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Pathogen resistance and gene frequency stability of major histocompatibility complex class IIB alleles in the giant spiny frog Quasipaa spinosa
Aquaculture 468 (2017) 410–416
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Pathogen resistance and gene frequency stability of major histocompatibility complex class IIB alleles in the giant spiny frog Quasipaa spinosa Wenfang Hu a, Baojuan Dong a, Shenshen Kong a, Yuanyuan Mao a, Rongquan Zheng a,b,⁎ a b
Institute of Ecology, Zhejiang Normal University, Jinhua, Zhejiang 321004, China Key Lab of Wildlife Biotechnology and Conservation and Utilization of Zhejiang Province, Jinhua, Zhejiang 321004, China
a r t i c l e
i n f o
Article history: Received 29 December 2015 Received in revised form 28 October 2016 Accepted 1 November 2016 Available online 03 November 2016 Keywords: Quasipaa spinosa Major histocompatibility complex Pathogen resistance
a b s t r a c t The major histocompatibility complex (MHC) is a highly polymorphic genomic region, which widely exists in vertebrates and plays a pivotal role in immune response. As such, the association between MHC alleles and pathogen resistance has been investigated in many vertebrates. However, limited information is reported about the heredity of alleles associated with disease resistance, and the related research has not been reported in amphibians. In this study, PCR primers were developed for a portion of the second exon of the MHC class II B gene in the giant spiny frog Quasipaa spinosa, which is an important aquaculture species with high economical value in China. 31 MHC class II B alleles were identified from 100 individuals intraperitoneally injected with Aeromonas hydrophila. We selected six high-frequency alleles that exist in death and survival to investigate the association between MHC class II B alleles and disease resistance/susceptibility. Q. spinosa with qasp-DAB*h and qasp-DAB*l alleles exhibited a higher resistance to A. hydrophila than Q. spinosa with other alleles. By contrast, Q. spinosa with qasp-DAB*e alleles displayed a higher susceptibility to A. hydrophila than Q. spinosa with other alleles. We also determined an array of immunological and haematological parameters of Q. spinosa offspring after this organism was intraperitoneally injected with A. hydrophila, Acinetobacter baumannii and Citrobacter braakii. We found that the resistant offspring group showed higher immunity than the control offspring group. Twenty-six MHC class II B alleles were identified from 100 resistant offspring individuals. Two alleles qasp-DAB*0701 and qasp-DAB*1001 that is same to qasp-DAB*h and qaspDAB*l of parents were also determined in the resistant offspring. The constant transmission of the qasp-DAB*h and qasp-DAB*l alleles were observed. This study confirmed the association between MHC class II B gene alleles and disease resistance. This study also detected the two alleles correlated with high resistance to bacterial infection in Q. spinosa parents and offspring. Statement of relevance: Q. spinosa is highly valued in Chinese markets because of its medicinal and nutritional values. The paper plays an important role on the selective breeding of resistant and genetically stable Q. spinosa strains. © 2016 Published by Elsevier B.V.
1. Introduction The giant spiny frog (Quasipaa spinosa) is distributed in China and Vietnam, particularly in southern and southeast parts of China. It's an economically important frog aquaculture species in China which is highly valued in Chinese markets because of its nutritional and medicinal values (Ye et al., 2013; Yu et al., 2010; Chen, 2013). For example, 205 giant spiny frog farmers reach a certain scale and achieve an annual production value of 58.0276 million yuan in Zhejiang Province alone (Mei et al., 2015). However, frequent outbreaks of viral and bacterial diseases limit the profitability and development of cultured Q. spinosa. Various diseases, such as ⁎ Corresponding author at: Institute of Ecology, Zhejiang Normal University, Jinhua, Zhejiang 321004, China. E-mail address: [email protected] (R. Zheng).
http://dx.doi.org/10.1016/j.aquaculture.2016.11.001 0044-8486/© 2016 Published by Elsevier B.V.
