Skin Mucus Proteome Analysis Reveals Disease-Resistant Biomarker Signatures in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) against Vibrio alginolyticus

Abstract

Fish skin mucus is the first line of defense that provides physical and chemical barriers against pathogens and toxins. The mucus is produced continuously and sloughed off regularly from the skin to defend against infections through the skin. However, the molecular properties of the mucus content that prevent pathogen invasion are yet to be fully understood. In this study, a proteomic approach using liquid chromatography–mass spectrometry (LCMS) was applied to explore the changes in the mucus protein content of resistant and susceptible groupers in response to Vibrio alginolyticus. The Vibrio-resistant groupers showed no observable clinical sign of infection after the immersion challenge, while the Vibrio-susceptible groupers presented either hemorrhagic- or non-hemorrhagic ulceration of the skin. A comparative proteome analysis on the mucus samples yielded 1488 identified proteins. The immune-related proteins, namely Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1 and Annexin A1, that were significantly abundant in the resistant group responded to V. alginolyticus infection. Interestingly, there was an expression of immune-related proteins that possibly could be the non-invasive biomarkers, namely 3-hydroxybutyrate dehydrogenase type 2 and L-rhamnose-binding lectin SML.

1. Introduction

The world aquaculture production in 2018 surpassed capture fisheries with a record of 114.5 million tons of production. Fish protein primarily derived from aquaculture is expected to comprise 59% of human consumption by 2030 [1]. Grouper is one of the most abundant fishery species due to its significant commercial value that has been established in Singapore, Malaysia, Hong Kong, Thailand and Taiwan since the 1970s. Among the most common grouper species are the Epinephelus fuscoguttatus (Brown Marble grouper), Epinephelus coiodes (Orange Spotted grouper), Epinephelus lanceolatus (Giant grouper), Epimetheus tauvina (Greasy grouper), Mycteroperca tigris (Tiger grouper) and Cromileptes altivelis (Mouse grouper). However, throughout the years, the transition of the aquaculture technique from extensive to semi-intensive and intensive farming has led to the emergence and outbreak of infectious diseases. In order to mitigate the outbreak, several approaches have been taken to improve the quality of fish seeds produced in the hatchery. Hybridization, an approach to crossbreeding between two species, has been practiced in aquaculture to improve the quality of the fish seeds. The hybrid offspring inherited the desirable traits of the parental species, such as a superior growth rate, robustness and disease resistance [2,3,4]. The hybrid of E. fuscoguttatus × E. lanceolatus represents 70% of the total cultured grouper population in Indonesia, and this hybrid has become the second target species for aquaculture production in Hong Kong [5,6,7,8].

Although the novel hybrid of E. fuscoguttatus × E. lanceolatus yielded a significant and superior growth performance over its parental species [9], the inheritance of the desirable disease-resistant trait requires further evaluation [9,10,11]. Notwithstanding the reported improved immunological parameters in this hybrid grouper [9], the aquaculture of the hybrid species is still being affected by the prevailing infectious diseases, such as grouper iridovirus [12], vibriosis [13] and marine leech infestation [14]. Vibriosis is among the most common infectious diseases that affect grouper aquaculture. This disease is caused by the Gram-negative Vibrio sp., which includes V. alginolyticus, V. vulnificus, V. harveyi and V. parahaemolyticus [15]. Dark skin, pale gills, excessive mucus production, hemorrhage and ulceration of the affected areas, such as body, mouth and fins, are the common clinical signs of infection. The outbreak of vibriosis commonly resulted in a high mortality rate of the affected farms, causing an inevitable slump in marine aquaculture production [16,17].

Proteomics has been widely applied in studies related to infectious diseases, which include drug discovery, biomarker identification, disease pathogenesis, vaccine development and disease diagnosis, as well as in fundamental research to comprehend the host’s physiological and immunological responses against pathogens [18,19]. The characterization of the immunological responses against pathogen invasion using the proteomics approach facilitates the identification of novel immune-related proteins and potential biomarkers for disease diagnosis and therapy [20,21]. This approach has successfully identified differentially expressed proteins in different immune organs of the disease-resistant/tolerant and the susceptible fish [22]. The fish skin mucus is rich in biochemicals, especially proteins and metabolites [23,24]; hence, a comparative analysis of the mucus has become of great interest in understanding the immunological responses of skin- and mucosal-associated lymphoid tissues. The functional skin mucus proteome of disease-resistant grouper is hypothesized to provide new insights into the disease-resistant/tolerant phenotype, which could assist in the screening of broodstock/fish seed quality. This study aimed to identify the proteome changes in the grouper skin mucus in response to Vibrio alginolyticus infection and the potential protein biomarkers that correspond to the disease-resistant phenotype.

2. Materials and Methods

2.1. Fish and Bacterial Infection, Mucus Sample Collection and Preparation

All animal handling and experimental protocol were performed in accordance with the ethics guidelines approved by Universiti Putra Malaysia Animal Ethics Committee (Approval number: UPM/IACUC/AUP-R054/2022). Hybrid grouper fingerlings (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) in 9.6 ± 0.4 cm size were obtained in April 2021 from a local hatchery in Kuala Selangor, Malaysia, and transported to the laboratory using plastic bags containing oxygen. The fingerlings were screened for health issues, from which only healthy fish were subjected to acclimatization under laboratory conditions for 2 weeks in 25 ppt saltwater with continuous aeration and fed with commercial feed twice daily on an ad libitum basis. The water temperature was maintained at 27 to 34 °C throughout the day.

Vibrio alginolyticus isolate was obtained from the Laboratory of Fish Health and Disease, Department of Aquaculture, Universiti Putra Malaysia. The isolate was cultured on marine agar for 24 h at 16 °C. A single colony was inoculated in 10 mL marine broth, and then the culture was up-scaled to 1 L. Bacterial cells were then collected through centrifugation at 11,000× g for 15 min. Bacterial cell suspension of 9.4 × 10⁷ CFU/mL was prepared in 25 ppt saltwater for subsequent experimental infection in grouper fingerlings.

Eighty healthy grouper fingerlings with a mean total body length of 9.6 ± 0.4 cm were randomly selected. For experimental infection, 60 fingerlings were exposed to V. alginolyticus at a dose of 9.4 × 10⁷ CFU/mL by immersion for 4 h, whereas the control group consisted of 20 fingerlings were not exposed to V. alginolyticus. The fingerlings were then transferred to clean saltwater with continuous aeration for observation, and the control group was kept in a separated aquarium. On day 7 post-infection, fingerlings were categorized into Vibrio-susceptible group according to the presence of clinical signs of infection that include red spots, hemorrhagic and/or non-hemorrhagic ulceration on the body, while the Vibrio-resistant fingerlings presented no observable clinical sign of infection. The fish were fasted 12 h before harvesting.

In the pre-harvesting process, all (46 samples) of the fish mucus from resistant and susceptible groups, including those from the dead fish, were spread individually on marine agar and thiosulfate citrate bile salts sucrose agar (TCBS: selective media for Vibrio sp.) to confirm the presence of Vibrio. The bacteria from the dead fish spleen were also detected by plating on the designated agar. All the subjected samples showed a positive Vibrio growth on the TCBS agar and thus confirmed the presence of Vibrio through the immersion method.

