A new potential therapeutic remedy against Aeromonas hydrophila infection in rainbow trout (Oncorhynchus mykiss) using tetra,Cotinus coggygria

Different antibiotic‐based drugs are being used for the treatment of Aeromonas hydrophila infection in rainbow trout, and several studies emphasize the use of medicinal plants as immunostimulants for prophylactic measure against Aeromoniasis disease. However, therapeutic effects of aqueous methanolic extracts of tetra (Cotinus coggygria) against A. hydrophila in rainbow trout were not investigated. Four different concentrations of tetra extract (0 [control], 4, 8 and 12 mg/100 µl) and also two different positive control groups (florfenicol and doxycycline antibiotics) were administered orally using feeding needles to individual rainbow trout, Oncorhynchus mykiss of all experimental groups twice a day after intramuscular inoculation of A. hydrophila. The study period was for 10 days. On 0th, 3rd, 7th and 10th day, blood and tissues were collected from the fish and changes in humoral immune responses, haematology and immune‐related gene expressions were determined. In the study, superoxide radical production was decreased generally in all experimental groups except in 12 mg tetra and florfenicol treatments compared to control (p < .05). Lysozyme activity was generally decreased (p < .05), or no differences were observed in all experimental groups compared to the control. Myeloperoxidase activity was significantly increased in florfenicol‐treated fish group on 7th day (p < .05). Generally, myeloperoxidase activity showed an increase in almost all tetra‐treated groups. Haematological parameters increased but were not significantly high enough in treatments. Almost all immune‐related gene expressions were significantly enhanced on 3rd and 10th day of the study. Survival rate of 53.33% was found in control group. There were no significant differences in survival between control and 4 mg tetra‐treated group (p > .05). All the other groups' survival rate was significantly increased compared to control. The highest survival rate was found in florfenicol group (80%). In 12 mg tetra‐, doxycycline‐ and 8 mg tetra‐treated groups, survival rate was recorded as 74.44%, 70% and 70%, respectively. Our results suggest that tetra methanolic extract is an effective therapeutic remedy against A. hydrophila infection in rainbow trout at the dose of 24 mg/32.34 g body weight/day.     

Natural co‐infection of cultured Nile tilapia Oreochromis niloticus with Aeromonas hydrophila and Gyrodactylus cichlidarum experiencing high mortality during summer

Aeromonas hydrophila and Gyrodactylus cichlidarum are common pathogens that induce significant economic losses in farm‐reared Nile tilapia. Nowadays, the sudden appearance of fish mortalities was exaggerated due to mixed and multiple infections. During summer 2016, mass mortality among earthen pond‐farmed Nile tilapia was reported. Clinico‐pathological, bacteriological and parasitological examinations have been demonstrated. As well, the water quality parameters were assessed. The clinical and histopathological findings of the moribund and recently dead fish were characterized by generalized septicaemic signs. The water quality parameters were significantly elevated over the permissible levels, whereas there was an elevation in nitrite (0.04 mg/L), un‐ionized ammonia (0.8 mg/L), hydrogen sulphide levels (153.1 mg/L) and organic matter content (3.79 mg/L). A. hydrophila was identified based on phenotypic characterization, API 20E features and the homology of 16S rRNA gene sequence analysis. In addition, PCR data confirmed the presence of aerolysin (aerA) and haemolysin (hly) genes in the identified A. hydrophila isolates. Gene sequencing and phylogenetic analysis based on 16S rRNA sequence confirmed that A. hydrophila H/A (accession No. MN726928) of the present study displayed 98%–99% identity with the 16S rRNA gene of A. hydrophila. Furthermore, the monogenetic trematode, G. cichlidarum was identified in the wet mounts from the skin and gills of the examined fish with a high infestation rate. In this context, it was reported that the synergistic co‐infection of A. hydrophila and G. cichlidarum with deteriorated water quality parameters could induce exaggerated fish mortalities during hot weather.     

