily involve consuming zooplankton and small crustaceans, increase their likelihood of ingesting microplastics (MPs) of similar size, which makes them particularly susceptible to MP contamination (Domínguez‐López et al. 2022). The big‐belly seahorse Hippocampus abdominalis is typically distributed in coastal habitats, including seagrass beds and sheltered nearshore environments, where land‐based plastic inputs and aquaculture activities may elevate local MP and pathogen loads (Cohen et al. 2017; Gonsilou et al. 2025).

In recent years, marine pollution caused by MPs has emerged as a serious global issue threatening the health of marine ecosystems (Ghosh et al. 2023). MPs, defined as plastic fragments smaller than 5 mm in diameter, enter marine ecosystems through multiple sources. Due to their minute size, marine organisms can easily ingest these particles, often mistaking them for prey or food items (Ziajahromi et al. 2017; Gopal et al. 2022). According to Min et al. (2024), MPs ranging from 20 to 300 μm were found in Gwangyang Bay, South Korea, at an average concentration of 2.86 particles per liter. Likewise, previous studies have indicated that smaller‐sized MPs (less than 300 μm) generally occur at considerably higher levels in marine environments (Medina Faull et al. 2021). In semi‐enclosed coastal areas and aquaculture‐influenced waters, MP concentrations can potentially increase, which may pose exposure risks for benthic and site‐attached species (Eshom‐Arzadon et al. 2025; Zhang et al. 2025).

MPs not only cause physical damage to marine organisms but also act as carriers for environmental contaminants such as polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and various pathogens, facilitating their accumulation through the food chain (Pittura et al. 2018; Zhao et al. 2024). Consequently, numerous studies have reported that MP exposure negatively affects physiological functions, growth, reproduction, and immune responses in marine organisms (Vo and Pham 2021; Yang et al. 2023).

Marine environmental changes driven by global warming and various anthropogenic activities have accelerated the spread of pathogens (Combe et al. 2023). Among these, Vibrio spp. are known to form biofilms, enabling their long‐term survival on surfaces in aquaculture facilities and water tanks, posing a persistent source of infection for marine organisms (Cholewińska et al. 2022). In particular, Vibrio spp., which cause severe economic losses in seahorse aquaculture, are recognized as major pathogens responsible for large‐scale mortality events, such as septicemia and ulcerative diseases (Shafiee et al. 2024). Infections caused by Vibrio harveyi result in mass mortality of seahorses, leading to not only significant economic losses but also restrictions on the trade of infected individuals, ultimately affecting the ornamental fish market as well as pharmaceutical and food‐related industries (Cholewińska et al. 2022; Shafiee et al. 2024).

MPs can enter the circulatory system of fish through the digestive tract as well as the gills, leading to various physiological impairments (Zheng and Wang 2023). During this process, plasma serves as an essential body fluid reflecting changes in metabolic and physiological homeostasis, and can be used as a sensitive indicator for assessing MP‐induced toxicity (Pastorino et al. 2022). In addition, the biological response to external environmental stressors, including pathogenic infections, involves the activation of immune systems, stress responses, and apoptotic pathways (Liu et al. 2022). Lysozyme G (lysG) is a key antimicrobial enzyme in the innate immune system that is mainly found in leukocytes and liver tissue, and hydrolyzes bacterial cell walls to initiate the first line of defense against pathogen invasion (Ferraboschi et al. 2021). LysG is rapidly activated during the early stages of infection, contributing to host defense and the regulation of inflammatory responses (Ferraboschi et al. 2021; Matwiejczyk et al. 2025). Interferon regulatory factor‐8 (IRF8) is a transcription factor essential for both innate and adaptive immunity and regulates the differentiation and activation of macrophages and dendritic cells (Cai and Chen 2022; Moorman et al. 2022). IRF8 also mediates pathogen recognition signaling and controls cytokine secretion (Shi et al. 2021). Interleukin‐10 (IL10), one of the most potent anti‐inflammatory cytokines, plays a crucial role in preventing tissue damage by suppressing excessive immune responses (Islam et al. 2021). IL10 maintains immune homeostasis through intercellular signaling, providing protection against pathogenic bacteria and environmental stress (Mahapatro et al. 2021). Changes in the expression of these immune‐related genes serve as indicators of the immune defense and regulatory capacity of seahorses in response to pathogen infection and inflammatory stress.

Heat shock protein 75 (hsp75) is a molecular chaperone that maintains proper protein folding and stability, regulates mitochondrial function, and protects cells through antioxidant responses (Hu et al. 2022). The expression of hsp75 increases in response to various environmental stimuli such as elevated temperature, oxidative stress, and exposure to pathogens, playing an important role in minimizing cellular damage and maintaining homeostasis (Hu et al. 2022). Closely associated with these stress defense mechanisms is the regulation of apoptosis, in which caspase‐8 (casp8) serves as a key mediator. Casp8 acts as an initiator in the extrinsic pathway of apoptosis and is responsible for eliminating damaged or abnormal cells, thus contributing to tissue homeostasis (Orning and Lien 2021).