Fish were anesthetized using tricaine methanesulfonate (MS222) before skin mucus sampling. Skin mucus (n = 5) was collected by gently scraping the body using a sterile glass slide avoiding the collection of blood and the anal area. The 5 mucus samples of fish from the same group were pooled to represent one biological replicate due to the minute amount of mucus from each fish. All samples were stored at −80 °C until further use. The mucus samples were reconstituted in 1× phosphate-buffered saline and sample buffer (7 M Urea, 2 M Thiourea, 2 mM PMSF). The samples were mixed well and sonicated on ice at 20% amplitude for 3 min (10 s pulses on, 15 s pulses off). The samples were then centrifuged at 15,000× g for 15 min to collect supernatant containing skin mucus total protein which concentration was quantified using Bradford assay (Solarbio, Beijing, China) before subsequent analysis. The total protein in 9 µg from each sample was electrophoretically separated on 12% SDS polyacrylamide gel and double stained with Coomassie-silver.

2.2. Liquid Chromatography–Mass Spectrometry (Hybrid Quadrupole-Orbitrap Technology)

Nine samples of 3 biological replicates from each group were submitted to proteomic analysis using LCMS. Prior to LCMS, protein samples of 75 µg were loaded into 12% SDS polyacrylamide gel. Protein bands were then excised and in-gel digested using trypsin gold (Promega, Madison, WI, USA). Subsequently, clean-up of the digested samples was performed using Ziptip (Millipore, Burlington, MA, USA). LCMS analysis of the peptide samples was conducted according to the previous report [25]. In brief, peptide samples were reconstituted in 30 μL of 0.1% formic acid and 5% acetonitrile, before 2 μL of the samples were loaded onto an Acclaim PepMap 100 C18 column (2 μm, 0.075 × 150 mm) (Thermo Scientific, Waltham, MA, USA). The reverse phase column was equilibrated with 0.1% aqueous formic acid (mobile phase A) and 80% acetonitrile containing 0.1% formic acid (mobile phase B). Elution of gradient 5–35% mobile phase B was performed at a flow rate of 0.3 mL/min for 75 min using EASY-nano liquid chromatography (EASY-nLC) 1200 System (Thermo Scientific, MA, USA). An online Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer system (Thermo Scientific, MA, USA) was used to generate the peptide ions with a spray voltage of 1900 V in positive mode. A precursor ion scan was conducted with a resolution of 70,000 and a mass range of m/z 310–1800. Precursors containing charge states from 2+ to 8+ were further fragmented via collision induced and high-energy collision induced (CID and HCD) at normalized energy of 28%. Precursor mass was scanned at the range of 110–1800 m/z.

2.3. Data Processing and Protein Annotation

Mass spectra of the peptides were acquired using Tune (Ver. 2.11 QF1 Build 3006; Thermo Scientific, Waltham, MA, USA) and deconvoluted with Proteome Discoverer Ver. 2.4 (Thermo Scientific, Waltham, MA, USA) to create the peptide mass list. SEQUEST HT search engine, incorporated in the Proteome Discoverer, was used to match the generated mass list against Epinephelus FASTA sequences downloaded from NCBI. Mass tolerance for the peptides and their fragments was fixed at 10 ppm and 0.02 Da, respectively. Trypsin was indicated as the digestion enzyme with up to two miscleavages allowed during the search. Carbamidomethylation (CAM) modification on cysteine residues was set as a static modification, while variable amino acid modifications were set as deamidation for asparagine and glutamine residues, and oxidation for methionine residues. The mass list was also searched against a decoy database generated from the randomized protein sequences. The identified proteins were accepted with at least a Rank 1 peptide and a false discovery rate of 1%. Spectra that matched the sequences were further validated with the Percolator algorithm (Ver. 2.04) where q-value at 1% false discovery rate (FDR).

2.4. Statistics and Protein Enrichment

The statistical analysis was performed using Perseus [26] to identify differentially expressed proteins. One-way ANOVA was applied and the comparison between groups was analyzed by applying a two-tailed Student’s t-test (p ≤ 0.05). Proteins with fold change ≥2 were identified to be differentially expressed [27], and the differentially expressed proteins identified were mapped to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analyses. Fisher’s exact test was applied to perform enrichment analysis (p-value ≤ 0.05).

3. Results

3.1. Experimental Infection of Hybrid Grouper with Vibrio alginolyticus

The hybrid grouper fingerlings were exposed to V. alginolyticus by immersion in the saltwater of 25 ppt containing approximately 9.4 × 10⁷ CFU/mL of V. alginolyticus for 4 h. On day 7 post-infection, 10 mortalities out of 60 infected fingerlings were recorded (16.7% of cumulative mortality), 28 fingerlings (46.7%) were resistant to V. alginolyticus and 18 fingerlings (30%) were susceptible to the infection, exhibiting clinical signs of infection where ulceration and red spots were observed on the mouth, body and fins (Figure 1). The resistant fingerlings recorded no observable clinical signs of infection.

The infection of V. alginolyticus on the selected healthy hybrid grouper was confirmed by applying the bacteria plating method. The fish mucus from resistant and susceptible groups was spread individually on marine agar and TCBS agar to confirm the presence of Vibrio in the fish. All the subjected samples, including the spleen of the dead fish, showed positive for Vibrio infection on the TCBS agar (Appendix A Figure A1). However, the bacterial load was not quantitatively analyzed. The immersion in the same concentration of bacterial suspension in controlled conditions and exposure time might have given similar bacterial exposure to the fish.

The evaluation on the skin mucus protein of the susceptible and resistant groups showed viscosity variation. The susceptible group secreted much more and a viscous mucus than the resistant fish. The protein composition of the skin mucus between the infected group and control group was significantly different with the former showing more variable intensities of the protein bands (Appendix A Figure A2).

3.2. Skin Mucus Proteome Profiling and Differential Expression Analysis

The label-free shotgun proteomic analysis using LCMS identified a total of 1488 proteins in the skin mucus samples. Out of the 1488 proteins, 3, 2 and 8 proteins were found to be exclusively expressed in the control, resistant and susceptible groups, respectively (Figure 2). The identified putative proteins are listed in

Table 1. Putative proteins were found to be exclusively expressed in the control, resistant and susceptible groups, respectively.

Group	Accession	Description
Control	834906564	cysteine desulfhydrase
	1832645835	ruvB-like 2
	1832690337	chitinase
Resistant	1832634797	transmembrane emp24 domain-containing protein 2 isoform X1
	1832622809	RWD domain-containing protein 1 isoform X2
Susceptible	1832685063	hydroperoxide isomerase ALOXE3
	1832644368	peroxiredoxin-6
	1832670971	LSM8 homolog, U6 small nuclear RNA associated
	1832638785	leucine-rich alpha-2-glycoprotein
	1832664977	cytochrome c oxidase subunit 6B1
	1832612090	heat shock 70 kDa protein 4b
	1042045193	glycine dehydrogenase (aminomethyl-transferring)
	1832627111	nesprin-2-like isoform X4

.

3.3. Differentially Expressed Proteins (DEPs) in Response to V. alginolyticus Experimental Infection

A pairwise comparison between the control, resistant and susceptible groups revealed different numbers of DEPs in response to the V. alginolyticus experimental infection (Figure 3). A comparison of the protein abundance between the control and resistant groups reveals 99 DEPs (

Table 2. Differentially expressed protein was found to be resistant when compared to the control.

