MHC polymorphism and disease resistance to Singapore grouper iridovirus (SGIV) in the orange-spotted grouper, Epinephelus coioides

Sci. Bull. (2016) 61(9):693–699 DOI 10.1007/s11434-016-1055-5

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Article

Life & Medical Sciences

MHC polymorphism and disease resistance to Singapore grouper iridovirus (SGIV) in the orange-spotted grouper, Epinephelus coioides Min Yang • Jingguang Wei • Pengfei Li • Shina Wei • Youhua Huang • Qiwei Qin

Received: 14 January 2016 / Revised: 26 February 2016 / Accepted: 3 March 2016 / Published online: 4 April 2016 Ó Science China Press and Springer-Verlag Berlin Heidelberg 2016

Abstract Major histocompatibility complex (MHC) genes are critical members in both innate and adaptive immunity, and the association between their polymorphism and disease resistance has been reported in many teleosts. In the present study, we first investigated the genetic variation at the MHC II b gene in orange-spotted grouper (Epinephelus coioides) after a challenge with Singapore grouper iridovirus (SGIV). The results reveal that a high polymorphism level of the MHC II b gene (H = 1.000; K = 20.206; p = 0.081) and at least three loci exist in grouper. The rate of dN/dS in the peptide-binding region (PBR) and non-PBR were both [1, suggesting the loci were evolving under positive selection. A high ratio of heterozygous individuals (37.26 %) and high rate of dN/dS were discovered, suggesting that both heterozygote advantage and frequency-dependent selection might result in the high polymorphism levels in MHC II b genes in grouper. A total of 33 MHC II b alleles were identified from 40 high-susceptibility (HS) and 40 high-resistance group (HR) individuals, and 15 alleles were used in the association analysis. Three alleles, EPCO-DBB*0302, EPCO-DBB*0307, EPCO-DBB*0603, and EPCODBB*1001 were significantly associated with resistance ability to SGIV, and the EPCO-DBB*0607 and EPCOElectronic supplementary material The online version of this article (doi:10.1007/s11434-016-1055-5) contains supplementary material, which is available to authorized users. M. Yang  J. Wei  P. Li  S. Wei  Y. Huang  Q. Qin (&) Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China e-mail: [email protected] P. Li University of Chinese Academy of Sciences, Beijing 100049, China

DBB*1303 alleles were associated with susceptibility (P \ 0.05). To further confirm the association, another independent challenge experiment was performed. The result of association analysis in the verification test found that only EPCO-DBB*1001 alleles were significantly associated with resistance to SGIV (P \ 0.05), while the other alleles showed no significance (P [ 0.05) in the frequency distribution between HR and HS groups. Therefore, the EPCO-DBB*1001 alleles could be used as a disease resistance-related MHC marker in the molecular marker-assisted selective breeding program of grouper. Keywords Orange-spotted grouper  Major histocompatibility complex II b  Singapore grouper iridovirus  Disease resistance

1 Introduction The major histocompatibility complex (MHC) is a multiple gene family in the vertebral immune system; these genes encode highly polymorphic polypeptides and serve as cell surface receptors in the pathogen-derived peptide-MHC complex. According to character and molecular function, MHC genes are generally divided into two types in fish: MHC class I and MHC class II. The MHC I molecules are commonly expressed in nucleated cells, and bind peptides from intracellular pathogens to CD8? T cells receptors, while the MHC II molecules are commonly expressed by specialized antigen-presenting cells, and bind peptides from extracellular pathogens to helper CD4? T cells receptors [1]. The MHC II molecule contains one a chain and one b chain, these two domains constitute the peptidebinding region (PBR), which forms a closed groove to bind the foreign peptide [2]. Hence, MHC class II genes are