Marine organisms inhabit diverse aquatic environments; however, those dwelling in coastal regions are frequently exposed to a complex array of conditions where MPs and pathogenic bacteria coexist (Trevisan et al. 2022). MPs provide a favorable substrate for microbial attachment because of their large surface area, forming a novel microbial community referred to as the “plastisphere” (Zhai et al. 2023; Lombardo et al. 2025). Consequently, combined exposure to MPs and pathogenic bacteria may result in synergistic effects that exceed the impacts of individual factors, posing potential risks to marine organisms. Understanding these combined exposure effects is crucial not only for safeguarding marine organismal health but also for ensuring the sustainability of seahorse aquaculture (Albadawi 2025).

In the present study, we investigated the physiological responses of big‐belly seahorses, a coastal species, under controlled laboratory conditions following exposure to MPs and the pathogenic bacterium V. harveyi individually and in combination, focusing on immune‐ and stress‐related responses. The copy number of V. harveyi in the liver and kidney tissues was quantified, and MP accumulation in the gill and intestinal tissues was assessed. Furthermore, plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicators of hepatic integrity and function, were analyzed. In addition, changes in the expression of immune and stress‐related genes were examined, and apoptotic responses were assessed by quantifying casp8 mRNA expression and visualizing casp8 signals using in situ hybridization.

2. Materials and Methods

2.1. Experimental Seahorse

Big‐belly seahorses (mean length: 14.5 ± 1.1 cm; mean weight: 8.8 ± 1.5 g) were obtained from the Seahorse Research Center located on Jeju Island, South Korea. After collection, they were transported to the laboratory in Busan in an aerated seawater transport vehicle and acclimated for 1 week in a 350‐L recirculating aquarium system. During the acclimation period, the seahorses were fed frozen Neomysis awatschensis three times daily. Water quality conditions were maintained at a salinity of 33.5 ± 0.5 psu, temperature of 20.0° ± 0.6°C, pH of 8.0 ± 0.3, and dissolved oxygen of 7.5 ± 0.4 mg/L. A light/dark cycle of 14 h light and 10 h dark was maintained. Prior to the experimental exposure, all seahorses were fasted for 24 h to standardize physiological conditions and minimize dietary influences on MP exposure. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Korea Maritime and Ocean University (Approval No. KMOU IACUC 2024–07).

2.2. Bacterial Strain Preparation and Injection

V. harveyi was obtained from a diseased rockfish (Sebastes schlegelii) that displayed skin ulcer symptoms. The isolate was grown overnight at 25°C in Brain Heart Infusion broth (Difco, Franklin Lakes, NJ, USA). Species identification was conducted through polymerase chain reaction (PCR) amplification of the 16S rRNA gene using universal primers (27F and 1492R), as well as V. harveyi‐specific primers (Costa et al. 2022). Following three washes with phosphate‐buffered saline (PBS), the bacterial suspension was adjusted to 1 × 10³ CFU/mL for use in infection trials (Qin et al. 2016). Qin et al. (2016) reported a mortality rate of 46.7% ± 13.3% following exposure to V. harveyi at a concentration of 10⁴ CFU/mL. To control for injection‐related artifacts, fish in the control group received PBS injections, and preliminary testing indicated that PBS‐injected fish showed no significant differences compared with non‐injected controls.

2.3. Treatment Conditions and Sampling Procedures

Polystyrene MPs used for exposure included 0.2 μm beads (SMP; F8811) and 1.0 μm beads (LMP; 13080) (Thermo Scientific, Waltham, USA). To investigate changes in immune and stress responses, seahorses were divided into six experimental groups: PBS injection (Cont), SMP exposure, LMP exposure, V. harveyi injection (VH), and combined exposures of SMP/VH and LMP/VH. Each group was maintained in a single tank containing 20 individuals, yielding five biological replicates (n = 5) per treatment. The MP exposure trial was conducted at a concentration of 50 beads/L under continuous aeration to ensure uniform suspension within the tank. The MP concentration (50 beads/L) was selected to reflect environmentally relevant levels previously reported in coastal and nearshore environments, where MP abundance can vary widely depending on local pollution sources (Cohen et al. 2019). For V. harveyi exposure, each fish was intraperitoneally injected with 10 μL of a bacterial suspension (1 × 10³ CFU/mL), and no additional injections were administered during the 4‐day exposure period.

Each experimental group was subjected to either individual or combined exposure to MPs and V. harveyi for a period of 4 days. At 0, 1, 2, and 4 days post‐exposure, five seahorses were randomly selected from each treatment group and anesthetized with clove oil (100 ppm; Sigma‐Aldrich, St. Louis, MO, USA) prior to sample collection. No mortality occurred in any group during the experimental period. Blood was drawn from the caudal vein using heparinized syringes and centrifuged at 6000 × g for 10 min at 4°C to separate the plasma. The obtained plasma, along with liver and kidney samples, was stored at −80°C until subsequent analyses. Liver tissues intended for in situ hybridization were fixed in 4% paraformaldehyde and kept refrigerated at 4°C.

