The residue of salinomycin in the muscles of olive flounder (Paralichthys olivaceus) and black rockfish (Sebastes Schlegeli) after oral administration analyzed by LC-Tandem-MS.

BACKGROUND: Salinomycin, an antibiotic, have potential as a veterinary drug for fish due to its anti-parasitic activity against several fish parasites. Thus the residual levels of salinomycin in muscles of two significant aquaculture species in Korea, olive flounder and black rockfish, were analyzed using HPLC-MS-MS. RESULTS: The proper method to analyze the residual salinomycin in fish muscles using LC-MS-MS was settled and the method was validated according to CODEX guidelines. The residues in three distinct groups for two fish species were analyzed using the matrix match calibration curves at points of five different times following oral administration. After oral administration, salinomycin rapidly breaks down in both olive flounder and black rockfish. After 7th days, the average residue in all groups of two fish spp. decreased below limit of quantitation (LOQ). CONCLUSION: Due to low residue levels in fish muscles, salinomycin may therefore be a treatment that is safe for both fish and humans. This result could contribute to establishment of MRL (minimal residual limit) for approval of salinomycin for use in aquaculture.

Characterization of the complete mitochondrial genome of Miamiensis avidus causing flatfish scuticociliatosis.

Miamiensis avidus is a parasitic pathogen that causes the disease scuticociliatosis in teleost fish species. It is a ciliate and a free-living marine protozoan belonging to the order Philasterida, subclass Scuticociliatida, class Oligohymenophorea, and phylum Ciliophora. The complete mt-genome of M. avidus was linear and 38,695 bp in length with 47 genes, including 40 protein-coding genes, two ribosomal RNA (rRNA) genes, and five transfer RNA (tRNA) genes. Of these, 20 genes typically belong to the clusters of orthologous groups, playing roles in energy production and conversion, translation, ribosomal structure and biogenesis, and defense mechanisms. This is the first report of sequencing and characterization of the mt-genome of M. avidus, which was observed to be linear and possessing the typical ciliate mitochondrial genome organization and phylogenetic relationships. Remarkable differences were observed between M. avidus and other ciliates in the mitochondrially encoded rRNAs, extensive gene loss in ribosomal genes and tRNAs, terminal repeat sequences, and stop codon usage. A comparative and phylogenetic analysis of M. avidus and Uronema marinum of the order Hymenostomatida, which is most closely related to the order Philasterida, signified the promise of the mitogenome data of M. avidus as a valuable genetic marker in species detection and taxonomic research. The present study has potential applications in epidemiological studies and host-parasite interaction investigations facilitating disease control.

Inorganic nitrogen compounds reduce immunity and induce oxidative stress in red seabream.

In this study, the effect of ammonia derived from different stocking densities on immunological, hematological, and oxidative stress parameters was analyzed in the blood or liver of red seabream. Density- and time-dependent increases in inorganic nitrogen compounds were measured for 20 days by analyzing the three major inorganic nitrogen compounds, total ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. Three immunity parameters, alternative complement activity, lysozyme activity, and total immunoglobulin content were significantly decreased in the blood at the highest stocking density (10 kg m-3). The concentrations of hemoglobin and white blood cells were significantly decreased at 10 kg m-3, while there was no significant change in red blood cells. The significant increases in cortisol level and the enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase at 10 kg m-3 clearly supported inorganic nitrogen compounds-triggered stress. A significant elevation of lipid peroxidation value and depletion of intracellular glutathione were observed at 5 and/or 10 kg m-3 in the liver tissue. The hepatic enzymatic activities of antioxidant defense enzymes, catalase and superoxide dismutase were also significantly increased. When a protein skimmer removes the inorganic nitrogen compounds at the highest density, most parameters showed no significant change. Taken together, these results suggest that accumulated inorganic nitrogen compounds at the highest stocking density inhibit innate immunity and induce oxidative stress in red seabream. This information will be helpful to maintain homeostasis of red seabream by controlling immunity and oxidative status through inorganic nitrogen compounds removal in intensive culture condition.

Current use and management of commercial fish vaccines in Korea.