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considered to be intimately related to disease resistance in vertebrates [3, 4]. MHC II genes are the most variable functional genes known to date; each MHC molecule has the ability to recognize and present short peptides and activate specific immune responses. The polymorphism of MHC genes is displayed not only in allelic diversity at a single locus, but also in gene duplication [5]. Among the PBR sites of the MHC molecule, a single amino acid mutation may directly affect the binding ability to certain pathogen peptides. Therefore, variations of the PBR sites can lead to higher resistance or susceptibility to disease. The association between MHC polymorphism and resistance or susceptibility to many diseases have been described in several species of teleosts, such as Atlantic salmon [6], common carp [7], grass carp [8], Japanese flounder [9], turbot [10], half smooth tongue sole [11] and Nile tilapia [12]. Orange-spotted grouper, Epinephelus coioides, is one of the most important seawater aquaculture species in China and Southeast Asian countries, for its taste and nutritional value [13]. In recent years, the grouper aquaculture industry has been threatened by frequent outbreaks of viral disease. Singapore grouper iridovirus (SGIV), first isolated from diseased groupers, is one of the major pathogens [14]. To solve this problem, chemical therapeutics such as antibiotics is used in the process of cultivation. However, this method will usually lead to food safety problems. The disease resistant breeding program based on marker-assisted selection (MAS) seems to be an effective approach to control the threat of disease. The MHC II genes are candidate gene markers for disease-resistant breeding programs [15, 16]. In this study, we analyzed the polymorphism of exon 2 of the MHC IIb gene in orangespotted grouper which were challenged with SGIV; as the exon 2 region is the most polymorphic fragment among the whole MHC IIb gene sequence. This study has the following objectives: (1) to estimate the genetic variation of MHC IIb gene in grouper infected with SGIV; and (2) to investigate the association between MHC allele diversity and resistance or susceptibility to SGIV in grouper populations.

2 Materials and methods 2.1 Fish and virus challenge Grouper (E. coioides) with a body weight of 40–60 g and body length 10–14 cm were collected from Leizhou Aqua Culture Farm in Zhanjiang city (Guangdong Province, China). The fish were acclimated in a recirculating aquaculture system for 7 d; the temperature was maintained in the range of 25–30 °C. All the fish were fed with a diet of 3 % body weight daily, and the

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dissolved oxygen, pH and ammonia concentration of the sea water were closely monitored daily. To obtain a concentration of SGIV which could cause 50 %–80 % mortality, different concentrations of the virus were tested on fish of the same size as a pre-challenge experiment. Five groups (15 fish in each group) were used in this pre-challenge experiment; one of them was a control group. During the 2 weeks of the pre-challenge experiment, the mortality of each group was recorded every day. A SGIV concentration of 1 9 106 TCID50/mL which caused a mortality of 65 % was considered as the satisfactory lethal concentration for the formal challenge experiment. In the challenge experiment, 240 fish were intraperitoneally injected with 100 lL (1 9 106 TCID50/mL) SGIV suspension, 30 fish were intraperitoneally injected with 100 lL saline (0.9 %) as the control group. After the treatment, all the fish were returned to seawater tanks and then reared for 2 weeks. Mortality was calculated daily during this period, and no fish died in the control group. 2.2 Sample collection and DNA isolation The fish that died in the first 120 h during the challenge experiment were defined as the high-susceptibility (HS) group; the fish still alive after the end of the challenge experiment were defined as the high-resistance (HR) group. Fin clips of the two groups were collected and stored in 95 % ethanol (samples were collected at individual levels). Genomic DNA was extracted from grouper fin samples using a EzgeneTM tissue gDNA kit (Biomiga, USA), according to the manufacturer’s instructions. The quality of genomic DNA was estimated in 1 % agarose gel by horizontal electrophoresis. All the genomic DNA samples were adjusted to 100 ng/lL, then stored at -20 °C for later use. 2.3 Primer design and PCR amplification A pair of specific primers, (OSPMF: 50 -TGTTTTTGTTCC TCAGATGGATTTCC-30 , OSPMR: 50 -ATCAATAAAGA CCGTCAGATGAATC-30 ) were designed according to the E. coioides MHC II b gene sequence in the NCBI database (GenBank accession number FJ598317). This primer pair was designed to amplify part of intron 1, exon 2 and part of intron 2. The PCR reaction system contained 36.5 lL H2O, 5 lL MgCl2 (25 mmol/L), 5 lL 109 PCR buffer, 1 lL dNTP (10 mmol/L each), 1 lL each primer (20 lmol/L), 0.5 lL (5 U/lL) Taq polymerase (TaKaRa Biotech, Japan), and 1 lL DNA template in a final volume of 10 lL. The PCR conditions were 94 °C for 4 min, followed by 35 cycles at 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 45 s with a final elongation step of 10 min at 72 °C. PCR products were analyzed on 1 % agarose gel by electrophoresis and purified using a PCR purification kit