2.4. Determination of V. harveyi Copy Number by Quantitative Real‐Time PCR (qRT‐PCR)

Following exposure to MPs and V. harveyi, genomic DNA was isolated from liver tissues using the Patho Gene‐spin DNA/RNA Extraction Kit (iNtRON Biotechnology, Korea) in accordance with the manufacturer's protocol. Quantitative PCR was performed on a CronoSTAR 96 Real‐Time PCR System (Clontech, USA) with a 20 μL total reaction volume containing 10 μL of TOPreal SYBR Green qPCR PreMIX (2×) (Enzynomics, Korea), 7 μL of DEPC‐treated water, 1 μL each of the groEL‐F and groEL‐R primers (Costa et al. 2022), and 1 μL of DNA template. The amplification protocol consisted of an initial denaturation step at 95°C for 3 min, followed by 50 cycles of denaturation at 95°C for 20 s and annealing/extension at 63°C for 45 s. A melt curve analysis was subsequently performed by ramping the temperature from 65°C to 98°C at 0.5°C/s for 5 s. Standard curves were generated from serial dilutions of plasmid DNA containing the groEL gene, which encodes a molecular chaperone of V. harveyi. The plasmid copy number was calculated using the Copy Number Calculator provided by Integrated DNA Technologies (IDT, Coralville, IA, USA).

2.5. Detection of MP Accumulation

MP accumulation in the gill and intestinal tissues was quantified following a protocol reported by Yu et al. (2023), with some modifications. Tissue samples collected at 1, 2, and 4 days after exposure were digested in 10% potassium hydroxide solution and incubated at 60°C with continuous agitation for 48 h to remove organic material. Following digestion, MP particles (diameters 0.2 and 1.0 µm) were separated using filtration through sieves and membrane filters with pore sizes of 0.1 and 1.2 µm, respectively. The retained particles were subsequently subjected to density separation by immersion in zinc chloride solution (density 1.8 g/cm³) for 24 h. The supernatant was then vacuum‐filtered through a polycarbonate track‐etched membrane filter (47‐mm diameter, 0.1‐µm pore size; GVS, Rome, Italy). The membrane filters were transferred to Petri dishes and air‐dried at room temperature (20°C) for 24 h. After drying, the filters were stained with 200 μL Nile red solution and incubated in the dark for 30 min. Excess dye was removed by gently rinsing the filters with 100 μL n‐hexane. Finally, fluorescently labeled MP particles were visualized and quantified using a fluorescence microscope (Eclipse Ci; Nikon Instruments, Tokyo, Japan). To ensure quality control during MP detection, several measures were implemented. To assess background contamination, procedural blanks were prepared using tissues from unexposed control animals processed through an identical extraction and staining procedure. No MP particles were detected in the procedural blanks, confirming the absence of contamination introduced during sample processing. Furthermore, the morphology and fluorescence characteristics of the detected particles were verified against reference images of the original MP beads obtained prior to exposure, confirming their identity as the experimentally introduced particles, rather than environmental contaminants or tissue debris.

2.6. Measurement of ALT, AST, and Glucose Levels

Plasma biochemical parameters, including AST (GOT/AST‐PIII), ALT (GPT/ALT‐PIII), and glucose (GLU; GLU‐PIII), were quantified using an automated biochemistry analyzer (Fuji Dri‐Chem 4000, Fujifilm, Tokyo, Japan) based on the dry multilayer analytical slide method. For each experimental group, three technical replicates were analyzed to ensure accuracy and assess statistical significance.

2.7. Total RNA Extraction and qRT‐PCR

Total RNA was extracted from seahorse liver and kidney tissues using TRI Reagent (TR188, Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. The concentration and purity of extracted RNA (A260/A280 ratio of approximately 1.8–2.0) were verified using a spectrophotometer (BioDrop, Cambridge, UK). Subsequently, 2 µg of total RNA was reverse‐transcribed into complementary DNA with an oligo(dT)₁₅ anchor primer, and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). The synthesized cDNA was then diluted (1:100) and stored at −20°C until quantitative PCR analysis.

qPCR was carried out using a CFX96 Real‐Time PCR Detection System (Bio‐Rad Laboratories, Hercules, CA, USA) and iQ SYBR Green Supermix (Bio‐Rad) to determine the relative expression levels of lysG, IL10, IRF8, hsp75, and casp8. Primer sequences were designed based on corresponding gene sequences retrieved from the National Center for Biotechnology Information (NCBI) database, and are detailed in Table 1. The amplification protocol consisted of an initial denaturation step at 95°C for 3–5 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at gene‐specific temperatures for 15 s, and extension at 72°C for 20 s. Relative gene expression levels were calculated using the ΔΔC _t method, expressed as ΔΔC _t = $2^{\left[‐\left(∆{C_{\mathrm{t}}}^{\mathrm{sample}}‐∆{C_{\mathrm{t}}}^{\mathrm{internal}\mathrm{control}}\right)\right]}$, with normalization to 40S ribosomal protein S7 (40S RPS7) as the internal reference. Primer efficiencies for both target and reference genes are provided in Table 1.

Table 1.

Primers used in real‐time PCR amplification.