The aquaculture industry in Korea has grown rapidly since the 1960s, and it is a major food source. However, the expansion of aquaculture systems has increased the chances of infectious disease outbreaks, and vaccination plays an important role in commercial fish farming. This is the first comprehensive review of commercial fish vaccines in Korea. It not only provides an overview of commercially available fish vaccines and their associated approval processes and laws, but also some perspectives on research advances regarding fish vaccines in Korea. In Korea, fish vaccines are approved only after their safety and effectiveness have been verified according to the Pharmaceutical Affairs Act, and after approval, each vaccine lot must pass the national evaluation criteria. As of the end of 2019, 29 vaccines were approved for 10 fish pathogens, including both single and combination vaccines containing more than two inactivated pathogens. The approved fish vaccines consist of 2 immersion vaccines, as well as 1 intramuscular and 26 intraperitoneal vaccines, which require syringe injection. All the 29 vaccines are manufactured as formalin-inactivated vaccines; 1 is an adjuvant vaccine and 28 are non-adjuvant vaccines; 25 are bacterial vaccines, 2 are viral vaccines, 1 is a parasite vaccine, and 1 is a parasite and bacterial vaccine. In terms of the target fish species, 27 vaccines are used in the olive flounder (Paralichthys olivaceus), 1 in the starry flounder (Platichthys stellatus), and 1 in the red seabream (Pagrus major), striped beakfish (Oplegnathus fasciatus), and amberjack (Seriola quinqueradiata). This imbalance exists mostly because the olive flounder is the main farmed fish species in Korea. In 2018, 67.71 million vaccine doses were distributed following satisfactory performance in the national evaluation. They were used to vaccinate approximately 80.6% of farmed olive flounders.

Two short antimicrobial peptides derived from prosaposin-like proteins in the starry flounder (Platichthys stellatus).

Prosaposin (PSAP) is a precursor of saposin (SAP), which is present in lysosomal and secreted proteins. PSAP is a member of the SAP-like protein families, which comprise multifunctional proteins. In particular, their antimicrobial activity has been reported. We identified PSAP-like (PsPSAPL) sequences from starry flounder and analysed their expression and antimicrobial activity based on cDNA and amino acid sequences. PsPSAPL showed conservation of three saposin B type domains at high levels, and PsPSAPL mRNA was relatively abundantly distributed in the brain and gills of healthy starry founders. PsPSAPL mRNA showed significant expression changes in response to viral haemorrhagic septicaemia virus and Streptococcus parauberis. Synthetic peptides (PsPSAPL-1 and -2), prepared based on amino acid sequences, were used to confirm as well as analyse the antimicrobial activity against bacteria and parasites. Consequently, PsPSAPL-1 and -2 were found to significantly inhibit the growth of various bacteria and kill the Miamiensis avidus. In addition, bacterial biofilm formation was significantly inhibited. Safety was also confirmed by analysing cell haemolysis. These results indicate the immunological function of PsPSAP and the potential antimicrobial activity of the AMPs PsPSAPL-1 and -2.

Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major).

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.

Whole-genome next-generation sequencing and phylogenetic characterization of viral haemorrhagic septicaemia virus in Korea.

Whole-genome next-generation sequencing was used to investigate the local evolution of viral haemorrhagic septicaemia virus, a serious pathogen affecting economically important fish such as rainbow trout and turbot in Europe and olive flounder in Asia. Sequence analysis showed that all isolates were genotype IVa, but could be classified further into four subgroups (K1-K4). In addition, genomic regions encompassing the nucleoprotein, phosphoprotein, matrix protein and non-virion protein genes, as well as the seven non-coding regions, were relatively conserved, whereas glycoprotein and RNA-dependent RNA polymerase genes were variable in the coding region. Taken together, the data demonstrate that whole-genome next-generation sequencing may be useful for future surveillance, prevention and control strategies against viral haemorrhagic septicaemia.

The atypical chemokine receptor 4 in red sea bream (Pagrus major): Molecular characterization and gene expression analysis.

Atypical chemokine receptor 4 (ACKR4) is regulated by cytokines, binds chemokines and regulates the chemokine gradient. We verified the cDNA sequence by confirming ACKR4 from red sea bream (PmACKR4) by next generation sequencing (NGS) and analysed the molecular characteristics and gene expression profile. In the analysis using the predicted amino acid sequence of PmACKR4, a highly conserved G protein-coupled receptor 1 region and two cysteine residues were identified and included in the ACKR4 teleost cluster in the phylogenetic analysis. In healthy red sea bream, PmACKR4 mRNA was expressed at the highest levels in head kidney and was upregulated in all immune -related tissues used in the experiment after challenges with Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV). These results suggest that ACKR4 is highly conserved in red sea bream and may play an important role in the immune system as previously reported. It is thought that ACKR4 acts as a regulator of immune -related cells via immune reactions after pathogenic infection.