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(TaKaRa Biotech). The PCR purified products were cloned into a pMD-18T vector (TaKaRa Biotech), and five clones from each individual were sequenced using the ABI 3730 automated sequencer. 2.4 Sequence alignment and statistical analysis The alignments of sequence data were analyzed by Clustal W software [17]. The DNAsp 5.0 software package was used to analyze the polymorphism of these sequence data [18]. The rates of synonymous substitution (dS) and nonsynonymous substitution (dN) were calculated by the software MEGA 5.0 using the Nei and Gojobori method [19]. The v2 test was used to analyze the significance of the MHC IIb allele frequency between HS and HR groups by the SPSS 13.0 software (IBM, USA). The difference was considered statistically significant at P \ 0.05. 2.5 Confirmation of significant alleles in another independent population Another independent challenge experiment was performed to further confirm the association between specific alleles and the susceptibility or resistance to SGIV. Three hundred grouper from the grouper aquaculture farm of Wenchang City in Hainan Province, China were collected and maintained in the same conditions as Sect. 2.1, with an average weight of 50 g and an average length of 12 cm. The same challenge treatment to SGIV was taken to acquire new susceptible and resistant groups. Forty fish that died first were regarded as the Wenchang HS (WC-HS) group, 48 fish that survived on day 14 were regarded as the Wenchang HR (WC-HR) group. The PCR system was the same as Sect. 2.3, statistical analysis was the same as Sect. 2.4. 3 Results

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of the expected fragment was about 610 bp based on the sequence alignment with the E. coioides MHC II b gene complete sequence [20], a fragment of about 610 bp spanned the complete exon 2 of MHC IIb, partial intron 1 (31 bp), intron 2 (about 273 bp, existing as a polymorphic dinucleotide (TG)n microsatellite repeat), and a 34 bp fragment. Four hundred fragments of 251 bp of exon 2 of the MHC II b gene were subsequently used in the analysis. These fragments revealed 33 different alleles, which belong to 13 major allele types following established allele nomenclature methods (Table S1) [21, 22]. The polymorphic values were calculated by DnaSP v5.0 software, the average number of nucleotide diversity pi (p) of the 33 sequences was 0.081, and the Theta–W value per site was 0.076. The haplotype diversity (H) was 1.0, the number of variable sites was 77 (30.7 %) and the average number of nucleotide differences (K) was 20.206, these values revealing a high level of nucleotide diversity in the grouper MHC II b gene. Figure 1 displays the spatial distribution of the nucleotide diversity in the MHC II b exon 2, the peak appeared at the downstream of the sequences respectively, indicating the hot mutation regions of the sequences. The full alignment of the 251 bp exon 2 of the MHC IIb gene did not show any gaps. The nucleotide sequence was deduced to be an 82 amino acid peptide according to the full length of the grouper MHC IIb cDNA sequence reported by Lu et al. [20]. Analysis of all the amino acid sequences showed that 52.44 % (43 out of 82) amino acid site were variable (Fig. 2). There were 78 SNP variation sites in 67 regions (Table S2), the substitution types of these SNP sites including both transitions and transversions. Three or four kinds of mutation per site were observed in positions 37, 124. 133, 134, 166, 167, 173, 200, 209, 221 revealing the mutation hotspots. Eleven out of 67 mutation regions were

