LC-qTOF-MS analysis of fish immune organs reveals the distribution of amino acids in response to metabolic adaptation of the survival phenotype in grouper against Vibrio infection

Abstract

Epinephelus fuscoguttatus is economically crucial to various Southeast Asia countries where they are reared in fish farms to meet the demand for supply. However, a systemic infectious disease known as vibriosis has steadily and extensively affected the fish farming industry. The disease is caused by Vibrio spp., which are pathogenic gram-negative bacteria. This study focused on understanding the host's metabolic adaptation against Vibrio vulnificus infection, which features a survival phenotype, by profiling the metabolites in grouper fingerlings that survived the experimental infection. Mapping of the pathways is crucial to explain the roles of metabolites in fish immunity. A solvent extraction method was used on the grouper's immune organs (gills, liver and spleen) prior to Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-qTOF-MS) analysis. The metabolites identified in fingerlings that survived experimental infections were mostly amino acids (primary metabolites). Glutamine (0.44%), alanine (0.68%), phenylalanine (2.63%) and tyrosine (2.60%) were highly abundant in survived-infected gills. Aspartic acid (13.57%) and leucine (4.01%) were highly abundant in the livers of the survived-infected fish and lysine was highly abundant in both gills (2.94%) and liver (3.64%) of the survived-infected fish. Subsequent bioinformatics analysis revealed the involvement of the identified functional amino acids in various immune-related pathways. The current findings facilitate the comprehension of the metabolic adaptation of grouper fingerlings that exhibited a survival phenotype against Vibrio infection.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03269-1.

Keywords: Amino acids; Grouper; LC–qTOF-MS analysis; Metabolic adaptation; Vibriosis.

Fig. 1

Comparison of the control fingerling (a) and survived-challenged fingerling (b). Skin discoloration was examined on the survivors (b). Swab culture of the dead fingerling skin revealed high density of Vibrio grown on TCBS agar (c1), compared to the survivor (c2, 3) 29 days post-infection

Fig. 2

PCA score plot that has a value of 88% (A) where the control samples are labeled in blue (1), and the survived-infected samples are labeled in red (2)

Fig. 3

PCA derived loading plot analysis of control and survived-infected grouper. Compounds marked red indicates distinct distribution of metabolites between these two groups. Control samples are labeled in blue, and the survived-infected samples are labeled in red

Fig. 4

Classification of putatively identified metabolites obtained from the analysis of immune organ and body tissue samples from control and survived-infected grouper using LC–qTOF-MS analysis. The figure shows the breakdown of the different groups of compounds present in each of the immune organ and body tissue

Fig. 5

Heat map depicting the metabolite concentration in control and survivor samples. Classes 1–4 are control samples and are arranged in this order: gills, liver, spleen, body tissue. Classes 5–8 are survived-infected samples and are arranged in the same order as control samples

Fig. 6

Integrated amino acids pathways that contribute to the immune response of groupers towards vibriosis based on the metabolites present in high intensities in gills and liver samples from survivor grouper samples. Yellow indicates pathways contributed by survivor gills, green indicates pathways contributed by survivor liver, orange indicates pathways contributed by both survivor gills and liver while the blue pathways are connecting pathways that show the connection between all the pathways involved. The box represents the average replicates used in the study and their corresponding intensities