Characterization of gene expression profiles and functional analysis of peptidoglycan recognition protein 2 from rock bream (Oplegnathus fasciatus).

Peptidoglycan recognition protein 2 (PGRP2) is a Zn2+-dependent peptidase that plays important roles in binding to microbial components of the cell membrane, inducing phagocytosis and antimicrobial activity. Rock bream (Oplegnathus fasciatus) PGRP2 (RbPGRP2) was identified in the intestine by next generation sequencing (NGS) analysis. The open reading frame (ORF) the RbPGRP2 cDNA (470 amino acid residues) contains a peptidoglycan recognition protein domain (residues 300 to 446). Alignment analysis revealed that RbPGRP2 shares 37.6-53.5% overall sequence identity with the PGRP2s of other species. Phylogenetic analysis revealed that RbPGRP2 clustered together with PGRP2s from teleosts. In healthy rock bream, RbPGRP2 was found to be ubiquitously expressed in all of the examined tissues, especially in the liver. RbPGRP2 expression was significantly upregulated in all of the examined tissues of rock bream after infection with Edwardsiella piscicida, Streptococcus iniae and red sea bream iridovirus (RSIV) compared with the control. Purified rRbPGRP2 interactions with bacteria and inhibited the growth of bacteria in the presence of Zn2+. These results indicate that RbPGRP2 plays an important role in the innate immune response against bacterial infection.

Transcriptome analysis of olive flounder (Paralichthys olivaceus) head kidney infected with moderate and high virulent strains of infectious viral hemorrhagic septicaemia virus (VHSV).

Olive flounder (Paralichthys olivaceus) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease-causing VHSV isolates, ADC-VHS2012-11 and ADC-VHS2014-5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post-infection using Illumina sequencing. After removing low-quality sequences, we obtained 144,933,160 high quality reads from thirty-six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut-off e-value of 10-5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV.

Olive flounder CD276 (B7-H3) a coinhibitory molecule for T cells: Responses during viral hemorrhagic septicemia virus (VHSV) stimulation.

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response.

Complete genome sequence and phylogenetic analysis of spring viremia of carp virus isolated from leather carp (Cyprinus carpio nudus) in Korea in 2016.

Spring viremia of carp (SVC) is listed as a notifiable viral disease by the World Organization for Animal Health (OIE). In 2016, the first official SVC outbreak was detected in the city of Gyeongsan, Korea. The present study reports the first complete genome analysis of SVC virus (SVCV, ADC-SVC2016-5) isolated from leather carp (Cyprinus carpio nudus). The results revealed that ADC-SVC2016-5 has a 11,029-bp genome containing five genes: N, P, M, G, and L. Phylogenetic analysis indicated that ADC-SVC2016-5 (accession number MG663512), isolated from leather carp, was closely related to genogroup Ia isolates of the Asian clade. This report provides additional information for studying the molecular epidemiology and evolution of spring viremia of carp virus.

Nanoplastic Ingestion Enhances Toxicity of Persistent Organic Pollutants (POPs) in the Monogonont Rotifer Brachionus koreanus via Multixenobiotic Resistance (MXR) Disruption.

Among the various materials found inside microplastic pollution, nanosized microplastics are of particular concern due to difficulties in quantification and detection; moreover, they are predicted to be abundant in aquatic environments with stronger toxicity than microsized microplastics. Here, we demonstrated a stronger accumulation of nanosized microbeads in the marine rotifer Brachionus koreanus compared to microsized ones, which was associated with oxidative stress-induced damages on lipid membranes. In addition, multixenobiotic resistance conferred by P-glycoproteins and multidrug resistance proteins, as a first line of membrane defense, was inhibited by nanoplastic pre-exposure, leading to enhanced toxicity of 2,2',4,4'-tetrabromodiphenyl ether and triclosan in B. koreanus. Our study provides a molecular mechanistic insight into the toxicity of nanosized microplastics toward aquatic invertebrates and further implies the significance of synergetic effects of microplastics with other environmental persistent organic pollutants.

Interferon-induced protein 56 (IFI56) is induced by VHSV infection but not by bacterial infection in olive flounder (Paralichthys olivaceus).