3.1 Identification of susceptible and resistant groupers In the virus challenge experiment, the first dead grouper was observed at 72 h after the SGIV challenge, and in the following period, the mortality rate of groupers increased gradually and reached a peak at 168 h. Forty-six groupers in total survived after SGIV challenge until they were sampled at 324 h, these fish were selected as HR individuals. And 46 groupers that died in the first 120 h were selected as HS individuals. During the whole period, all groupers in the control group were still alive. 3.2 Polymorphisms of MHC II b exon 2 in grouper Forty HS and forty HR individuals were analyzed, and five positive clones were each sequenced individually. The size

Fig. 1 The nucleotide diversity within exon 2 sequences of MHC IIb genes at the 33 alleles denoted by Theta–W. Sliding window length: 50; step size 25

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Fig. 2 Amino acid polymorphic sites and variation within exon 2 of the 33 MHC IIb alleles in orange-spotted grouper. Dots indicates identical amino acid; asterisks indicated the codons involved in antigen binding region

multi-nucleotide commutations, ranging from two nucleotides per region. The frequency ratio of SNP types ranged from 0.030 to 0.970. The PBR sites in grouper MHC II b were based on the corresponding peptide binding sites identified in humans [23]. The 20 positions were used as PBRs for in-depth study: 12, 14, 16, 21, 22, 30, 39, 43, 44, 48, 52, 54, 57, 61, 64, 65, 68, 69, 71, 72 (Fig. 2). In the putative PBR, the dN was 1.59 times higher than the dS. And the rate of dN was 1.55 times higher than the dS in the non-PBR region. In the complete MHC II b exon 2 sequence, the ratio of dN/dS was 3.28. 3.3 The association between alleles and resistance to SGIV Five positive clones, each from individuals in the HS and HR groups, were sequenced. The numbers of alleles from each individual ranged from 1 to 5, indicating at least five alleles and three loci of the MHC IIb gene exist in E. coioides. Among the HS and HR groups, 32 out of 80 (37.26 %) individuals were heterozygous in MHC II b exon 2.

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A total of 33 different alleles were identified from the 80 individuals, their distribution numbers and frequencies in each group are displayed in supplementary material (Table S3). The allele frequencies were distributed unequally, some alleles were low frequency or only existed in one group, these alleles were not suitable to statistical requirements and were excluded from the association analysis. Fifteen alleles with relatively high frequencies were then used for subsequent distribution analysis (Fig. 3). The alleles EPCO-DBB*0302, EPCO-DBB*0307, EPCO-DBB*0603, and EPCO-DBB*1001 were significantly more frequent in individuals of the HS group (P = 0.035, 0.002, 0.007 and 0.023, respectively, n = 80), while the alleles EPCO-DBB*0607 and EPCO-DBB*1303 were significantly more frequent in individuals from the HR group than in individuals from HS families (P = 0.007 and 0.003, n = 80). From this result we deduced that the EPCO-DBB*0302, EPCO-DBB*0307, EPCO-DBB*0603, and EPCO-DBB*1001 alleles were associated with the ability of resistance to SGIV in grouper, and the EPCODBB*0607 and EPCO-DBB*1303 alleles were associated with susceptibility.

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Fig. 3 Distribution of MHC II b alleles in high-resistance group (HR) and high-susceptibility group (HS) of orange-spotted grouper during challenge test. The asterisk indicated statistically significant difference (P \ 0.05)

3.4 Confirmation of the association between alleles and the susceptibility or resistance to SGIV In order to detect whether the association between the suggested alleles and the susceptibility or resistance to SGIV is universal or not, another independent challenge experiment was performed. The cumulative mortality rate of the Wenchang population was 71 %, the daily mortality of this population was similar to values in the Zhangjiang population (the population which was tested in Sect. 2.1). The frequent distribution of inferred associated alleles between the WC-HS group and WC-HR group were showed in supplementary materials (Table S4). The results of a v2 test showed that the allele frequency of EPCO-DBB*1001 was significantly higher in the HR group than in the HS group (P = 0.002), while the frequency of other alleles showed no significant between the two groups (P [ 0.05) (Fig. 4).