Genes (Accession no.)	Forward and reverse primer sequences	Tm (°C)	E (%)
For qPCR
groEL‐F (CP014038.2)	5ʹ‐ ATC ACT GTT GAA GAA GGT CAA ‐3ʹ	63	98.2
	5ʹ‐ ATG ATC AGT AGT GGG CGA GA ‐3ʹ
lysG (KX611130)	5ʹ‐ ACG TTG ATC CTG CTA TCA TC ‐3ʹ	51.4	99.5
	5ʹ‐ CTT GTG GAG TGT GAT ACC TT ‐3ʹ
IRF8 (MN046396)	5ʹ‐ TCA GGA CCA TTT ACC AAC TG ‐3ʹ	51.4	102.4
	5ʹ‐ TGA GTC ACC AAT ACG TTC TC ‐3ʹ
IL10 (MG053180)	5ʹ‐ CAA GAG GGA TGT CAC TCA AT ‐3ʹ	50.2	98.7
	5ʹ‐ TTG TAC ACG CCT TTG TTT TG ‐3ʹ
hsp75 (KJ756322)	5ʹ‐ TGA TCA CGG TAC TGG AAA TG ‐3ʹ	51.4	101.5
	5ʹ‐ CAG TTC ATT GTT GGA GTC CT ‐3ʹ
casp8 (KU363232)	5ʹ‐ GGA AGA AAG AGA TTG CCT GA ‐3ʹ	51.4	97.6
	5ʹ‐ ACA GCA TCA CCC TGT AAT TT‐3ʹ
40s RPS7 (KP780177)	5ʹ‐ AAG CCA TCA TCA TCT TCG TT ‐3ʹ	50.2	101.5
	5ʹ‐ TTA GGC AGG ATT CTC CTC TG ‐3ʹ
For in situ hybridization
casp8 (KU363232)	5ʹ‐ GGA AGA AAG AGA TTG CCT GA ‐3ʹ	51.4	97.6
	5ʹ‐ ACA GCA TCA CCC TGT AAT TT‐3ʹ

Note: E was calculated as E = (10^−1/slope − 1) × 100.

Abbreviations: E, qPCR efficiency; Tm, annealing temperature.

2.8. Detection of casp8 mRNA Using In Situ Hybridization

In situ hybridization was performed to localize casp8 mRNA expression in the liver following the protocol described by Kim et al. (2023) with minor modifications. Liver tissues were fixed in 4% paraformaldehyde at 4°C for at least 24 h, rinsed with PBS, and cryoprotected in 30% sucrose to prevent ice crystal formation. The samples were then embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and cut into 4‐µm sections using a cryostat (Leica CM1510 S; Leica Biosystems, Germany). The sections were mounted on glass slides and stored at −20°C until further processing.

To generate in situ hybridization probes specific for the casp8 sequence (Table 1), PCR amplification was performed using the corresponding casp8 primers. DIG‐labeled RNA probes were synthesized with T7 RNA polymerase (Merck, Darmstadt, Germany), and hybridization was conducted overnight at 65°C. Hybridization signals were detected using an alkaline phosphatase–conjugated anti‐DIG antibody, and color development was achieved with NBT/BCIP. Once adequate color intensity was observed, slides were washed with PBST, mounted using Aqua Polymount (Warrington, PA, USA), and sealed with cover glasses. Images of the hybridization signals were captured under a stereomicroscope (Nikon Eclipse).

2.9. Statistical Analysis

All statistical analyses were conducted using SPSS Statistics (version 29.0; IBM Corp., Armonk, NY, USA). Data normality was examined using the Shapiro–Wilk test, and the homogeneity of variances was verified with Levene's test. Differences among treatment groups and exposure periods were determined by two‐way analysis of variance, followed by Tukey's multiple comparison post hoc test. Statistical significance was considered at a confidence level exceeding 95% (p < 0.05). All results are presented as the mean ± standard error.

3. Results

3.1. Copy Number of V. harveyi in the Liver and Kidney

To investigate bacterial proliferation in seahorse, we examined V. harveyi copy number in the liver after infection with MP and V. harveyi (Table 2). V. harveyi was not detected in the control group and the group that was exposed to only MP. When the seahorse liver was simultaneously exposed to both MP and V. harveyi, a higher V. harveyi copy number was identified in the liver than in the group that was exposed to only V. harveyi (p < 0.05). The highest copy number was observed in the LMP/VH group in liver tissue during the 4‐day exposure period. In addition, the number of V. harveyi replicates in the LMP/VH group (3080.67 ± 236.08 copies/g) was significantly higher than in the VH (638.83 ± 140.11 copies/g) and SMP/VH (1561.33 ± 97.71 copies/g) groups on the 4th day after exposure. When compared with liver tissue, a higher V. harveyi copy number was observed in the kidney tissue. On Day 1 post‐exposure, the kidney of seahorses in the SMP/VH group exhibited the highest bacterial copy number (p < 0.05). On Day 2, the LMP/VH group showed the highest kidney copy number (p < 0.05). By Day 4, the VH group displayed a higher copy number than the SMP/VH group; however, the LMP/VH group exhibited the most significantly elevated copy number among all groups (p < 0.05).

Table 2.

Vibrio harveyi copy numbers in the liver and kidney of big‐belly seahorses following 4‐day exposure.

Experimental group		Exposure day
		1 day	2 days	4 days
Liver (copies/g)	VH	260.23 ± 67.60a1	427.43 ± 126.30a1	638.83 ± 140.11a1
	SMP/VH	368.57 ± 28.10a1	1194.63 ± 243.09b2	1561.33 ± 97.71b2
	LMP/VH	1144.67 ± 237.39a2	1278.00 ± 26.23b2	3080.67 ± 236.08b3
Kidney (copies/g)	VH	12,248 ± 3553.0a1	7162 ± 728.5a1	12,813.33 ± 916.6a2
	SMP/VH	13,571.33 ± 728.5b1	20,851.33 ± 121.0c2	8589.33 ± 888.6a1
	LMP/VH	21,826.27 ± 1335b2	14,826.67 ± 3802.5a12	18,731.67 ± 344.1ab3

Note: Different letters indicate significant differences among exposure days within the same experimental group (p < 0.05). Different numbers indicate significant differences among experimental groups on the same exposure day (p < 0.05). Values are presented as mean ± standard deviation (n = 5).