Accession	Description	MW (kDa)	Fold Change	p-Value	Abundances Control	Abundances Resistant
Upregulated
1832619927	elongation factor 1-alpha	50.3	−6.64	0.000		31.5
1832702676	von Willebrand factor A domain-containing protein 5A-like isoform X1	69.9	−6.64	0.000		102.0
1832606995	tropomyosin alpha-4 chain-like isoform X4	28.4	−6.64	0.000		172.3
1832633330	protein phosphatase 1, catalytic subunit, alpha isozyme a	37.5	−6.64	0.000	24.9	125.1
1832671158	23 kDa integral membrane protein	26.7	−6.64	0.000		49.9
1832663676	catechol O-methyltransferase domain-containing protein 1-like isoform X1	28.6	−6.64	0.000		86.7
1832613275	cytochrome c oxidase subunit 5A, mitochondrial	16	−6.64	0.000		118.8
1832681058	antithrombin-III	51.2	−6.64	0.000	44.5	92.2
1832628613	EH domain-containing protein 4	62.3	−6.64	0.000		98.1
1832617553	small nuclear ribonucleoprotein Sm D1	13.3	−6.64	0.000		146.4
1832651057	uncharacterized protein si:ch1073-126c3.2	23.2	−6.64	0.000		194.3
1832634797	transmembrane emp24 domain-containing protein 2 isoform X1	23.3	−6.64	0.000		300.0
1832661391	complement factor I	77.7	−6.64	0.000		89.6
1832671044	deoxyribose-phosphate aldolase	35	−6.64	0.000	34.0	134.8
1832645324	Arginine-tRNA ligase, cytoplasmic	75.6	−6.64	0.000		108.9
1832640714	L-threonine dehydrogenase	41.7	−6.64	0.000	33.9	137.4
1832610610	60S ribosomal protein L39	6.4	−6.64	0.000		126.1
1832685492	39S ribosomal protein L45, mitochondrial	35.9	−6.64	0.000	72.9	168.9
1832700793	TATA-binding protein-associated factor 2N isoform X1	43.2	−6.64	0.000	19.6	147.4
1832687225	3-hydroxybutyrate dehydrogenase type 2	26.4	−6.64	0.000	121.5	178.5
1832690325	uncharacterized protein LOC117256382 isoform X3	72.6	−6.64	0.000	130.4	169.6
1832622809	RWD domain-containing protein 1 isoform X2	23.2	−6.64	0.000		300.0
1996377847	DUF2850 domain-containing protein	16.9	−4.8	0.000	41.2	206.2
1832685958	arachidonate 15-lipoxygenase B-like isoform X1	76.9	−4.44	0.000	6.9	45.6
1832683648	nucleolin	74.2	−3.43	0.000	71.9	105.2
1832680261	low molecular weight neuronal intermediate filament	59.2	−3.3	0.000	66.6	152.2
1832613023	transmembrane protein 258	9.1	−3.15	0.000	90.0	104.2
1832671502	basic leucine zipper and W2 domain-containing protein 1-A	48.1	−3.15	0.000	103.3	116.1
145105480	interleukin enhancer-binding factor 2	42.8	−2.63	0.001	62.4	174.9
161935902	MHC class I alpha antigen	41.6	−2.63	0.000	23.1	176.2
1832684689	trypsin-3	30.1	−2.44	0.000	88.5	163.5
1832608120	cytochrome c oxidase subunit 4 isoform 1, mitochondrial	19.4	−2.42	0.001	30.0	194.5
1832640175	FAU ubiquitin-like and ribosomal protein S30 fusion a	14.5	−2.04	0.004	37.7	171.2
1832678052	CD59B glycoprotein-like isoform X1	11.5	−2.04	0.000	31.5	105.5
343459105	NADH dehydrogenase 1 alpha, partial	9.5	−2.01	0.000	81.3	139.3
1832688344	ADP/ATP translocase 1	32.9	2.26	0.001	28.7	215.2
1832676130	brain acid soluble protein 1	36.1	2.27	0.004	76.4	137.1
1832620903	cystatin-B	11.9	2.41	0.003	17.5	89.8
1832667755	elongation factor 1-alpha 1a	50.5	3.18	0.000	54.3	99.7
327239696	complement component C6, partial	11.4	3.18	0.000	14.4	19.4
1832625921	uncharacterized protein LOC117266161	62.4	3.2	0.000	64.4	120.6
1832674019	deleted in malignant brain tumors 1 protein-like isoform X1	141.3	3.72	0.000	122.9	135.4
1832691617	ubiquinol-cytochrome-c reductase complex assembly factor 2	15	6.64	0.000	104.7	113.7
1832614309	very-long-chain 3-oxoacyl-CoA reductase-A	37.2	6.64	0.000	48.9	187.2
1832627296	isovaleryl-CoA dehydrogenase, mitochondrial	45.9	6.64	0.000	95.8	116.4
1832635569	annexin A1	39.2	6.64	0.000	58.0	125.4
1832617105	NADH dehydrogenase (ubiquinone) iron-sulfur protein 6, mitochondrial	14.1	6.64	0.000	114.8	117.4
Downregulated
1832631564	E3 ubiquitin-protein ligase RBX1	12.3	−6.64	0.000	102.9	83.5
197725770	mitochondrial cytochrome C oxidase subunit Vb precursor	14.9	−3.44	0.000	74.5	73.5
1832626150	C-reactive protein	25.5	−3.11	0.000	65.4	53.7
2067150115	Thioredoxin domain-containing protein 12	22.7	2.01	0.021	125.9	60.8
1832680021	Betaine-homocysteine S-methyltransferase 1	44.1	2.02	0.003	207.7	86.8
1832680525	ubiquitin-conjugating enzyme E2 L3	17.8	2.04	0.022	95.7	27.5
1832606043	ras-related protein Rab-27A	24.8	2.06	0.017	103.4	83.7
1710371347	electron transfer flavoprotein subunit beta	31.4	2.25	0.009	130.4	54.0
1832702451	LOW-QUALITY PROTEIN: apolipoprotein(a)-	122.9	2.26	0.000	132.7	27.3
1832645146	pigment epithelium-derived factor	44.7	2.38	0.000	228.9	46.5
1832639949	collagen alpha-1(X) chain	62.9	2.56	0.000	200.3	42.5
1832701092	latexin	33	2.58	0.000	136.3	24.0
1832662114	parvalbumin alpha	11.5	2.6	0.001	269.9	13.3
1832699882	peptidyl-prolyl cis-trans isomerase FKBP10	62.4	2.64	0.000	241.4	32.8
343458999	nattectin, partial	18.1	2.79	0.000	229.8	50.1
1832696854	protein bassoon	431.2	2.9	0.000	159.2	43.0
1832670505	coatomer subunit gamma-2	97.4	3	0.000	22.2	17.8
1379027526	ribonuclease G	55.4	3.21	0.000	142.9	15.5
209981964	alpha-1-antitrypsin	46.1	3.37	0.000	199.3	22.4
1832702196	vasodilator-stimulated phosphoprotein isoform X1	44.6	3.87	0.000	177.5	122.5
834904058	malate dehydrogenase	32.4	4.52	0.000	200.3	54.0
1832627647	PTTG1-interacting protein a	18.8	4.52	0.000	147.0	30.5
1832656308	neural cell adhesion molecule 1a isoform X4	109.2	5	0.000	155.3	14.2
1393214856	type I glyceraldehyde-3-phosphate dehydrogenase	36	5.03	0.000	130.0	65.7
306008591	heat shock protein	61.2	6.64	0.000	84.3
295792244	fructose-bisphosphate aldolase A	39.6	6.64	0.000	221.9	29.6
328677149	hypothetical protein, partial	33.1	6.64	0.000	132.9	18.3
343459175	ATP synthase, H+ transporting, mitochondrial F0 complex, partial	18.2	6.64	0.000	156.9
1832625753	glyceraldehyde-3-phosphate dehydrogenase	36	6.64	0.000	188.4	87.4
1832630592	glucosidase 2 subunit beta	59.5	6.64	0.000	151.8
1832669039	aldo-keto reductase family 1 member B1 isoform X1	35.7	6.64	0.000	105.8
1832632244	parvalbumin beta	11.7	6.64	0.000	180.6	96.3
1832620905	stefin-C	11.7	6.64	0.000	171.2	51.2
1832677686	NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial	19	6.64	0.000	89.3	12.3
1832638111	hyaluronan and proteoglycan link protein 1-like	40	6.64	0.000	271.7
1832619571	tetranectin	22.4	6.64	0.000	193.6	84.8
1832616138	testin	62.2	6.64	0.000	72.2
1832604382	histone H1	27.4	6.64	0.000	48.3
1832692739	protein SSUH2 homolog	39.7	6.64	0.000	66.0
834904842	phosphoglycerate kinase	41	6.64	0.000	286.0
1832645835	ruvB-like 2	50.7	6.64	0.000	300.0
1832635997	proteasome subunit beta type-7 isoform X1	32.9	6.64	0.000	300.0
1832640826	papilin b, proteoglycan-like sulfated glycoprotein	63	6.64	0.000	161.6	54.5
1832641526	uroporphyrinogen decarboxylase	41.3	6.64	0.000	157.7
1832690337	chitinase	50.6	6.64	0.000	300.0
1832673253	60S ribosomal protein L8	28	6.64	0.000	101.8
834904288	TDP-fucosamine acetyltransferase	25.4	6.64	0.000	199.5	6.5
1832638138	transcription factor BTF3	17.7	6.64	0.000	79.7	14.6
1832643512	rho GTPase-activating protein 12-like isoform X1	100.9	6.64	0.000	129.7
1832644997	coatomer subunit beta’ isoform X1	105.9	6.64	0.000	70.5
1832642084	neurogenic differentiation factor 6-B	37.7	6.64	0.000	193.0	107.0
834906564	cysteine desulfhydrase	52.8	6.64	0.000	300.0