Valsa sordida Nits, red leg disease and bacterial meningitis, influence the healthy development of the breeding industry of Q. spinosa. Consider to the negative impacts of antibiotic application, such as antibiotic residue accumulation in frogs, environmental pollution antibiotic resistance development and food safety problem (Xu et al., 2010; Zhou et al., 2013; Zhang et al., 2008). Therefore, frog strains with enhanced resistance to major viral and bacterial diseases should be cultured, disease resistance genes should be determined at a molecular level and molecular markerassisted selective breeding should be applied to cultivate resistant Q. spinosa varieties (Yu et al., 2014). The major histocompatibility complex (MHC) is a genomic region with high polymorphism, and it is implicated in innate and adaptive immune responses by presenting self and foreign peptides to T cells, and a specific immune response is initiated in vertebrates (Germain, 1994; Sheng et al., 2011). The major histocompatibility complex genes influence disease
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dynamics for encoding cell-surface glycoproteins, which regulate the acquired immune response in vertebrate organism and affect the occurrence and development of diseases (Bernatchez and Landry, 2003). The major histocompatibility complex genes are classified into three classes, namely, I, II and III, which are genomically linked among tetrapods (Savage and Zamudio, 2011). Antigen-presenting MHC molecules are encoded by classes I and II, which are two distinct gene families in structure and function (Klein and Horejsi, 1997; Marsh et al., 2000). High levels of polymorphism are found in antigen binding sites (ABS) of MHC classes I and II, and these sites are in the most dynamic coding regions of genome (Kelley et al., 2005). Specific MHC alleles of classes I and II genes have been discovered in some species and have been linked to some diseases (Briles et al., 1983; Medina and North, 1998; Paterson et al., 1998; Hill et al., 1991; Flores-Villanueva et al., 2003). The major histocompatibility complex class I genes and class II genes are predominantly implicated in immune responses to intracellular pathogens and extracellular pathogens respectively (Yu et al., 2014). The major histocompatibility complex class II loci are expressed on antigen-presenting cells and epithelial cells, and primarily present peptides derived from extracellular pathogens (Braciale et al., 1987; Kaufman et al., 1985). Therefore, the major histocompatibility complex class II genes are the most likely candidate in bacterial immunity because these genes are the main presenters of extracellular fungal pathogens (Braciale et al., 1987), and the dendritic and Langerhans lymphocytes of major histocompatibility complex class II-expression are also presented in amphibian skin (Du Pasquier and Flajnik, 1990; Carrillo-Farga et al., 1990). Moreover, the exon 2 sequence of the MHC class II B gene has been considered as a candidate molecular marker of the association between MHC class II B alleles and susceptibility/ resistance to diseases because the sequence has a high polymorphism (NikolichŽugich et al., 2004). The major histocompatibility complex genes are extensively investigated regions in vertebrate genomes (Horton et al., 2004) because of their important roles in immunity and fitness; the associations between MHC alleles and disease resistance or susceptibility in a large number of species have been documented (Sommer, 2005; Grimholt et al., 2003; Langefors et al., 2001; Paterson et al., 1998; Savage and Zamudio, 2011). However, research has focused on mammals, birds and fish. In amphibians, the whole MHC loci have been analysed and identified in model organisms, such as Xenopus laevis and Silurana (Xenopus) tropicalis (Kaufman et al., 1985; Sato et al., 1993; Flajnik et al., 1991; Liu et al., 2002). Attention for amphibian immunogenetics has been precipitated because of the emergence of amphibian infectious diseases (Berger et al., 1998; Gray et al., 2009). Studies have focused on the complete molecular structural analysis (Yu et al., 2014; Zeisset and Beebee, 2009), characterisation (Hauswaldt et al., 2007; Kiemnec-Tyburczy et al., 2010; May and Beebee, 2009; Shu et al., 2013; Zeisset and Beebee, 2009), disease association of MHC class II B in non-model amphibian species (Yu et al., 2014; Barribeau et al., 2008; Savage and Zamudio, 2011; Bataille et al., 2015). However, the disease association combine with genetic stability of MHC class II B in amphibians have been rarely investigated. In this study, the association between specific MHC class II B alleles and disease resistance to bacterial infections was detected in Q. spinosa. The immunity and disease resistance of Q. spinosa offspring were determined. The frequency of MHC class II B was also verified to evaluate the frequency stability of specific MHC class II B alleles between parent and next generation. This study helps elucidate MHC as a molecular marker of breeding. This study also provides a basis to improve disease resistance by applying molecular techniques and by selectively breeding resistant and genetically stable Q. spinosa strains. 2. Materials and methods 2.1. Animals A total of 100 individuals provided by an experimental aquaculture farm (Lanxi, China) were exposed to Aeromonas hydrophila to induce
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infection. After the infection experiment was completed, the surviving individuals were cultivated and reproduced in the farm. A total of about 5600 frog spawns were successfully obtained, 150 individuals were randomly selected from offspring (60 ± 10 g) and divide into three groups for A. hydrophila, Acinetobacter baumannii and Citrobacter braakii infection experiments. A. hydrophila strain QuSp1, Acinetobacter baumannii strain QuSp2 and Citrobacter braakii strain QuSp3 were isolated from an infected Q. spinosa individual collected from a farm in 2013.