Fig. 4 Distribution of significant MHC II b alleles in Wenchang high-resistance group (WC-HR) and high-susceptibility group (WCHS) of orange-spotted grouper during independent challenge test. The asterisk indicated statistically significant difference (P \ 0.05)

4 Discussion Selective breeding of resistant strains is one of the potential solutions to disease control in aquaculture. The MHC genes have demonstrated to be associated with resistance to a variety of diseases in teleosts [8, 11, 24–26]. In the present study, we detected the polymorphism of MHC IIb exon 2 in grouper populations which were challenged with SGIV, and examined differences in the allele frequencies between the HS and HR group. To our knowledge, this is the first study on the association between MHC II b polymorphism and disease resistance or susceptibility to a viral pathogen in grouper. 4.1 Polymorphism and evolution mechanism of grouper MHC II b alleles The sequence analysis of 33 MHC II b alleles revealed a high level of polymorphism in grouper (H = 1.000; K = 20.206; p = 0.081), this is similar to the polymorphism of MHC II b in Atlantic salmon and cyprinid fish [27, 28]. The dN/dS ratio (x) is a common parameter used to detect which selection pattern have acted on protein coding genes. A ratio of x [ 1 suggesting that the coding region was positively selected. And x = 1 is interpreted to be caused by neutral amino acid changes, while purify selection will result in a ratio of x \ 1 [29]. In this study, the values of x in PBR and non-PBR were both [1, suggesting the loci were evolving under positive selection. The position of PBR sites in fish MHC genes has not been defined, they are generally indentified by comparing with the HLA II molecule in humans [28]. The PBR sites of the MHC II b gene in the present study were also identified by this method, and with the MHC II b gene in half-smooth tongue sole [23].

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Many hypotheses predict that pathogen-driven balancing selection is the main reason to maintain high levels of genetic diversity in MHC genes [30]. It functions in two ways: (1) heterozygote advantage and (2) negative frequency-dependent selection [31]. In the present study, the high ratio of heterozygous individuals (37.26 %) and high rate of dN/dS revealed that ways result in high polymorphism levels in MHC II b genes in grouper. 4.2 Association of MHC class II b alleles with resistance or susceptibility to SGIV The association between MHC genetic polymorphisms and disease resistance or susceptibility has been reported in many teleosts, such as Atlantic salmon [32] and common carp [6]. In the present study, the analysis results of the first challenge experiment and the confirmation challenge experiment showed, remarkably, that EPCO-DBB*0302, EPCO-DBB*0307, EPCO-DBB*0603 and EPCODBB*1001 alleles were significantly associated with resistance to SGIV, as well as a significant associations between the EPCO-DBB*0607, EPCO-DBB*0307 alleles and susceptibility to SGIV. And the second challenge experiment confirmed that the EPCO-DBB*1001 allele was associated with resistance to SGIV. Therefore, the EPCO-DBB*1001 alleles could be used for a disease resistance-related MHC marker in the molecular MAS breeding program of grouper. The next part of our study will be to construct grouper families, then to detect the associate alleles in these families. In addition, the study will deal with single viruses, other viruses or bacteria that are likely to be associated with other MHC alleles. There are many different strains within the species iridovirus [33], and further studies will detect whether the associated alleles are also associated with resistance or susceptibility to other species of iridovirus. Acknowledgments This work was supported by China Postdoctoral Science Foundation Funded Project (2015M572380) and National Basic Research Program of China (973) (2012CB114402). Compliance with ethical standards Conflict of interest of interest.

The authors declare that they have no conflict

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