3.2. Accumulation of MP in the Gill and Intestinal Tissues

To investigate MP accumulation in seahorses, we examined MP levels in the gill and intestinal tissues following exposure to MPs and V. harveyi individually or in combination (Table 3). In the gill tissues, MP accumulation increased over time in all MP‐exposed groups. In the SMP and LMP groups, the accumulation levels on Day 4 were significantly higher than those on Days 1 and 2 (p < 0.05). In the SMP/VH and LMP/VH groups, MP accumulation on Day 4 was significantly higher than that in the corresponding MP‐only exposure group (p < 0.05).

Table 3.

Accumulation of microplastic particles in the gill and intestinal tissues of big‐belly seahorses following 4‐day exposure.

Experimental group		Exposure day
		1 day	2 days	4 days
Gills (numbers/g of tissue)	Cont	N.D	N.D	N.D
	SMP	12.5 ± 3.1a	16.0 ± 2.1a	25.9 ± 2.0b
	LMP	12.3 ± 2.1a	13.3 ± 2.0a	21.0 ± 2.4b
	VH	N.D	N.D	N.D
	SMP/VH	14.8 ± 1.9a	20.7 ± 2.6a1	35.7 ± 1.8c1
	LMP/VH	13.1 ± 2.0a	18.0 ± 1.2ab	32.3 ± 2.2b1
Intestine (numbers/g of tissue)	Cont	N.D	N.D	N.D
	SMP	31.4 ± 3.7a1	36.0 ± 1.1a	54.2 ± 2.1b1
	LMP	28.4 ± 3.3a	30.3 ± 0.9a	48.9 ± 1.9b
	VH	N.D	N.D	N.D
	SMP/VH	33.1 ± 2.8a	43.7 ± 1.8b1	61.8 ± 5.4c1
	LMP/VH	30.4 ± 3.5a2	40.5 ± 2.6ab1	56.3 ± 3.5b

Note: Different letters indicate significant differences among exposure days within the same experimental group (p < 0.05). Different numbers indicate significant differences among experimental groups on the same exposure day (p < 0.05). Values are presented as mean ± standard deviation (n = 5).

Abbreviation: N.D, no detection.

MP accumulation in the intestinal tissues was higher than that in the gills. Both the SMP and LMP groups exhibited significantly elevated accumulation on Day 4 relative to that on Days 1 and 2 (p < 0.05). Furthermore, MP accumulation in the SMP/VH group was higher than that in the SMP group on Days 2 and 4 after exposure (p < 0.05), suggesting that co‐exposure to V. harveyi enhanced MP retention in intestinal tissues.

3.3. Changes in Plasma Biochemical Parameters

From Day 2 of exposure, plasma ALT and AST levels were significantly higher in the MP/VH co‐exposure groups compared with the MP individual‐exposure group (p < 0.05) (Figure 1a,b). Notably, the VH, SMP/VH, and LMP/VH groups exhibited the highest ALT and AST levels on Day 4 of exposure. In contrast, glucose concentrations showed no significant changes in response to either individual or combined exposure to MP and VH, and remained stable throughout the 4‐day exposure period (p > 0.05) (Figure 1c).

Figure 1.

Changes in (a) alanine transaminase (ALT), (b) aspartate transaminase (AST) levels, and (c) glucose in the plasma of big‐belly seahorse exposed to microplastic and Vibrio harveyi for 4 days. Different letters indicate significant differences among exposure days within the same experimental group (p < 0.05). Different numbers indicate significant differences among experimental groups on the same exposure day (p < 0.05). Values are presented as mean ± standard error (n = 5).

3.4. Changes in Plasma Biochemical Parameters

From Day 2 of exposure, plasma ALT and AST levels were significantly higher in the MP/VH co‐exposure groups compared with the MP individual‐exposure group (p < 0.05) (Figure 1a,b). Notably, the VH, SMP/VH, and LMP/VH groups exhibited the highest ALT and AST levels on Day 4 of exposure. In contrast, glucose concentrations showed no significant changes in response to either individual or combined exposure to MP and VH, and remained stable throughout the 4‐day exposure period (p > 0.05) (Figure 1c).

3.5. Changes in the Expression of Immune‐Related Genes lysG, IRF8, and IL10

We determined the lysG, IRF8, and IL10 mRNA expression in the liver and kidney following exposure to MP and V. harveyi. In the liver, lysG mRNA expression significantly increased starting from Day 2 of exposure in all groups subjected to either individual or combined exposure to MP and V. harveyi, compared with the control group (p < 0.05) (Figure 2). In the kidney, significant increases in lysG expression were observed as early as Day 1 in the SMP and SMP/VH. On Day 4, lysG mRNA levels in the kidney were significantly higher in the SMP than in the LMP group (p < 0.05), whereas no significant differences were found between the SMP and MP/VH co‐exposure groups (p > 0.05).

Figure 2.

Changes in mRNA expression of lysozyme G (LysG) in the (a) liver and (b) kidney of big‐belly seahorse exposed to microplastic and Vibrio harveyi for 4 days. Different letters indicate significant differences among exposure days within the same experimental group (p < 0.05). Different numbers indicate significant differences among experimental groups on the same exposure day (p < 0.05). Values are presented as mean ± standard error (n = 5).