2.2. Animal challenge The experimental frogs were temporarily placed in new rearing pond with a fresh water supply at 20 ± 0.5 °C (PH 6.7 ± 0.5) for a week to adapt to the surrounding environment before artificial infection was induced. In the infection experiment with A. hydrophila, the test individuals were inoculated with A. hydrophila were infected by intraperitoneal injection as described previously (Cosma et al., 2006) with 0.45 mm syringe needle. Control group trials were prepared under the same conditions. Different A. hydrophila concentrations were examined in a pre-challenge experiment with same-sized frogs as test organisms to determine the infective concentration. The median lethal concentration was determined according to Xu (Xu et al., 2008). The results revealed that the infective concentration was 4 × 107 cfu/ml. 100 individuals approximately weighing 200 ± 50 g were inoculated intraperitoneally with 0.2 ml of A. hydrophila colony forming units. The control group was inoculated intraperitoneally with the same dose of 0.65% sterile saline solution. The test lasted 18 days, and mortality and active state were recorded every 6 h for the succeeding test period. All of the dead individuals were stored at −20 °C. The dead individuals were considered to be susceptible organisms, and the surviving frogs were considered as disease-resistant individuals. Male and female surviving individuals were all selected as parental frogs by haphazard mating to produce offspring in an aquaculture farm. For the bacterial challenge of the offspring, the median lethal concentration was determined also according to Xu (Xu et al., 2008). Three bacteria, namely, A. hydrophila, Acinetobacter baumannii and Citrobacter braakii which isolated by our laboratory, were used for our challenge experiment. Bacteriums were inoculated on nutrient agar. Bacterial cells were obtained in sterile saline solution after overnight growth at 37 °C. The densities of A. hydrophila, Acinetobacter baumannii and Citrobacter braakin suspensions were respectively adjusted to 4 × 107, 1 × 108 and 1 × 109 cfu/ml, which were determined by colony counting. The 150 offsprings were divided into three groups and injected with 0.05 ml of each bacterial suspension (50 individuals per bacterium), and placed in different and separated rearing ponds. The control group comprised 150 ordinary and contemporary Q. spinosa, which were injected with same dose of bacterial suspension. In order to eliminate the wound affect individual normal breeding after injection, the blank control group was consisted of 150 ordinary and contemporary Q. spinosa, which were injected with the same dose of sterile saline solution. The test lasted 18 days.The survival rate was calculated at the end of experiment.
2.3. DNA extraction Genomic DNA was extracted from muscle or toe clip samples of 200 individuals (100 individuals of parent and 100 of offspring) through phenol-chloroform extraction in accordance with previously described methods (Ye et al., 2013). The quality and concentration of DNA were assessed through agarose gel electrophoresis. These parameters were determined by using a DNA spectrophotometer. The DNA was stored at − 20 °C for future use.
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2.4. Polymerase chain reaction (PCR) amplification and purification
3. Results
On the basis of the sequence primers of X. laevis (Hauswaldt et al., 2007), we used a degenerate MHC class II B forward primer MHC-F CCSCAGAKGATTWCGTGWMTCA and a reverse primer MHC-5R TGTCTGCAGACTGTYTCCACCHCAGCC to amplify exon 2. Polymerase chain reaction (PCR) was conducted to amplify exon 2 of the MHC class II B gene in a 25 μl reaction mixture under the following conditions: initial denaturation for 5 min at 95 °C; 34 cycles of denaturation for 30 s at 95 °C, annealing for 1 min at 58 °C and extension for 40s at 72 °C; and final extension for 10 min at 72 °C. PCR products were assessed through 1% agarose gel electrophoresis. The PCR products were then purified by using a Wizard PCR Preps DNA purification kit (Promega).The DNA data of 200 individuals (100 individuals of parent and 100 of offspring) were used for PCR and sequencing.