), where 47 upregulated and 52 downregulated proteins were from the resistant group, while 60 upregulated and 42 downregulated proteins were identified in the susceptible group compared to the control group (

Table 3. Differentially expressed protein was found to be susceptible when compared to the control.

Accession	Description	MW (kDa)	Fold Change	p-Value	Abundances Control	Abundances Susceptible
Upregulated
306008591	heat shock protein	61.2	−6.64	0.000	84.3	215.7
1832619927	elongation factor 1-alpha	50.3	−6.64	0.000		268.5
1832702676	von Willebrand factor A domain-containing protein 5A-like isoform X1	69.9	−6.64	0.000		198.0
1832606995	tropomyosin alpha-4 chain-like isoform X4	28.4	−6.64	0.000		127.7
1832685063	hydroperoxide isomerase ALOXE3	82.7	−6.64	0.000		300.0
1832633330	protein phosphatase 1, catalytic subunit, alpha isozyme a	37.5	−6.64	0.000	24.9	150.0
1832683648	nucleolin	74.2	−6.64	0.000	71.9	122.8
1832671158	23 kDa integral membrane protein	26.7	−6.64	0.000		250.1
1832663676	catechol O-methyltransferase domain-containing protein 1-like isoform X1	28.6	−6.64	0.000		213.3
1832644368	peroxiredoxin-6	24.6	−6.64	0.000		300.0
1832613275	cytochrome c oxidase subunit 5A, mitochondrial	16	−6.64	0.000		181.2
1832681058	antithrombin-III	51.2	−6.64	0.000	44.5	163.2
1832628613	EH domain-containing protein 4	62.3	−6.64	0.000		201.9
1832617553	small nuclear ribonucleoprotein Sm D1	13.3	−6.64	0.000		153.6
1832651057	uncharacterized protein si:ch1073-126c3.2	23.2	−6.64	0.000		105.7
1832661391	complement factor I	77.7	−6.64	0.000		210.4
1832670971	LSM8 homolog, U6 small nuclear RNA associated	10.4	−6.64	0.000		300.0
1832638785	leucine-rich alpha-2-glycoprotein	38.9	−6.64	0.000		300.0
1832671044	deoxyribose-phosphate aldolase	35	−6.64	0.000	34.0	131.3
1832664977	cytochrome c oxidase subunit 6B1	10.2	−6.64	0.000		300.0
1832613023	transmembrane protein 258	9.1	−6.64	0.000	90.0	105.8
1832645324	arginine--tRNA ligase, cytoplasmic	75.6	−6.64	0.000		191.1
1832612090	heat shock 70 kDa protein 4b	95.1	−6.64	0.000		300.0
1832640714	L-threonine dehydrogenase	41.7	−6.64	0.000	33.9	128.6
1832626150	C-reactive protein	25.5	−6.64	0.000	65.4	180.9
1832610610	60S ribosomal protein L39	6.4	−6.64	0.000		173.9
1042045193	glycine dehydrogenase (aminomethyl-transferring)	104.1	−6.64	0.000		300.0
1832622572	acyl-CoA thioesterase 9, tandem duplicate 1 isoform X1	49.1	−6.64	0.000	79.4	106.4
1832631564	E3 ubiquitin-protein ligase RBX1	12.3	−6.64	0.000	102.9	113.6
1832700793	TATA-binding protein-associated factor 2N isoform X1	43.2	−6.64	0.000	19.6	133.0
1832644997	coatomer subunit beta’ isoform X1	105.9	−6.64	0.000	70.5	229.5
1832627111	nesprin-2-like isoform X4	250.6	−6.64	0.000		300.0
1832670505	coatomer subunit gamma-2	97.4	−6.26	0.000	22.2	260.0
327239696	complement component C6	11.4	−5.36	0.000	14.4	266.2
1832685958	arachidonate 15-lipoxygenase B-like isoform X1	76.9	−5.04	0.000	6.9	247.5
1832620903	cystatin-B	11.9	−4.32	0.000	17.5	192.7
1832656769	vesicle-associated membrane protein 2	12	−4.29	0.000	45.4	236.3
45269063	immunoglobulin light-chain variable region	11.9	−3.91	0.000	54.4	202.5
1832638138	transcription factor BTF3	17.7	−3.64	0.000	79.7	205.7
197725770	mitochondrial cytochrome C oxidase subunit Vb precursor	14.9	−3.61	0.000	74.5	152.0
328677247	glyceraldehyde 3-phosphate dehydrogenase isoform, partial	24.3	−3.13	0.002	101.5	169.7
1832679851	drebrin-like b isoform X1	53.6	−3.05	0.003	31.0	256.8
1832650400	prefoldin subunit 1 isoform X1	15.6	−2.96	0.000	49.6	195.5
221048061	fructose-bisphosphate aldolase	33.4	−2.92	0.000	56.5	199.4
1832658469	fibrinogen beta chain	40.2	−2.83	0.000	27.7	224.7
1832612579	60S ribosomal protein L27a	16.6	−2.65	0.005	26.6	166.9
222087999	fibrinogen beta chain precursor	16.7	−2.61	0.000	28.6	197.6
1832689402	fibrinogen gamma chain	48.5	−2.49	0.000	33.5	221.5
1832662038	leukocyte cell-derived chemotaxin-2	17.1	−2.39	0.000	39.6	145.9