In the liver, IRF8 mRNA expression significantly increased in the V. harveyi exposure and the MP and V. harveyi co‐exposure groups from Day 1 (p < 0.05) (Figure 3a). This elevation persisted until Day 4, showing significantly higher levels compared with the control and MP‐only groups. In the kidney, IRF8 mRNA expression in the SMP and SMP/VH groups was significantly higher than that in the other groups from Day 1 (p < 0.05), with the SMP/VH group exhibiting the highest expression level among all groups at Day 4 (Figure 3b). Similarly, IL10 mRNA expression in the liver was significantly higher in the SMP/VH and LMP/VH groups than in the MP‐ or V. harveyi‐only groups from Day 1 (p < 0.05), with both liver and kidney showing the highest expression levels at Day 4 (Figure 3c,d). In the kidney, IL10 mRNA expression was also significantly higher in the co‐exposure groups compared with the MP‐only group (p < 0.05).

Figure 3.

Changes in mRNA expression of (a, b) interferon regulatory factor‐8 (Irf8) and (c, d) interleukin 10 (Il10) in the (a) liver and (b) kidney of big‐belly seahorse exposed to microplastics and Vibrio harveyi for 4 days. Different letters indicate significant differences among exposure days within the same experimental group (p < 0.05). Different numbers indicate significant differences among experimental groups on the same exposure day (p < 0.05). Values are presented as mean ± standard error (n = 5).

3.6. Changes in Expression of Stress Response Genes

Hsp75 mRNA expression in the liver and kidney was significantly increased on Day 1 in all groups except the LMP group, compared with the control (p < 0.05) (Figure 4). From Day 2, co‐exposure groups (MP and V. harveyi) showed significantly higher hsp75 mRNA expression in both liver and kidney than the MP‐only group (p < 0.05). Hsp75 mRNA expression in the kidney of the SMP group was comparable to that observed in the co‐exposure groups, showing no significant differences (p > 0.05).

Figure 4.

Changes in mRNA expression of heat shock protein 75 (hsp75) in the (a) liver and (b) kidney of big‐belly seahorse exposed to microplastics and Vibrio harveyi for 4 days. Different letters indicate significant differences among exposure days within the same experimental group (p < 0.05). Different numbers indicate significant differences among experimental groups on the same exposure day (p < 0.05). Values are presented as mean ± standard error (n = 5).

3.7. Changes in Casp8 Expression

To investigate the impact of MP and V. harveyi exposure, casp8 mRNA expression was examined using qPCR (Figure 5a,b). In the kidney, casp8 mRNA expression was significantly increased in the LMP/VH group compared with the control on Day 1 (p < 0.05). In the liver, all experimental groups except the control showed a significant increase in casp8 mRNA expression. On Day 2, casp8 mRNA expression in the kidney of the SMP/VH was significantly higher than that in the other groups (p < 0.05). By Day 4, casp8 mRNA expression in both liver and kidney was significantly higher in the VH than in the LMP (p < 0.05), while no significant differences were observed compared with the SMP group (p > 0.05). Moreover, co‐exposure to MP and V. harveyi resulted in significantly higher casp8 mRNA expression than MP‐only exposure.

Figure 5.

Changes in mRNA expression of Caspase‐8 in the (a) liver and (b) kidney of big‐belly seahorse exposed to microplastics and Vibrio harveyi for 4 days. Different letters indicate significant differences among exposure days within the same experimental group (p < 0.05). Different numbers indicate significant differences among experimental groups on the same exposure day (p < 0.05). Values are presented as mean ± standard error (n = 5).

Additionally, in situ hybridization analysis was performed on liver samples from the Cont, SMP, VH, and SMP/VH groups at 4 days post‐exposure to investigate changes in casp8 mRNA expression (Figure 6). Casp8 mRNA signal was stronger in groups exposed to MPs and V. harveyi compared to the control group. SMP and V. harveyi co‐exposure group (SMP/VH) exhibited more intense casp8 mRNA signals than either of the individual‐exposure groups (SMP and VH).

Figure 6.

Caspase‐8 mRNA expression in the liver on 4 days of exposure, detected using in situ hybridization. Image showing localized Caspase‐8 mRNA expression in the (a) control, (b) SMP, (c) VH, and (d) SMP/VH groups on Day 4. Dark areas (black arrow) indicate the mRNA expression of Caspase‐8 in the liver. Scale bars: 50 µm.

4. Discussion

In the present study, we investigated the physiological responses of seahorses exposed to a combined environment of MP and V. harveyi to determine how co‐exposure to multiple external factors affects their internal physiology. Wright et al. (2020) reported that the size and surface properties of MPs influence microbial attachment and growth. Similarly, Zhao et al. (2024) demonstrated that MPs can facilitate the transmission of pathogenic bacteria. In the present study, changes in the V. harveyi copy number in the liver and kidney were observed depending on the exposure period and MP size, suggesting that MP size may influence bacterial accumulation within host tissues. In addition, the V. harveyi copy number in the liver and kidney of seahorses exposed to both MPs and V. harveyi was significantly higher than in the individual exposure group (VH). Although the absolute bacterial copy numbers were relatively low and exhibited inter‐individual variability, such levels may still be biologically meaningful. In teleost fish, even low‐level or subclinical bacterial persistence can induce measurable immune modulation and oxidative stress responses without causing overt mortality. The observed variability may reflect differences in individual immune competence and bacterial‐clearance capacity under co‐exposure conditions.