3.1. MHC II B exon 2 alleles in parents
2.5. Cloning and sequencing The PCR products were cloned into a pMD18-T vector (TaKaRa), which was subsequently used to transform Escherichia coli DH5α competent cells. Five to eight colonies from each transformed cell were selected to identify MHC class II B alleles in 100 individuals. Positive clones were screened through PCR with M13± primers. An average of 4 clones per individual was sequenced using an ABI 3730 automated sequencer with the M13 primer. 2.6. Sequence analysis We used translated amino acid queries in GenBank to confirm the MHC class II B homology. The alignment of the deduced amino acid sequence of the MHC class II B peptide was performed using Clustalx and DNAstar (http://www.dnastar.com). 2.7. Blood sampling and immune analysis After the offspring were subjected to bacterial challenge, 100 individuals were chosen randomly from surviving individuals (50 experimental group and 50 from control group) to collect blood sample. Afterward, 1.5–2 ml of blood sample was obtained from 100 individual frogs from each treatment via a puncture of the sciatic artery. The control group comprised ordinary and contemporary giant spiny frogs. Heparinised blood was used to determine haematological and immunological parameters. Haematological analyses included total and differential leucocyte counts and erythrocyte counts; immunological parameters consisted of the phagocytic capacity and phagocytic index of peritoneal phagocytes. 1.5–2.0 ml of blood were collected for the determination of several haematological parameters: the number of erythrocytes was calculated using a Neubauer chamber, and the erythrocyte were counted as described by Wintrobe (1933). Leucocyte counts were obtained from the smears stained with May-Grünwald and Giemsa in accordance with the methods described by Rosenfeld (1947). Phagocytes were calculated by a phase-contrast microscope. The phagocytic capacity was measured in accordance with the method described by Silva et al. (2002, 2005). The phagocytic index (PI) were calculated using the following equations (Zhang et al., 2008): PI = N1 / 100 × 100%, where N1 is the total number of bacteria engulfed by 100 random phagocytes. 2.8. Statistical analysis Data were expressed as means ± SE. Statistical analysis was performed in SPSS18.0. Different bacteria infected survival rate between experiment group and control group and allele frequency discrepancies were verified through Chi-square test. U test was conducted to analyze the trend in the changes in immune parameters. Differences were considered significant at P b 0.05.
After 100 individual parents were subjected to A. hydrophila challenge, 47 individuals survived, and the dead individuals appeared corresponding symptoms that infected with A. hydrophila disease. No mortality was observed in the control frogs. 27 females and 20 males of 47 surviving individuals used as parents by haphazard mating to produce offspring, and 20 females and 20 males contributed to offspring groups which could be ensured from the number and location of the frogs' spawns. The genomic DNA from 47 resistant frog and 53 susceptible individuals was amplified using a pair of primers, namely, MHC-F and MHC-5R. Afterward, 186 bp MHC II B exon 2 sequence was obtained. According to the accepted nomenclature rules, these alleles were designated as qasp-DAB*a to qasp-DAB*v. 31 distinct amino acid sequences of Q. spinosa MHC class II B is shown in Fig.1. Alleles that differ by less than five amino acid substitutions are considered as subtype within a single major type (Xu et al., 2007; Xu et al., 2005). Among these sequences, 16 were the same as those in a previous report (Yu et al., 2014). The 16 sequences include qasp-DAB*a, qasp-DAB*c, qaspDAB*e, qasp-DAB*g, qasp-DAB*h, qasp-DAB*i, qasp-DAB*l, qaspDAB*n1, qasp-DAB*n2, qasp-DAB*p1, qasp-DAB*p2, qasp-DAB*p3, qasp-DAB*p4, qasp-DAB*s, qasp-DAB*u and qasp-DAB*v. Furthermore, 15 sequences were newly discovered in this study. 