Growth and immune response of giant freshwater prawn Macrobrachium rosenbergii (De Man) postlarvae fed diets containing Chlorella vulgaris (Beijerinck)

A 50‐day growth trial was conducted to evaluate the efficacy of Chlorella vulgaris (Beijerinck) as an ingredient in the diets of giant freshwater prawn Macrobrachium rosenbergii (De Man) postlarvae (PL30). Immune response (total haemocyte count and prophenoloxidase activity) was also assessed by subjecting postlarvae to a challenge test against Aeromonas hydrophila (Chester) for 14 days. Iso‐nitrogenous and iso‐lipidic test diets were prepared using a fishmeal‐based‐positive control diet (D0) and four basal diets with inclusion levels of 2% (D2), 4% (D4), 6% (D6) and 8% (D8) C. vulgaris. Postlarvae of M. rosenbergii were randomly stocked (mean initial body weight of 0.19 ± 0.02 g) in 30‐L tanks in three replicates per dietary treatment for evaluation of growth performance. Another set of postlarvae (mean initial body weight of 1.25 ± 0.02 g) was randomly distributed in 95‐L tanks in three replicates per dietary treatment for the assessment of immune response. Results showed that specific growth rate was significantly higher (P < 0.05) in postlarvae fed D4 and D6. Variations in values for carcass protein, lipid, moisture and ash were also evident. Postlarvae fed diets with Chlorella showed increased prophenol oxidase activity and total haemocyte counts. Moreover, survival rate after challenge with A. hydrophila was significantly increased (P < 0.05). Inclusion of C. vulgaris in diets enhanced immune response and resistance of M. rosenbergii postlarvae against A. hydrophila infection.     

Molecular isolation and characterization of a haemocyanin of Macrobrachium rosenbergii reveal its antibacterial activities

Haemocyanin is a multi‐subunit protein complex found in the haemolymph and is involved in the immune system of crustaceans. In this study, a haemocyanin gene of Macrobrachium rosenbergii, designated MrHc, was successfully isolated. The MrHc gene contained an open reading frame (ORF) of 1,992 nucleotides, encoding a protein of 663 amino acid residues with a molecular mass of 76.5 kDa. The deduced amino acid sequence contained distinct structural motifs of the haemocyanin superfamily, including an all‐alpha domain, a copper‐containing domain and an immunoglobulin‐like domain. Based on the phylogenetic analysis, the MrHC protein demonstrated a close relationship with the haemocyanins of Palaemon carinicauda and Macrobrachium nipponense. The MrHc gene was expressed in various shrimp tissues, including the hepatopancreas, gill, haemocytes, stomach and muscle. After Macrobrachium rosenbergii nodavirus (MrNV) challenge tests, the MrHc gene was up‐regulated 237‐fold at day 2. A recombinant protein of the MrHc immunoglobulin‐like domain exhibited antibacterial activity against Vibrio vulnificus, V. parahaemolyticus, Aeromonas caviae, A. veronii, A. hydrophila and Bacillus cereus. This study suggested that MrHc may play important roles in the shrimp innate immune response to MrNV infection and bacterial infection.     

Effect of Ocimum sanctum Linn. (Tulsi) extract on the immunity and survival of Labeo rohita (Hamilton) infected with Aeromonas hydrophila

A 60 days study was conducted to evaluate the efficacy of water extract of Ocimum sanctum Linn. leaf on the immune response and disease resistance of Labeo rohita fingerlings against the Aeromonas hydrophila infection. Ocimum sanctum extract was incorporated in the diets (at 0.0%, 0.05%, 0.1%, 0.2%, 0.5% and 1%) of Labeo rohita, rohu fingerlings (6.6 ± 0.013 g). After 42 days blood, plasma and serum were sampled to determine super oxide anion production, lysozyme activity, total immunoglobulin in plasma, blood glucose, serum total protein, albumin, globulin, albumin:globulin ratio, WBC, RBC, haemoglobin content. Fish were challenged with A. hydrophila after 42 days and mortalities were recorded over 18 days post infection. The results demonstrate enhanced super oxide anion production, lysozyme activity, total immunoglobulin in plasma, serum total protein, globulin, total RBC counts, total WBC counts and haemoglobin content (P < 0.05) in treatments group compared with control group. Dietary O. sanctum extracts of 0.2% showed significantly (P < 0.05) higher protection relative percentage survival (RPS 40.00 ± 5.773%) against A. hydrophila infection than control. These results indicate that O. sanctum leaf extract stimulates the immunity and makes L. rohita more resistant to bacterial infection (A. hydrophila).     