To directly verify MP uptake and internal burden, we quantified MP accumulation in the gill and intestinal tissues (Table 3). MPs were detected in both tissues following waterborne exposure, confirming that the observed physiological and immunological responses were associated with actual tissue‐level accumulation rather than indirect exposure effects alone. MP accumulation increased over time and was generally higher in the intestinal tissues than in the gills, indicating MP ingestion and retention within the digestive tract. Furthermore, particle size influenced tissue accumulation patterns. Previous studies have reported that smaller MPs exhibit enhanced bioavailability and interactions with epithelial tissues in aquatic organisms (Wright et al. 2013; Jeong et al. 2016; Lu et al. 2016). Smaller particles may exhibit enhanced interaction with epithelial surfaces and prolonged tissue retention, potentially facilitating closer contact among MPs, host tissues, and co‐occurring pathogens under laboratory exposure conditions.

Shan et al. (2023) reported that exposure to 500 μg/L of polyvinyl chloride MPs increased the replication of white spot syndrome virus (WSSV) in white leg shrimp (Litopenaeus vannamei), leading to high mortality in shrimp larvae and promoting horizontal transmission of WSSV. Consistent with these findings, the increased copy number observed in the MP and V. harveyi co‐exposure group suggests that MPs may facilitate bacterial accumulation and potentially increase disease susceptibility in big‐belly seahorses. MPs provide a surface for microbial attachment and biofilm formation, which can increase the local abundance and persistence of bacteria and act as a carrier that brings microbes in close contact with host tissues (Yang et al. 2023). In addition, MP exposure may indirectly enhance bacterial colonization by compromising epithelial barriers and modulating immune responses, thereby reducing the ability of hosts to control infection; this mechanism warrants further investigation under natural waterborne exposure conditions (Liang et al. 2025). These results suggest that MPs are not merely physical pollutants, but may also interact with pathogens to enhance infection susceptibility under controlled experimental conditions, ultimately aggravating the vulnerability of seahorses to disease. However, whether such interactions occur similarly under natural waterborne exposure conditions should be confirmed in future studies.

The kidney is a major immune organ where immune cells are actively generated and activated, and in fish, it plays a key role in immune cell distribution and activation (Bjørgen and Koppang 2022). The higher V. harveyi copy number observed in the kidney tissue of seahorses in this study may not necessarily reflect a more active initial immune response in this organ alone. It may be associated with tissue tropism or a higher bacterial affinity for renal tissue, as several Vibrio species have been reported to preferentially colonize hematopoietic or highly vascularized organs in marine fish (Austin and Zhang 2006; Bjørgen and Koppang 2022). Moreover, MPs can act as a carrier that facilitates bacterial attachment and transport within an organism (Jiang et al. 2020). Thus, interactions between MPs and pathogens may enhance bacterial persistence in immune‐related organs such as the kidney, potentially triggering immune activation, intracellular stress, and apoptotic pathways, although these mechanisms should be interpreted within the context of the intraperitoneal injection model used in this study.

The liver is one of the primary organs used to assess the degree of oxidative damage caused by external pollutants, and ALT and AST are key indicators for evaluating liver function (Dong et al. 2022). In the present study, plasma analysis of big‐belly seahorses exposed to MP and V. harveyi showed that ALT and AST levels were significantly higher in the co‐exposure groups (SMP/VH and LMP/VH) than in the MP‐only exposure group. Meanwhile, no significant differences in ALT and AST levels were observed between the VH individual exposure group and the SMP/VH and LMP/VH co‐exposure groups. These results suggest that the presence of MPs, rather than V. harveyi infection alone, had a greater influence on hepatic enzyme activity (ALT and AST) in seahorses. Banihashemi et al. (2021) reported that in rainbow trout (Oncorhynchus mykiss) exposed to MPs and Yersinia ruckeri, exposure to Y. ruckeri alone did not significantly alter plasma ALT activity; however, high MP concentrations (1000 mg/kg) significantly increased ALT activity, confirming the hepatotoxic effects of MPs themselves. ALT activity was further elevated in the co‐exposure group, indicating a potential enhancement of toxicity under combined exposure conditions (Banihashemi et al. 2021). Based on these findings, co‐exposure to MP and V. harveyi may increase the likelihood of hepatic damage in seahorses compared with MP exposure alone.

The innate immune response plays a crucial role as the first line of defense during disease infection or when the immune system is weakened by external pollutants (Ferraboschi et al. 2021). Niemcharoen et al. (2022) reported that the expression of lys mRNA in the hepatopancreas of L. vannamei significantly increased after 7 days of feeding with a diet containing 50 µm MPs, while Ceseña et al. (2021) found that V. parahaemolyticus infection significantly upregulated the expression of lys in Penaeus vannamei. Similarly, in the present study, lysG mRNA expression in the liver and kidney of seahorses co‐exposed to MP and V. harveyi was significantly higher than that of the control group, with the SMP/VH group showing the highest expression. Following innate immune activation, analysis of IRF8 and IL10 mRNA—key genes involved in host immune regulation—also revealed significant upregulation in the MP and V. harveyi co‐exposure groups compared to the individual exposure groups. Gao et al. (2023) reported that infection by V. parahaemolyticus increased mRNA expression of IL‐related immune genes, including IL10, in the liver and spleen of S. schlegelii. The increased expression of immune‐related genes in this study indicates that exposure to MP and V. harveyi activated the immune system of seahorses, and that co‐exposure to both the stressors further enhanced immune activation, inducing a stronger immune response.