3.2. Association between allele frequency and resistance/susceptibility to A. hydrophila A total of 31 different MHC II exon 2 alleles were identified from the sampled individuals. The frequency of alleles was not distributed equally. Six high-frequency alleles were selected to investigate the association between MHC class II B allele polymorphism and disease resistance/susceptibility. The frequencies of qasp-DAB*e (χ2 = 8.999, df = 1, P = 0.025), qasp-DAB*h (χ2 = 15.468, df = 1, P = 0.001) and qasp-DAB*l (χ2 = 16.329, df = 1, P = 0.001) were significantly different between the dead individuals (Dd) and the surviving individuals (Fig. 2). qasp-DAB*h and qasp-DAB*l may be associated with the increased resistance to A. hydrophila of Q. spinosa (Sv). qasp-DAB*e may be associated with the susceptibility to A. hydrophila of Q. spinosa (Dd). 3.3. Disease resistance and immunity of the next generation After the pathogenic bacterial challenge was conducted in offsprings, the control Q. spinosa manifested various symptoms, such as poor activity and appetite loss. The experimental Q. spinosa also displayed these symptoms, but these symptoms appeared at a later time than those of the control group. The survival rate of the resistant group was higher than that of the control group (Table 1). The survival rate of the blank control group was 100%. There is a significantly difference between the resistant groups and control group which were injected with A. hydrophila (χ2 = 3.841 , df = 1, P = 0.035), The groups are also with significantly difference which were injected with Citrobacter braakii (χ2 = 4.233, df = 1, P = 0.023). The survival rate of the blank control group was 100%. The blood samples obtained from the resistant group and the control group were evaluated to determine the immune status of the next generation. Haematological and immunological parameters were detected in the resistant offspring and the control Q. spinosa (Table 2). The numbers of leucocytes in blood of bacterium-injected offspring were observed to be significantly higher than those in the control group (u = 11.913, df = 1, P = 0.002), and a sharp increase in the number of erythrocyte in bacterium-injected offspring was observed compared with control group (u = 34.474, df = 1, P = 0.001).There was a slight increase in the phagocytosis activity and phagocytic index of bacteriuminjected offspring, so the phagocytosis of phagocytes of Q. spinosa in
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Fig. 1. Putative amino acid sequences for MHCIIB exon2 alleles of Q. spinosa. Dots indicate identity with the top sequences; the putative peptide binding region is indicated with asterisks.
the resistant offspring group was stronger than those in the control group. 3.4. Inheritance of the alleles in the next generation In this study, 300 MHC class II nucleotide sequences and 24 distinct MHC II exon 2 alleles were identified from the 100 randomly selected offspring of the disease-resistant individuals (Fig.3). The full alignment of 186 bp exon 2 of the MHC II B gene did not show any gap with the
aligned MHC II exon 2 alleles of their parent (Fig. 1). A total of 24 distinct MHC II exon 2 alleles were found in the parents. qasp-DAB*h and qaspDAB*l were associated with the disease resistance allele of the parents; these alleles also corresponded to qasp-DAB*0701 and qasp-DAB*1001, respectively. This finding revealed that the MHC II B alleles were transmitted to the progeny and the frequency of the alleles was not distributed equally. To confirm the hereditary stability of the two disease resistance alleles, we calculated the frequency of the alleles. We found that the frequencies of qasp-DAB*l and qasp-DAB*h were 0.19 and 0.15 in the parents. The frequencies of qasp-DAB*1001 and qaspDAB*0701 were 0.57 and 0.46 in the offspring. After Chi-square test, we found frequencies of two alleles were exist highly significant difference (p = 0.001). 4. Discussion
Fig. 2. Distribution of MHC class IIB alleles in dead individuals (Dd) and survival individuals (Sv) of the giant spiny frog. Note: * for p b 0.05; ** for p b 0.01.