1832604382	histone H1	27.4	−2.38	0.016	48.3	251.7
1832678052	CD59B glycoprotein-like isoform X1	11.5	−2.38	0.000	31.5	163.0
1832685624	microfibril-associated glycoprotein 4	28.1	−2.36	0.039	42.3	180.9
1832634516	keratin, type I cytoskeletal 13	48.2	−2.32	0.000	38.0	189.1
1832685241	beta-2-glycoprotein 1	40.1	−2.23	0.012	43.7	189.4
1832623025	apolipoprotein Eb	30.8	−2.2	0.000	35.0	159.4
1832688344	ADP/ATP translocase 1	32.9	3.23	0.000	28.7	56.1
405790938	MHC class II antigen, partial	18.4	6.64	0.000	57.5	137.5
1832677686	NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial	19	6.64	0.000	89.3	198.3
1832616138	testin	62.2	6.64	0.000	72.2	227.8
295792326	mitochondrial cytochrome c oxidase subunit 7C	7.1	6.64	0.000	99.8	121.7
Downregulated
343459175	ATP synthase, H+ transporting, mitochondrial F0 complex	18.2	−6.64	0.000	156.9	143.1
834904842	phosphoglycerate kinase	41	−6.64	0.000	286.0	14.0
1832671502	basic leucine zipper and W2 domain-containing protein 1-A	48.1	−6.64	0.000	103.3	80.5
1832630592	glucosidase 2 subunit beta	59.5	−3.16	0.002	151.8	148.2
1832626146	allograft inflammatory factor 1	16.6	−3.36	0.000	194.3	50.7
1832685492	39S ribosomal protein L45, mitochondrial	35.9	−3.36	0.000	72.9	58.3
1832610590	DNA-directed RNA polymerase I subunit RPA1	191	2.02	0.001	167.4	50.2
451931262	Multidrug resistance efflux pump	37.1	2.04	0.006	159.8	38.8
1832645146	pigment epithelium-derived factor	44.7	2.44	0.000	228.9	24.7
1832632244	parvalbumin beta	11.7	2.62	0.000	180.6	23.2
1832616865	receptor-transporting protein 4	18.9	2.14	0.000	154.5	68.8
1832625915	uncharacterized protein LOC117266159 isoform X1	70.4	2.14	0.000	145.3	47.5
295792242	nucleoside diphosphate kinase B	17	2.33	0.000	260.4	9.1
1832699882	peptidyl-prolyl cis-trans isomerase FKBP10	62.4	3.26	0.000	241.4	25.8
1832702726	serpin H1b	45.6	2.32	0.000	222.4	39.4
1832639949	collagen alpha-1(X) chain	62.9	2.34	0.000	200.3	57.2
1832662114	parvalbumin alpha	11.5	4.01	0.000	269.9	16.8
1832620905	stefin-C	11.7	3.97	0.000	171.2	77.6
295792244	fructose-bisphosphate aldolase A	39.6	3.61	0.000	221.9	48.4
1832619571	tetranectin	22.4	3.62	0.000	193.6	21.6
834906594	hypothetical protein QY76_16135	34.4	2.63	0.000	175.8	28.5
1832625753	glyceraldehyde-3-phosphate dehydrogenase	36	4.02	0.000	188.4	24.3
1832616029	three prime repair exonuclease 2	22.1	3.2	0.000	179.9	54.9
343458999	nattectin, partial	18.1	3.76	0.000	229.8	20.1
834906564	cysteine desulfhydrase	52.8	6.64	0.000	300.0
1832680021	betaine--homocysteine S-methyltransferase 1	44.1	6.64	0.000	207.7	5.6
1832702196	vasodilator-stimulated phosphoprotein isoform X1	44.6	6.64	0.000	177.5
1832665474	sulfotransferase 2B1	22.7	6.64	0.000	183.6	21.7
1832641369	phosphotriesterase-related protein	38.8	6.64	0.000	140.3
1832645835	ruvB-like 2	50.7	6.64	0.000	300.0
1832635997	proteasome subunit beta type-7 isoform X1	32.9	6.64	0.000	300.0
1832690337	chitinase	50.6	6.64	0.000	300.0
1832686354	puromycin-sensitive aminopeptidase	104.2	6.64	0.000	146.9
1393214856	type I glyceraldehyde-3-phosphate dehydrogenase	36	6.64	0.000	130.0	104.4
1832695641	N-alpha-acetyltransferase 10 isoform X1	24.8	6.64	0.000	109.3
1996375967	methionine ABC transporter substrate-binding protein	28.8	6.64	0.000	125.4
1832682447	dedicator of cytokinesis protein 7 isoform X1	244.1	6.64	0.000	176.4
1832697234	L-rhamnose-binding lectin SML	23.9	6.64	0.000	103.5
1832687225	3-hydroxybutyrate dehydremp24ogenase type 2	26.4	6.64	0.000	121.5
1832698896	uncharacterized protein KIAA2013 homolog isoform X1	69.5	6.64	0.000	160.6
1832690325	uncharacterized protein LOC117256382 isoform X3	72.6	6.64	0.000	130.4
1832642084	neurogenic differentiation factor 6-B	37.7	6.64	0.000	193.0

). The comparison between the resistant and susceptible groups revealed 94 DEPs (

Table 4. Differentially expressed proteins were found to be in resistant when compared to susceptible.