The expression of hsp75 in the organisms is known to reduce the occurrence of reactive oxygen species, help maintain mitochondrial function, and promote cell survival (Voloboueva et al. 2008). Hsp has also been widely used as a molecular marker for general stress responses induced by external contaminants such as MP (Roch et al. 2022). For instance, Daphnia magna exposed to 40 μm MP showed significant changes in the mRNA expression of hsp60 and hsp70, both belonging to the same HSP family (Imhof et al. 2017), strongly suggesting that MP induces cellular stress responses. In addition, environmental factors such as temperature and salinity have been reported to influence the expression of hsp family genes in Hippocampus erectus (Qin et al. 2018; Huang et al. 2020). In this study, consistent with previous findings, hsp75 expression increased not only under general stress conditions and MP exposure but also upon infection with pathogenic bacteria such as V. harveyi. Furthermore, in the combined exposure groups of MP and V. harveyi, hsp75 mRNA levels in the liver and kidney were significantly higher than those in the individual‐exposure groups. This indicates that hsp75 can be activated not only by physical stress but also by pathogenic infection, suggesting its potential as a more sensitive biomarker under combined stress conditions.

Excessive oxidative stress can disrupt homeostasis and induce apoptosis. Casp8 is a key initiator caspase in the extrinsic apoptotic pathway that activates downstream caspases, playing a central role in the programmed cell death process (Orning and Lien 2021). Kwon et al. (2025) reported that when S. schlegelii was co‐exposed to MP and Streptococcus iniae, casp3 mRNA expression was significantly increased compared to the individual‐exposure groups. In the present study, similar results were observed: casp8 mRNA levels in liver and kidney tissues of seahorses were significantly higher in the combined exposure groups of MP and V. harveyi than in the individual‐exposure groups. Notably, the SMP/VH group on Day 2 showed the highest expression levels, and in situ hybridization analysis of liver tissue also revealed more casp8 signals in the SMP/VH group. These results suggest that MP may enhance its interaction with V. harveyi, amplifying cellular toxicity and accelerating oxidative stress‐mediated apoptotic signaling.

In addition, this study demonstrated that exposure to MP and V. harveyi impaired liver function in seahorses, increased the expression of immune‐related genes (lysG, IRF8, and IL10), and promoted the proliferation of V. harveyi within the host in the presence of MP, thereby inducing greater stress and apoptosis. These findings suggest that the co‐occurrence of MP and pathogenic bacteria such as V. harveyi in marine environments can trigger physiological alterations beyond simple individual toxic responses, amplifying oxidative stress and cellular damage.

However, certain methodological considerations should be acknowledged. In this study, intraperitoneal injection was used to ensure consistent systemic infection and minimize inter‐individual variability in pathogen uptake. While this approach allows us to directly assess the interaction between confirmed internal bacterial infection and MP exposure, it bypasses natural infection routes such as waterborne transmission through epithelial surfaces. Further studies employing waterborne exposure models are required to confirm whether MPs act as infection vectors under ecologically realistic conditions. In addition, a single tank per treatment was used in this study, which constitutes pseudoreplication, as the tank rather than the individual fish represents the true experimental unit. This lack of independent tank‐level replication may confound treatment effects with potential tank‐specific environmental factors, thereby reducing the strength of statistical inference. Although consistent physiological patterns were observed among individuals within treatments, the findings should be interpreted with caution. Future studies should incorporate multiple independent tanks per treatment to ensure true replication.

Future studies should investigate how the physicochemical properties of MP, under various concentrations and prolonged exposure conditions, affect the interactions with pathogens and their infection pathways. In particular, studies employing a waterborne bacterial exposure approach rather than an intraperitoneal injection method will be essential to validate the role of MPs as potential infection vectors under natural conditions, to better understand the ecological relevance of MP–pathogen interactions in marine environments.

5. Conclusion

This study demonstrated that co‐exposure to MPs and V. harveyi induces more pronounced physiological disturbances in big‐belly seahorses than single exposures. Co‐exposure enhanced bacterial proliferation and exacerbated hepatic dysfunction, oxidative stress, and apoptotic responses. These findings highlight the ecological risk posed by the interaction between MPs and pathogens in marine environments. Given their sensitivity to environmental stressors, big‐belly seahorses may serve as a useful bioindicator for assessing combined MP–pathogen pressures in coastal ecosystems.

Author Contributions

Jin A Kim: designed the experiments, performed data curation, writing – original draft. Min‐Min Jung: formal analysis, investigation. Seong Don Hwang: statistical analysis. Jun‐Hwan Kim: writing – review and editing. Cheol Young Choi: writing – review and editing, supervision. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Kim, J. A. , Jung M.‐M., Hwang S. D., Kim J.‐H., and Choi C. Y.. 2026. “Synergistic Impacts of Co‐Exposure to Microplastics and Vibrio harveyi on the Immune and Stress Responses of the Big‐Belly Seahorse Hippocampus abdominalis .” Journal of Experimental Zoology Part A: Ecological and Integrative Physiology 345: 554–567. 10.1002/jez.70089.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.