The major histocompatibility complex genes have been extensively investigated because these genes are significant elements of the vertebrate immune system. In this study, 31 and 24 different MHC II exon 2 alleles were identified from the 100 parental individuals of Q. spinosa and 100 offspring, respectively. The associations between MHC class II B alleles and A. hydrophila resistance or susceptibility were presented. We also determined some immune parameters and the inheritance of the alleles associated with the disease resistance of their next generation. The major histocompatibility complex allele diversity is critical for resistance against parasites (Wegner et al., 2006). The high polymorphism of MHC genes in animals provides high disease resistance (Xu
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Table 1 The comparison of survival rate in resistant and control group of Q. spinosa. Method
Aeromonas hydrophila injection
group
Before /number
After /number
Survival rate
Before/number
Acinetobacter baumannii injection After/number
Survival rate
Citrobacter braakii injection Before/number
After /number
survival rate
E C
50 50
45 24
90% 48%
50 50
42 26
84% 52%
50 50
40 20
80% 40%
Note: E: Experiment group. C: Control group.
et al., 2008). In our study, sequence analysis revealed the high polymorphism of MHC II B gene in Q. spinosa parents. At least three loci of MHC II B gene were found in each individual. This result is similar to that reported by Chen et al. (2006) and Zhang and Chen (2006). Diseases are major factors seriously influencing the development of aquatic agriculture. Diseases pose a significant threat to commercial production in freshwater aquaculture in terms of the presence of ubiquitous pathogens and their frequent occurrence. A. hydrophila is considered as a secondary pathogen that is likely to infect immunocompromised animals (Barribeau et al., 2008; Taylor et al., 1999). A. hydrophila affects the growth and survival of tadpoles, and MHC mediates these responses (Barribeau et al., 2008; Song et al., 2014). A. hydrophila can also lead to the high mortality of farmed Q. spinosa with epidemic diseases (Ma et al., 2013). In this study, the different allele distributions indicated the association between MHC class II B alleles and resistance to A. hydrophila of Q. spinosa. qasp-DAB*h (χ2 = 15.468, df = 1, P = 0.001) and qasp-DAB*l (χ2 = 16.329, df = 1, P = 0.001) yielded a significantly higher frequency in the Sv individuals than in the Dd individuals. This result possibly demonstrated the association of the allele with the resistance to A. hydrophila of Q. spinosa parents. After sequence alignment was performed, the resistant alleles detected in our study are consistent with those described in a previous study (Yu et al., 2014). qasp-DAB*e alleles (χ2 = 8.999, df = 1, P = 0.025) were also associated with the susceptibility to A. hydrophila. The experimental result of the three bacterial challenges of the disease-resistant Q. spinosa offspring revealed that the immunity to bacterial pathogens of the disease-resistant offspring was significantly higher than that of the control group. The immunity of the resistance groups against different pathogens was not significantly different. Zhao (2010) confirmed that resistance to different pathogenic bacteria is controlled by different alleles. Nevertheless, a specific allele may exhibit resistance to various pathogens. Therefore, the survival of the individuals which was infected with A. hydrophila could be contributed to the resistance to other bacterial pathogens. Non-specific immunity is a natural trait of animals. This trait has evolved during the development of defense mechanisms to resist pathogenic microorganism infection. This trait can also remain stable and be transmitted to progenies. In amphibians, frogs have developed specific immunity (Marchalonis and Edelman, 1966), but specific immune responses are triggered several days or weeks after bacterial invasion occurs. Non-specific immunity plays a major role in the anti-infection immune response in the initial stage. Several immunological and haematological parameters have been introduced to evaluate the non-specific immune response of some aquatic animals to bacterial infection (Zhang et al., 2008; Dias et al., 2009; Yildiz, 1998; Benli and Yildiz,
Table 2 Values of leucocyte counts, erythrocytes counts, phagocytosis activity and phagocytic index of peritoneal phagocytes in the resistant offspring and control group of Q. spinosa. Category leucocyte counts erythrocytes counts phagocytosis activity phagocytic index
n 50 50 50 50
Disease-resistant offspring 3
−3
11.82 ± 0.16 × 10 mm 4.68 ± 0.03 × 104 mm−3 85 ± 0.32% 1.71 ± 0.02%
Note: Mean values and standard error for result.