Newly identified C‐type lysozyme in Chinese soft‐shelled turtle (Pelodiscus sinensis) exhibits potent antimicrobial activity

Lysozymes play vital roles in humoural immune response against bacterial invasion by its lytic activity. In the present study, a new C‐type lysozyme was identified and characterized from Chinese soft‐shelled turtle Pelodiscus sinensis. The full‐length cDNA of PslysC was of 923 bp, encoding a polypeptide of 148 amino acid residues. The multiple alignments and phylogenetic relationship analysis revealed the highly enzyme‐related conserved residues. The real‐time PCR analysis suggested that PslysC was constitutively expressed in a wide range of tissues with highest level in blood cells and liver. The expression of PslysC could be significantly up‐regulated under Aeromonas jandaei infection and ammonia exposure, while no significant changes were found under Poly I:C infection. The rPslysC protein was expressed in E. coli and purified by Ni‐NTA. The optimal pH and temperature for rPslysC protein lytic activities were determined at pH 7 and 30℃. rPslysC can inhibit the growth of eight kinds of Gram‐negative bacteria, and three kinds of Gram‐positive bacteria. The binding activity of rPslysC to different microbial polysaccharides and microorganism was analysed. The results showed that rPslysC could bind to selected bacteria, and exhibit a strong binding activity to lipopolysaccharide and peptidoglycan, but a weak binding activity to β‐glucan. This suggests that the binding activity might be the major mechanism of action to realize the antibacterial activity. The present study will provide helpful evidence to further understand the innate immunity of P. sinensis, and the interaction mechanisms of C‐type lysozymes with bacterial membranes.     

Effect of guava Psidium guajava (L.) aqueous extract diet on growth performance,intestinal morphology,immune response and survival of Oreochromis niloticus challenged with Aeromonas hydrophila

An 84‐day feeding trial was carried out to evaluate the effects of aqueous Psidium guajava leaf extract (PGE) on growth, intestinal morphology, physiology, immune response and susceptibility of Oreochromis niloticus to Aeromonas hydrophila. Diets containing 0% (P0), 0.25% (P1), 0.50% (P2), 0.75% (P3) and 1.00% (P4) PGE were fed to triplicate groups of fish (mean weight; 1.32 ± 0.04 g) for 84 days. After the 84‐day feeding trial, test fish were injected with pathogenic A. hydrophila and then fed for 14 days. More feed were consumed in groups of fish fed PGE‐treated diets and resulted in significantly higher weight gain and feed intake. Incidentally, there was an increase in the calculated area of absorption of fish fed PGE diets, as accounted for by marginally higher villi length and width. Antioxidant and immune response were improved with PGE inclusion in diets as total protein, superoxide dismutase, glutathione peroxidase and glutathione S‐transferase significantly increased (p < 0.05) in fish fed PGE diets. Results of the challenge test with A. hydrophila revealed that the highest mortality (100%) was recorded in P0. This study revealed that inclusion of P. guajava extract in the diet of O. niloticus improved growth, nutrient utilization, immune system and survival of O. niloticus fingerlings.     

In vivo and in vitro assessment of Lactobacillus acidophilus as probiotic for tilapia (Oreochromis niloticus,Perciformes:Cichlidae) culture improvement

In this study, the use of Lactobacillus acidophilus as a probiotic for Nile tilapia (Oreochromis niloticus) culture was studied. Fish survival and the expression of some genes involved in the immune response were assessed. Diet supplementation with L. acidophilus for 15 days caused a significant increase in fish survival during a challenge with Aeromonas hydrophila and variations in immune response related to IL‐1β and transferrin expression in Nile tilapia spleen and kidney. Moreover, extracellular products (ECPs) of L. acidophilus showed high antibacterial activity against fish pathogens such as A. hydrophila and Streptococcus agalactiae in vitro. It was also observed that viable L. acidophilus was able to disrupt quorum sensing activity in Chromobacterium violaceum.     

Indigenous AHL‐degrading bacterium Bacillus firmus sw40 affects virulence of pathogenic Aeromonas hydrophila and disease resistance of gibel carp

Interrupting quorum sensing represents a novel anti‐infective strategy to combat bacterial pathogen, and biodegradation of quorum sensing signal AHLs has been proved to be an efficient way to control pathogenic Gram‐negative bacteria in aquaculture. In this study, the effect of Bacillus firmus sw40 as efficient AHL‐degrading strain on virulence of fish pathogen Aeromonas hydrophila and disease resistance of gibel carp Carassius auratus gibelio was investigated. The results demonstrated that in vitro the B. firmus sw40 extracellular production (ECP) was able to significantly decrease protease production, haemolytic activity and biofilm formation in A. hydrophila. Dietary administration of B. firmus sw40 (10⁹ CFU/g) for 4 weeks significantly reduced the inflammatory cytokines TNF‐1a, TNF‐2a and IFN‐γ genes expression, antioxidant parameter MDA and GSH levels in serum and increased antioxidant enzyme SOD activity. Besides, B. firmus sw40 could significantly increase the survival of gibel carp with pathogenic A. hydrophila infection.     