Accession	Description	MW (kDa)	FC (S vs. R)	p-Value	Abundances Susceptible	Abundances Resistant
Upregulated
1832625921	uncharacterized protein LOC117266161	62.4	3.11	0.000	115.0	120.6
1832606995	tropomyosin alpha-4 chain-like isoform X4	28.4	3.23	0.000	127.7	172.3
1832625753	glyceraldehyde-3-phosphate dehydrogenase	36	3.25	0.000	24.3	87.4
1832626146	allograft inflammatory factor 1	16.6	4.27	0.000	50.7	55.0
1832632244	parvalbumin beta	11.7	6.64	0.000	23.2	96.3
1832691617	ubiquinol-cytochrome-c reductase complex assembly factor 2	15	6.64	0.000	81.5	113.7
1832614309	very-long-chain 3-oxoacyl-CoA reductase-A	37.2	6.64	0.000	64.0	187.2
1832627296	isovaleryl-CoA dehydrogenase, mitochondrial	45.9	6.64	0.000	87.8	116.4
1832635569	annexin A1	39.2	6.64	0.000	116.5	125.4
834904058	malate dehydrogenase	32.4	6.64	0.000	45.7	54.0
1832622572	acyl-CoA thioesterase 9, tandem duplicate 1 isoform X1	49.1	6.64	0.000	106.4	114.2
1832617105	NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial	14.1	6.64	0.000	67.8	117.4
1832667938	sideroflexin-3	35.5	−2.71	0.001	37.1	147.6
834906594	hypothetical protein QY76_16135	34.4	−2.26	0.000	28.5	95.7
1832634797	transmembrane emp24 domain-containing protein 2 isoform X1	23.3	−6.64	0.000		300.0
1832680021	betaine--homocysteine S-methyltransferase 1	44.1	−6.64	0.000	5.6	86.8
1832702196	vasodilator-stimulated phosphoprotein isoform X1	44.6	−6.64	0.000		122.5
1832665474	sulfotransferase 2B1	22.7	−6.64	0.000	21.7	94.7
1832641369	phosphotriesterase-related protein	38.8	−6.64	0.000		159.7
1832686354	puromycin-sensitive aminopeptidase	104.2	−6.64	0.000		153.1
1832695641	N-alpha-acetyltransferase 10 isoform X1	24.8	−6.64	0.000		190.7
1996375967	methionine ABC transporter substrate-binding protein	28.8	−6.64	0.000		174.6
1832682447	dedicator of cytokinesis protein 7 isoform X1	244.1	−6.64	0.000		123.6
1832697234	L-rhamnose-binding lectin SML	23.9	−6.64	0.000		196.5
1832687225	3-hydroxybutyrate dehydrogenase type 2	26.4	−6.64	0.000		178.5
1832698896	uncharacterized protein KIAA2013 homolog isoform X1	69.5	−6.64	0.000		139.4
1832690325	uncharacterized protein LOC117256382 isoform X3	72.6	−6.64	0.000		169.6
1832642084	neurogenic differentiation factor 6-B	37.7	−6.64	0.000		107.0
1832622809	RWD domain-containing protein 1 isoform X2	23.2	−6.64	0.000		300.0
Downregulated
1832702676	von Willebrand factor A domain-containing protein 5A-like isoform X1	69.9	−2.56	0.002	198.0	102.0
1393214856	type I glyceraldehyde-3-phosphate dehydrogenase	36	−3.16	0.000	104.4	65.7
405790938	MHC class II antigen, partial	18.4	−2.82	0.000	137.5	105.0
1832645324	arginine--tRNA ligase, cytoplasmic	75.6	−6.64	0.000	191.1	108.9
295792326	mitochondrial cytochrome c oxidase subunit 7C	7.1	−6.64	0.000	121.7	78.5
1832680525	ubiquitin-conjugating enzyme E2 L3	17.8	2.27	0.034	176.8	27.5
1832608362	AP-1 complex subunit gamma-1 isoform X1	91.3	2.48	0.022	161.8	74.6
1832621671	rRNA 2’-O-methyltransferase fibrillarin	33.5	2.35	0.012	177.5	76.4
45269063	immunoglobulin light-chain variable region, partial	11.9	2.43	0.010	202.5	43.1
1832611868	transmembrane 9 superfamily member 2 isoform X1	76	2.2	0.004	152.1	48.3
1832685958	arachidonate 15-lipoxygenase B-like isoform X1	76.9	2.06	0.004	247.5	45.6
1832671468	coagulation factor VIIi	50.6	2.29	0.004	162.7	32.2
1832667755	elongation factor 1-alpha 1a	50.5	2.96	0.003	146.0	99.7
1832702451	LOW-QUALITY PROTEIN: apolipoprotein(a)	122.9	2.34	0.001	140.0	27.3
1832618423	class I histocompatibility antigen, F10 alpha chain-like isoform X1	40.8	2.5	0.000	143.6	71.1
62255674	immunoglobulin mu heavy-chain	66.3	2.02	0.000	162.2	57.2
295792244	fructose-bisphosphate aldolase A	39.6	3.38	0.000	48.4	29.6
157929900	ribosomal protein L23	15	2.09	0.000	123.4	40.6
2022331196	immunoglobulin M heavy-chain constant mu variant 1	50.8	2.34	0.000	196.3	38.7
834906005	glyceraldehyde-3-phosphate dehydrogenase	35.5	3.65	0.000	3.2	0.9
1832671158	23 kDa integral membrane protein	26.7	2.98	0.000	250.1	49.9
1832604432	erythroblast NAD(P)(+)-arginine ADP-ribosyltransferase	36.7	3.26	0.000	198.8	56.8
1832697311	nucleobindin-2a isoform X1	59.4	2.93	0.000	163.7	24.8
1832604652	fibrinogen alpha chain	13.7	2.28	0.000	197.6	66.4
1832701092	latexin	33	2.94	0.000	139.7	24.0
326632479	MHC class II antigen	26.1	3.14	0.000	202.4	32.7
1832660271	macrosialin	38.8	2.67	0.000	179.2	27.2
1832689402	fibrinogen gamma chain	48.5	2.28	0.000	221.5	45.0
1832656769	vesicle-associated membrane protein 2	12	3.11	0.000	236.3	18.4
380006108	MHC class II antigen	10.2	3.19	0.000	112.0	100.2
1832658469	fibrinogen beta chain	40.2	2.36	0.000	224.7	47.6
1379027526	ribonuclease G	55.4	3.19	0.000	141.6	15.5
2022331198	immunoglobulin M heavy-chain constant mu variant 2	50.3	2.52	0.000	198.7	48.2
158602734	immunoglobulin light chain	26.4	3.6	0.000	172.9	15.8
1832627647	PTTG1-interacting protein a	18.8	3.53	0.000	122.5	30.5
1832677686	NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial	19	4.01	0.000	198.3	12.3
306008591	heat shock protein	61.2	6.64	0.000	215.7
328677149	hypothetical protein	33.1	6.64	0.000	148.8	18.3
343459175	ATP synthase, H+ transporting, mitochondrial F0 complex	18.2	6.64	0.000	143.1
1832685063	hydroperoxide isomerase ALOXE3	82.7	6.64	0.000	300.0
1832630592	glucosidase 2 subunit beta	59.5	6.64	0.000	148.2
1832644368	peroxiredoxin-6	24.6	6.64	0.000	300.0
1832669039	aldo-keto reductase family 1 member B1 isoform X1	35.7	6.64	0.000	194.2
1832638111	hyaluronan and proteoglycan link protein 1	40	6.64	0.000	28.3
1832616138	testin	62.2	6.64	0.000	227.8
1832670971	LSM8 homolog, U6 small nuclear RNA associated	10.4	6.64	0.000	300.0
1832638785	leucine-rich alpha-2-glycoprotein	38.9	6.64	0.000	300.0
1832604382	histone H1	27.4	6.64	0.000	251.7
327239696	complement component C6	11.4	6.64	0.000	266.2	19.4
1832664977	cytochrome c oxidase subunit 6B1	10.2	6.64	0.000	300.0
1832620903	cystatin B	11.9	6.64	0.000	192.7	89.8
1832692739	protein SSUH2 homolog	39.7	6.64	0.000	234.0
834904842	phosphoglycerate kinase	41	6.64	0.000	14.0
1832656308	neural cell adhesion molecule 1a isoform X4	109.2	6.64	0.000	130.5	14.2
1832670505	coatomer subunit gamma-2	97.4	6.64	0.000	260.0	17.8
1832612090	heat shock 70 kDa protein 4b	95.1	6.64	0.000	300.0
1832640826	papilin b, proteoglycan-like sulfated glycoprotein	63	6.64	0.000	83.8	54.5
1832641526	uroporphyrinogen decarboxylase	41.3	6.64	0.000	142.3
1042045193	glycine dehydrogenase (aminomethyl-transferring)	104.1	6.64	0.000	300.0
1832673253	60S ribosomal protein L8	28	6.64	0.000	198.2
834904288	TDP-fucosamine acetyltransferase	25.4	6.64	0.000	94.0	6.5
1832638138	transcription factor BTF3	17.7	6.64	0.000	205.7	14.6
1832643512	rho GTPase-activating protein 12-like isoform X1	100.9	6.64	0.000	170.3
1832644997	coatomer subunit beta’ isoform X1	105.9	6.64	0.000	229.5
1832627111	nesprin-2-like isoform X4	250.6	6.64	0.000	300.0