Interferon-inducible protein 56 (IFI56, also known as ISG56/IFIT1, interferon-induced protein with tetratricopeptide repeats 1) is strongly induced in response to interferon and a potent inhibitor of viral replication and translational initiation. Here, we describe the identification of IFI56 (OfIFI56) in olive flounder, its characteristic features, and expression levels in various tissues before and after viral hemorrhagic septicemia virus (VHSV) infection. The full-length OfIFI56 sequence was identified from rapid amplification of cDNA ends PCR. The complete coding sequence of OfIFI56 is 1971 bp in length and encodes 431 amino acids. The putative OfIFI56 protein has multiple tetratricopeptide (TPR) motifs, which regulate diverse biological processes, such as organelle targeting, protein import, and vesicle fusion. Based on sequence analysis, the Larimichthys crocea IFI56 protein (61%) had the highest sequence homology to OfIFI56. In healthy olive flounder, OfIFI56 mRNA expression was detected in many tissues such as intestine, gill, head kidney, heart, spleen, and trunk kidney tissues. After VHSV challenge, OfIFI56 mRNA was significantly up-regulated in these tissues. Additionally, OfIFI56 expression was induced by poly I:C but not by Streptococcus parauberis and S. iniae infection or lipopolysaccharide injection in kidney and spleen tissues of olive flounder. These results demonstrate that piscine OfIFI56 expression is not induced by bacterial infection but is selectively induced by viral infection, especially VHSV, and that OfIFI56 may play an important role in the host response against VHSV infection.

First Report of Clavinema mariae (Nematoda: Philometridae) in Cultured Rockfish, Sebastes schlegeli, in Cheonsuman (Bay), the Republic of Korea.

In July 2012, philometrid nematodes were discovered in cultured rockfish (Sebastes schlegeli) in Cheonsuman (Bay), the Republic of Korea. The nematodes were detected in the epithelial tissues of the rockfish and were identified as Clavinema mariae based on morphological studies using light and scanning electron microscopy. They revealed the characteristics same as previously identified C. mariae, notably having a long body with narrow posterior half, no caudal projection, a cylindrical-shaped esophagus, a well-developed anterior bulbous part of the esophagus, cephalic papillae, and a dorsal esophageal gland. This is the first confirmation of C. mariae infection in rockfish in Korea.

Molecular cloning and expression analysis of peptidase genes in the fish-pathogenic scuticociliate Miamiensis avidus.

BACKGROUND: Parasite peptidases have been actively studied as vaccine candidates or drug targets for prevention or treatment of parasitic diseases because of their important roles for survival and/or invasion in the host. Like other parasites, the facultative histophagous ciliate Miamiensis avidus would possess peptidases that are closely associated with the invasion into the host tissue and survival in the host. RESULTS: The 17 genes encoding peptidases, including seven cathepsin-like cysteine peptidases, four serine carboxypeptidases, a eukaryotic aspartyl protease family protein, an ATP-dependent metalloprotease FtsH family protein, three leishmanolysin family proteins and a peptidase family M49 protein were identified from a Miamiensis avidus cDNA library by BLAST X search. Expression of genes encoding two cysteine peptidases, three leishmanolysin-like peptidases and a peptidase family M49 protein was up-regulated in the cell-fed ciliates compared to the starved ciliates. Especially, one cysteine peptidase (MaPro 4) and one leishmanolysin-like peptidase (MaPro 14) were transcribed more than 100-folds in the cell-fed ciliates. CONCLUSIONS: The genetic information and transcriptional characteristics of the peptidases in the present results would be helpful to elucidate the role of peptidases in the invasion of scuticociliates into their hosts.

Influence of Intramuscular Injection Sites on Pharmacokinetics of Amoxicillin in Olive Flounder (Paralichthys olivaceus) and Its Implication for Antibacterial Efficacy.

This study aimed to investigate the effects of different injection sites, including dorsal, cheek, and pectoral fin muscles, on the pharmacological properties of amoxicillin (AMOX) in olive flounder (Paralichthys olivaceus) after a single intramuscular (IM) injection of 40 mg/kg. The AMOX concentration was measured using high-performance liquid chromatography-tandem mass spectrometry, followed by a non-compartmental model analysis. The peak serum concentrations (Cmax) achieved 3 h after dorsal, cheek, and pectoral fin IM injections were 202.79, 203.96, and 229.59 µg/mL, respectively. The area under the concentration-time curve (AUC) was 1697.23, 2006.71, and 1846.61 µg/mL·h, respectively. The terminal half-life (t1/2λZ) was prolonged for cheek and pectoral fin IM injections (10.12 and 10.33 h, respectively) compared to dorsal IM injection (8.89 h). In the pharmacokinetic-pharmacodynamic analysis, a higher T > minimum inhibitory concentration (MIC) and AUC/MIC values were observed after AMOX was injected into the cheek and pectoral fin muscles compared to the dorsal muscle. Muscle residue depletion was below the maximum residue level from day 7 after IM injection at all three sites. These findings suggest that the cheek and pectoral fin sites provide advantages regarding systemic drug exposure and prolonged action compared with the dorsal site.