Effect of Indian lotus (Nelumbo nucifera Gaertn.) leaf powder on immune status and disease resistance of Nile tilapia

This study assessed the immune response of Nile tilapia (Oreochromis niloticus L.) after feeding on different levels (0.1%, 0.2% and 0.4%) of dietary Indian lotus (Nelumbo nucifera) leaf powder for 45 days. We evaluated both the nonspecific immune response at the end of the feeding period and the resistance to Aeromonas hydrophila. Exposure to Indian lotus resulted in a significant elevation in serum total globulins, serum lysozyme activity, serum killing percentage and the phagocytic activity (p < 0.05). Total serum protein and albumin showed no remarkable variation between tilapia fed on 0.1% Indian lotus and the control group (p > 0.05). In addition, the relative expressions of immune‐related genes, namely interleukin–1β and tumour necrosis factor–α were significantly up‐regulated in tilapia fed on 0.4% Indian lotus as compared to the control group; their expressions were down‐regulated in the other tested groups (p < 0.05). The survival rate of Nile tilapia postchallenge to A. hydrophila reported a significant and dose‐dependent increase in the Indian lotus‐supplemented groups (p < 0.05). Therefore, dietary incorporation of Indian lotus leaves (0.4%, 0.2% and 0.1%) could strengthen the immunity of Nile tilapia and improve its resistance to A. hydrophila infection. Therefore, Indian lotus leaves could serve as potential feed supplements for Nile tilapia.     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.     

Effects of dietary soy isoflavones on growth,antioxidant status,immune response and resistance of juvenile grass carp (Ctenopharyngodon idella) to Aeromonas hydrophila challenge

The current study was conducted to evaluate the effect of dietary soy isoflavones (SI) on growth performance, antioxidant status, immune response and resistance to Aeromonas hydrophila in juvenile grass carp (Ctenopharyngodon idella). Six diets were formulated to contain 0 (control), 10, 50, 100, 500 or 1,000 mg SI per kg feed. Each diet was randomly allotted to triplicate net cages, and each net cage was stocked with 30 fish. The fish were fed one of the experimental diets to satiation twice per day for 60 days. The results showed that the WGR and DGC of the 500 mg/kg SI‐supplemented group were significantly higher than those of the non‐SI‐supplemented group (p < .05). Serum LZM and IgM activities in the SI‐supplemented groups were improved compared to the control group. SOD and GSH‐Px levels of fish fed the diet containing 500 mg/kg SI were significantly enhanced compared to those of fish fed the control diet (p < .05). Additionally, serum CAT, GSH‐Px and AKP activities in 50, 100 and 500 mg/kg SI‐supplemented groups were significantly higher than that in the control group (p < .05). The expression of most immune‐related genes (including IFN‐γ2, TNF‐α, M‐CSF2, IL‐6, IL‐12p40 and IL‐4) was significantly affected by dietary supplementation of SI. The group fed with 500 mg/kg SI had the highest 7‐day cumulative survival rate after challenge test (p < .05). The current results revealed that dietary inclusion of SI could improve the immune response and resistance against A. hydrophila and the supplementation level is suggested to be 500 mg/kg diet.     

Development of In situ hybridization and real‐time PCR assays for the detection of Hepatospora eriocheir,a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real‐time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue‐specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.     

Molecular cloning of sea perch (Lateolabrax japonicus) TLR1 and analysis of its expression pattern after stimulation with various bacteria