), wherein 28 upregulated and 66 downregulated proteins were recognized in the resistant group. Based on the list of DEPs, the immune changes in V. alginolyticus infection could be deciphered. These can be seen through the alteration of the expression of an immune-related protein, namely Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1, Annexin A1, 3-hydroxybutyrate dehydrogenase type 2 and L rhamnose-binding lectin SML.

3.4. Gene Ontology Analysis of DEPs

In order to gain further insight into the DEPs, they were categorized according to their biological process, molecular and cellular functions (Figure 4). The number of proteins annotated in most of the GO terms for the resistant group was relatively higher than the susceptible group, except for the developmental process (GO: 0032502), ATP-dependent activity (GO: 0140657), molecular function regulator (GO: 0098772) and transporter activity (GO: 0005215). The five GO terms annotated with the highest number of proteins were the cellular process, metabolic process, binding, cellular anatomical entity and protein-containing complex.

3.5. KEGG Enrichment

To ascertain the pathway-enriched proteins, the DEPs entailed in different biological pathways were annotated using the KEGG reference database. The DEPs were significantly (p-value ≤ 0.05) enriched in 17 pathways, with most of the DEPs highly associated with metabolic pathways followed by oxidative phosphorylation, glycolysis and carbon metabolism (

Table 5. KEGG pathways enrichment analysis of differentially expressed proteins that were associated with V. alginolyticus experimental infection.

Pathway ID	Description	Protein Count	p-Value
1100	Metabolic pathways	30	0.000
190	Oxidative phosphorylation	9	0.000
10	Glycolysis/Gluconeogenesis	7	0.000
1200	Carbon metabolism	8	0.000
1230	Biosynthesis of amino acids	6	0.000
4260	Cardiac muscle contraction	6	0.000
3010	Ribosome	5	0.001
30	Pentose phosphate pathway	3	0.001
4672	Intestinal immune network for IgA production	3	0.001
51	Fructose and mannose metabolism	3	0.001
260	Glycine, serine and threonine metabolism	3	0.002
4514	Cell adhesion molecules (CAMs)	4	0.005
4145	Phagosome	4	0.007
5168	Herpes simplex virus 1 infection	4	0.014
630	Glyoxylate and dicarboxylate metabolism	2	0.015
270	Cysteine and methionine metabolism	2	0.029
72	Synthesis and degradation of ketone bodies	1	0.047

Protein count is the differentially expressed proteins that were clustered in the significant enriched KEGG pathway.

).

More details of the differentially expressed proteins are presented in the appendix tables.

3.6. Identification of Potential Biomarker Candidates (PBCs)

The potential biomarkers that correspond to the Vibrio-resistant phenotype were identified and listed in

Table 6. Potential biomarker candidates.

Accession	Description	MW (kDa)	FC (S vs. R)	p-Value (S vs. R)	FC (C vs. R)	p-Value (C vs. R)	Abundances Susceptible	Abundances Resistant	Abundance Control
1832634797	transmembrane emp24 domain-containing protein 2 isoform X1	23.3	−6.64	0.00	−6.64	0.00		300.0
1832702196	vasodilator-stimulated phosphoprotein isoform X1	44.6	−6.64	0.00	3.87	0.00		122.5	177.5
1832641369	phosphotriesterase-related protein	38.8	−6.64	0.00	0.89	0.65		159.7	140.3
1832686354	puromycin-sensitive aminopeptidase	104.2	−6.64	0.00	−1.25	0.38		153.1	146.9
1832695641	N-alpha-acetyltransferase 10 isoform X1	24.8	−6.64	0.00	−0.80	0.65		190.7	109.3
1996375967	methionine ABC transporter substrate-binding protein	28.8	−6.64	0.00	1.70	0.13		174.6	125.4
1832682447	dedicator of cytokinesis protein 7 isoform X1	244.1	−6.64	0.00	0.81	0.65		123.6	176.4
1832697234	L-rhamnose-binding lectin SML	23.9	−6.64	0.00	−0.93	0.60		196.5	103.5
1832687225	3-hydroxybutyrate dehydrogenase type 2	26.4	−6.64	0.00	−6.64	0.00		178.5	121.5
1832698896	uncharacterized protein KIAA2013 homolog isoform X1	69.5	−6.64	0.00	0.47	0.78		139.4	160.6
1832690325	uncharacterized protein LOC117256382 isoform X3	72.6	−6.64	0.00	−6.64	0.00		169.6	130.4
1832642084	neurogenic differentiation factor 6-B	37.7	−6.64	0.00	6.64	0.00		107.0	193
1832622809	RWD domain-containing protein 1 isoform X2	23.2	−6.64	0.00	−6.64	0.00		300.0

. Thirteen proteins were found to be significantly altered in the resistant group when compared to the control, whereas no significant difference in these proteins was detected in the susceptible fish. This suggests disease-resistant properties against V. alginolyticus infection could be due to these identified non-invasive biomarkers.

4. Discussion

Skin mucus is known to be the first line of defense against diseases through the non-specific immune system [28]. Through proteome profiling, one can gain an understanding of molecular changes under certain events. Hence, it has been proven to be beneficial in gaining intuition about an organism’s biological response [29,30,31]. However, the immune response of hybrid groupers toward Vibrio sp. infection is poorly understood, owing to the little understanding of the underlying molecular mechanisms. This study assessed the acute inflammatory response of hybrid groupers by proteome analysis on skin mucus which allowed a better understanding of the fish immunity as well as to determine the possible disease biomarkers.

Currently, the execution of Vibrio sp. on hybrid groupers marked a positive effect on the innate immune response. Protein profiling on skin mucus showed several identified proteins in the susceptible group compared to the resistant group, which indicates a positive response to the infection [32]. The majority of DEPs were also found to be perturbed in the resistant and susceptible groups involved in the cellular process based on the GO classification and the metabolic and oxidoreductase phosphorylation activity as in the KEGG enrichment. In fish, skin mucus is crucial in providing respiratory and metabolic function [33]. In this regard, the present study exhibits most of the DEPs being enriched in metabolic pathways which could be classified as enzyme energy metabolisms, such as NADH dehydrogenase and ATP synthase. Moreover, two crucial proteins that may be involved in the pathological immune response, 3-hydroxybutyrate dehydrogenase type 2 (BDH2) and L rhamnose-binding lectin SML (L-RBL), were also observed.