Exploring the Efficacy and Safety of Levamisole Hydrochloride against Microcotyle sebastis in Korean Rockfish (Sebastes schlegelii): An In Vitro and In Vivo Approach.

Parasitic infections pose significant challenges in aquaculture, and the increasing resistance to conventional anthelmintics necessitates the exploration of alternative treatments. Levamisole hydrochloride (HCl) has demonstrated efficacy against monogenean infections in various fish species; however, research focused on Microcotyle sebastis infections in Korean rockfish (Sebastes schlegelii) remains limited. Therefore, this study aimed to evaluate the efficacy of levamisole HCl against M. sebastis infections in Korean rockfish with the goal of optimizing anthelmintic usage in aquaculture. In this study, we first assessed the susceptibility of M. sebastis to levamisole HCl in vitro. Subsequently, in vivo evaluations were conducted to assess the drug's efficacy, safety, and to identify optimal administration methods. In vitro experiments revealed concentration-dependent sensitivity of M. sebastis to levamisole HCl, with a minimum effective concentration (MEC) of 100 mg/L. In vivo experiments employed oral administration, intraperitoneal injection, and immersion treatments based on the MEC. Oral administration proved to be a safe method, yielding efficacy rates of 27.3% and 41.6% for 100 mg/kg and 200 mg/kg doses, respectively, in contrast to the immersion and injection methods, which induced symptoms of abnormal swimming, vomiting, and death. Biochemical analyses conducted to assess the safety of levamisole HCl revealed a transient, statistically significant elevation in the levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) on day three post-administration at 20 °C. Following this, no substantial differences were observed. However, at 13 °C, the enzyme levels remained relatively consistent, emphasizing the role of water temperature conditions in influencing the action of levamisole HCl. Our research findings substantiate the efficacy of levamisole HCl against M. sebastis in Korean rockfish, underscoring its potential for safe oral administration. These results provide valuable insights for developing parasite control strategies involving levamisole HCl in Korean rockfish populations while minimizing adverse impacts on fish health and the environment. However, this study bears limitations due to its controlled setting and narrow focus. Future research should expand on these findings by testing levamisole HCl in diverse environments, exploring different administration protocols, and examining wider temperature ranges.

Assessment of Salinomycin's Potential to Treat Microcotyle sebastis in Korean Rockfish (Sebastes schlegelii).

Aquaculture, a crucial sector of the global food industry, faces a myriad of issues due to parasitic invasions. One such parasite, Microcotyle sebastis, which afflicts Korean rockfish in South Korea, has a significant economic impact. The impending danger of resistance to traditional anthelmintics necessitates the exploration of new antiparasitic candidates. Although the efficacy of salinomycin against aquatic parasites such as ciliates and sporozoans is known, its influence on monogeneans has yet to be studied. Therefore, this study investigated the efficacy and safety of salinomycin for the treatment of M. sebastis infections, presenting the first exploration of salinomycin's therapeutic potential against monogeneans. In vitro examinations revealed a minimum effective concentration of salinomycin of 5 mg/kg, which led to necrosis of the haptor upon dislodging from the gill filaments. The one-time oral administration of the drug at concentrations of 5 mg/kg and 10 mg/kg showed a significant dose-dependent reduction in parasite counts, with no apparent behavioral side effects in Korean rockfish. Biochemical analyses monitored the liver, heart, and kidney enzymes, specifically aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatine kinase-myocardial band (CK-MB). At both 20 °C and 13 °C, no significant differences were observed in the levels of AST and ALT. However, at 20 °C, alterations in BUN levels were evident on Day 14, a deviation not observed at 13 °C. The CK-MB analysis revealed elevated enzyme levels at both temperatures when compared to the control group, reflecting the similar changes observed in terrestrial animals administered salinomycin. The biochemical data suggest that the oral administration of salinomycin is potentially more favorable at 13 °C than at 20 °C. Although our findings warrant further comprehensive studies, including on the long-term and potential effects on nontarget species and water quality, they also suggest that salinomycin could be considered as an alternative or adjunctive treatment if resistance to the currently used praziquantel against M. sebastis is confirmed.