Toll‐like receptors (TLRs) play an indispensable role in fish immunity, being involved in pathogen recognition and the triggering of immune reactions. Here, a member of the TLR family, TLR1, from Lateolabrax japonicus was characterized and its expression pattern and intracellular localization were analysed. The full‐length LjTLR1 cDNA (2,755 bp) was found to encode a polypeptide of 827 amino acids. The deduced amino acid sequence contained three main structural domains: an extracellular leucine‐rich repeat domain, a transmembrane domain and a Toll/IL‐1 receptor domain. Tissue distribution analysis indicated that LjTLR1 was expressed in all of the examined tissues to varying degrees, with the highest levels being measured in the head kidney. In order to assess the antibacterial functions of LjTLR1 during infection, the pathogenic bacteria Vibrio harveyi and Streptococcus agalactiae were used. LjTLR1 was significantly upregulated in the three immune organs (the head kidney, spleen and liver) following bacterial stimulation, and its expression was detected 6 hr after initial exposure. In mRNA in situ hybridization experiments, positive signals were more numerous in the treatment group than the control group, verifying the expression patterns observed. Assessment of the intracellular localization of LjTLR1 revealed it to be present in the cytoplasm. These results indicate the potential role of LjTLR1 in immune responses to bacterial infection. This study enriches our knowledge of L. japonicus immune genes and provides a theoretical basis for further research concerning the antibacterial functions of fish TLRs during infection.     

The extracts of Angelica sinensis restore the digestive and absorptive capacity through improving antioxidant status in digestive organs of fish treated with trichlorfon

Firstly, a linoleic acid emulsion and fish hepatopancreas homogenate were incubated with ethoxyquin and the extracts of Angelica sinensis. The results demonstrated that ethoxyquin showed the strongest protective effects against lipid oxidation of all of the examined compounds (p < 0.05). However, ethyl acetate extract of Angelica sinensis at high concentrations showed a stronger effect on lipid oxidation than that of ethoxyquin (p < 0.05). Next, seven experimental diets that contained 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 g/kg of ethyl acetate extract of Angelica sinensis were fed to seven groups of carp (Cyprinus carpio var. Jian) respectively. After 60 days, carp were exposed to 2.4 mg trichlorfon/L in water for 4 days. The results displayed that trichlorfon exposure increased the contents of malonaldehyde and protein carbonyl in digestive organs and the activities of glutamate‐oxaloacetate and glutamate‐pyruvate transaminase in plasma, and decreased feed intake, the level of reduced glutathione, and the activities of trypsin, chymotrypsin, lipase, alpha‐amylase, Na⁺,K⁺‐ATPase, alkaline phosphatase, antisuperoxide anion, antihydroxyl radical, superoxide dismutase, glutathione reductase, and glutathione S‐transferase in digestive organs of carp (p < 0.05). Moreover, the dietary ethyl acetate extract of Angelica sinensis prevented the decrease in the above parameters in carp treated with trichlorfon (p < 0.05). These results revealed that the dietary ethyl acetate extract of Angelica sinensis could quench the trichlorfon‐induced structural and functional damage by improving the antioxidative capacity of the digestive organs of fish. Therefore, the extract of Angelica sinensis could be used as an inhibitor of trichlorfon stress in fish.     

Functional identification of ToLAAO genes and polymorphism association analysis of Cryptocaryon irritans resistance in Trachinotus ovatus

‘Marine white spot disease’ is caused by Cryptocaryon irritans infection and can lead to high mortality in Trachinotus ovatus. L-Amino acid oxides (LAAOs) play a key role in antibacterial activity and parasitic activity. To investigate the function of the LAAO (ToLAAO) and LAAO-like (ToLAAO-like) genes of T. ovatus, this study explored the sequence characteristics and relationship between polymorphisms and traits of anti-C. irritans. The ToLAAO and ToLAAO-like ORF sequences obtained from the whole genome of T. ovatus were 1563 and 1584 bp, which encoded 520 and 527 amino acids respectively. Both sequences contained a highly conserved flavin adenine dinucleotide-binding domain and a similar amino oxidase domain. Sequence multiple alignment analysis showed that ToLAAO and ToLAAO-like had the highest homology to the LAAO sequence of Larimichthys crocea. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that ToLAAO and ToLAAO-like mRNA were generally expressed in 10 tissues. ToLAAO mRNA was highly expressed in the testis, while ToLAAO-like mRNA was highly expressed in muscle tissue. After C. irritans infection, ToLAAO and ToLAAO-like mRNA were significantly upregulated in the skin and spleen, while only ToLAAO mRNA was significantly upregulated in the liver and head kidney, and only ToLAAO-like mRNA was significantly upregulated in the gills. Five SNP sites were identified from the ToLAAO and ToLAAO-like genomic sequence fragments, and two sites (6200C/T and 6237G/A) of LAAO were significantly associated with resistance to C. irritans. These results suggest that ToLAAO and ToLAAO-like genes play crucial roles in defending against the immune response to C. irritans.