It is notable that the expression of BDH2, a regulatory molecule that was found to be enriched in metabolic pathways and the synthesis and degradation of ketone bodies pathways, is increased only in the resistant group. BDH2 is a key component of ketone bodies, an endogenous molecule that is responsible for maintaining cellular energy and modulating the signaling cascade through the cellular process. The cell can derive energy from BDH2 during disease infection [34,35]. A slight increase in BDH2 in concentrations was modulated by signaling pathways as they became involved in the cell growth, proliferation and oxidative stress resistance [36]. BDH2 can be activated in immune cells that lead to an anti-inflammatory effect [37]. The present findings support that BDH2 is involved in providing the disease-resistant properties of hybrid groupers, which is advantageous as a basis for developing the non-invasive biomarkers.

In addition, the L-RBL protein was also discovered in this study. It was anticipated that this immune-related protein would have a distinct role in providing disease resistance. Focusing on its high expression in resistant fish, it can offer a non-invasive biomarker during the segregation of disease-resistant and -susceptible groupers. L-RBL is a member of the rhamnose-binding lectin proteins (RBLs) that belong to the animal lectin group, a sugar-specific binding protein that recognizes the carbohydrate structure domain. RBLs are widely distributed in viruses, prokaryotes and eucaryotes [38], exclusively found in teleost and aquatic invertebrates which have been previously identified in eggs, serums and skin mucus [39,40,41]. It has been reported that RBLs can provide innate immunity as they inhibit proliferation, cytotoxicity and opsonize nonself-cells and particles [42]. The present study is consistent with the reported data on the clearance of Gram-positive and -negative bacteria by phagocytosis, which is enhanced by the expression of RBLs in sea bass [40].

The proteomic analysis also revealed some immune-related DEPs which are Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1 and Annexin A1. These DEPs have provided us with an understanding of the molecular changes during Vibrio infection.

Cystatin B was found to be upregulated in the infected fish only. This suggests the idea of fish with naive immunity may produce this protein in response to pathogen invasion to maintain their physiological state. The role of Cystatin B is consistent with the previous study [43], as the overexpression of Cystatin B in mice interacted with various types of proteins to activate the JAK/STAT signaling pathway for the removal of pathogens and the killing of infected cells. Cystatin B may also be present and translocated into mitochondria, which helps to protect the mitochondria activity and increase the ROS generation, enhancing the activity of pathogen removal [44,45]. Another protein that shares the same pattern of expression with Cystatin B is Complement component C6 and Complement factor 1. As components of the complement system, they are believed to play an important role in forming the membrane attack complex (MAC) and could assist in the cytolytic killing of the pathogen [46,47].

Other immune-related proteins were also found to be upregulated after infection in the resistant compared to the susceptible group. Allograft inflammatory factor 1 (AIF-1), Deleted in malignant brain tumors protein (DMBT1) and MHC class 1 alpha were detected. In this study, the resistance to infection was associated with high levels of Allograft inflammatory factor 1 (AIF-1) expression. AIF-1 is a 17 kDa type II interferon (IFN-y), for which expression is most likely limited to macrophages and monocytes. The high expression of AIF-1 was previously found in the spleen of E. awaora treated with lipopolysaccharides [48]. The present findings agree with the previous studies wherein AIF-1 was noted to provide innate immunity in various ways. According to a previous report [49], the presence of AIF-1 on the body wall of the infected area showed the migration and recruitment of numerous counts of macrophages after the lipopolysaccharides’ stimulation. AIF-1 is also claimed to be involved in wound healing and shell repair after the tissue injury of abalone [50]. Theoretically, the protein recognition receptor on the surface of Gram-negative bacteria is able to recognize the molecular pattern associated to the pathogen and hence initiate the defense mechanism of the cellular process. AIF-1 also has been proven to be involved in the innate immunity in the head kidney, spleen and various body tissues of vertebrates, but it was first established in the skin mucus of an aquatic organism [50,51,52].

Another detected protein is DMBT1 which belongs to a scavenger receptor protein that can bind to both Gram-negative and -positive bacteria. DMBT1 expression is obvious upon inflammation and epithelial cell differentiation and is involved in innate immunity. This protein was previously found to be upregulated in intestinal epithelial cells after lipopolysaccharide or tumor necrosis factor induction. However, it can also inhibit LPS-induced NF-B activation and cytokine secretion, leading to anti-inflammatory functions that can trigger the acquired immunity later [53,54].

MHC class 1 is a major histocompatibility complex class 1. The overexpression of MHC class I in a resistant group compared to the control that involved cell adhesion molecule and phagosome pathways was observed in the present study. In teleost, MHC class 1 normally displays and presents itself as a surface antigen-presenting cell on an infected cell. Upon entering the environment, an unknown pathogen will infect the cell which will then be degraded by innate immunity. As a result, MHC class 1 is produced within the reticulum and the pathogen is marked to trigger the cytotoxic T-cell wherein this process will help in the killing of the pathogen [55,56]. This finding is in line with the previous report in which the V. alginolyticus was found to be cytotoxic and lethal to fish cell lines and able to degrade the mucus of sea bream [57].

The present finding also reveals AnxA1 as the significantly expressed protein detected in the resistant group after infection. The AnxA1 protein was found to have dual functions in inducing inflammation. Over 100 types of annexins have been discovered in various tissues of vertebrates and invertebrates. The binding properties of this protein are influenced by cytoskeleton interactions, membrane fusion, anticoagulation, intracellular signaling and phospholipase inhibition. Molecular studies of annexin in humans are sparse, but some research could be found on teleost [58,59]. In humans, the expression of AnxA1 is a pro-inflammatory and anti-inflammatory response increased by exogenous and endogenous glucocorticoids, respectively. It is capable of controlling cell apoptosis in various pathways [59,60]. In the infected human, the cell will undergo apoptosis that may cause inflammation in the host [61]. Therefore, AnxA1 serves in apoptotic cell removal by acting as a highly specific signaling ligand of an endogenous danger molecule to enhance phagocytic clearance without causing any inflammation [61,62,63,64,65]. AnxA1 has been detected upregulated in the gill mucus of Atlantic salmon infected with amoebic gill disease with no observed lesion [66]. However, this protein was also expressed in serum and provided immunity to Chinook salmon with skin lesions [60]. The anti-inflammatory role of AnxA1 is still largely unknown, even though its pro-inflammatory-induced protein role is well studied in fish

5. Conclusions

This study has successfully generated new knowledge which strongly suggests that 3-hydroxybutyrate dehydrogenase type 2 and L-rhamnose-binding lectin SML protein, as new non-invasive biomarkers, could be used in the discrimination between Vibrio-resistant and -susceptible groupers. In addition, the biological context exhibited by the mucus is also provided, likely to aid in enhancing the understanding of fish immunity under natural infection. Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1 and Annexin A1 protein are signified as having distinct functions and roles toward V. alginolyticus infection.