Control group 9.408 ± 80.128 × 8103 mm−3 3.208 ± 80.038 × 8104 mm−3 71 ± 0.48% 1.56 ± 0.03%
2004). Bacteria-injected promote the transfer and accumulation of leukocytes to the infection site, and the continual proliferation of leucocytes can eliminate invasive bacteria (Lydyard et al., 2000). Increase in the numbers of leukocytes during fish defense against the invasive pathogenic bacteria was well known (Yildiz, 1998; Caruso et al., 2002) and many types of frog leucocytes have pseudopodia to ingest particles (phagocytosis) (Pan, 1999). Though the enhancement in the phagocytosis activity of fish peripheral blood neutrophil during bacterial infection has been reported (Park and Wakabayashi, 1992), erythrocyte, a type of immunocyte, has a great number of receptors of complement fragment (C3b),which can mediate the bonding of erythrocyte with pathogenic granule, and facilitate the bacteria clearing by phagocyte (Passantino et al., 2002). Phagocytes can excrete many types of molecules into blood which kill bacteria directly or indirectly during phagocytosis (Lydyard et al., 2000). So we can determine the animal's immune status by calculating the numbers of related immunocyte. In a previous study on the correlation of MHC genes with immune response, animals with different MHC alleles exhibit various immune responses (Liu, 2003). In our study, the number of erythrocytes, the number of leucocytes and the phagocytosis of phagocytes were verified between the resistant offspring group and the control group. The numbers of leucocytes and erythrocytes in blood of bacterium- injected offspring were observed to be higher than those in the control group, and the phagocytosis activity and phagocytic index of bacterium-injected offspring was increased in various degrees. These parameters significantly differed (P b 0.05) between the treatment groups and the control group. Major histocompatibility complex (MHC) class II antigens can activate different cellular functions in immune cells (Altomonte et al., 1999). We found larger numbers of two kinds of blood cells in injected offspring, but a hypothesis that whether the MHC-related resistance genes influenced the differentiation and expression of the blood cells which needed further study. In this study, the associations between MHC class II B alleles and resistance to bacterial infections were detected in the MHC II exon 2 alleles of the resistant offspring. The qasp-DAB*0701 and qaspDAB*1001 alleles were associated with the resistance to the three pathogens; these alleles corresponded to the qasp-DAB*h and qasp-DAB*l of the parents, respectively. The allele frequencies qasp-DAB*0701and qasp-DAB*1001 in the resistant offspring were 0.46 and 0.57 respectively. No new resistant alleles appeared. The constant heredity of the alleles observed in our study is similar to that reported by Xu et al. (2008). The frequencies of the two disease resistance alleles in the offspring were higher than those of the parents and no new resistant alleles were observed possibly because of the following. (i) The methods of data statistics and analysis. The allele frequency of individual which is lower than 1% of the sequences were eliminated because such alleles are generally consider to be almost impossible to be continued survival in the next generation, but maybe some of those alleles can survival in the next generation. (ii) Considering the condition of that we only determined the first generation and without any new genotype. The high frequency may be a result of sexual selection during mating, that is, individuals may prefer mates with “better alleles.” For instance, female tuco-tucos prefer males with special MHC loci considered as “good genes” (Ana et al., 2012). Therefore, Q. spinosa females may be more attracted to males with a disease-resistant gene than to males without this gene.
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Fig. 3. Putative amino acid sequences for MHC IIB exon2 alleles of Q. spinosa. Dots indicate identity with the top sequences.
The association between genes and diseases can be attributed to the effects of the gene or the linkage disequilibrium of the studied gene with a resistance gene (Langefors et al., 2001; Kjøglum et al., 2005). Nevertheless, another linked gene possibly causes the observed association; in our study, qasp-DAB*h and qasp-DAB*l alleles exhibited a higher resistance to A. hydrophila than other alleles. The qasp-DAB*h and qaspDAB*l alleles were continuously passed on from the parents to the offspring. Thus, our research may be used as a basis to develop disease resistance-related MHC markers for the molecular marker-assisted selective breeding of Q. spinosa with enhanced resistance to diseases caused by bacterial infections. This marker will be used in our future research to select individuals for mating. This study also helps improve the selective breeding of Q. spinosa with enhanced disease resistance. Acknowledgments The research was supported by the National Natural Science Foundation of China (Nos. 31172116 and 31472015), the Major Science and Technology Specific Projects Zhejiang Province, China (No. 2012C12907-9), the Agricultural Science and technology achievement transformation project of China (No. 2014GB2C200199), and Science and Technology Innovation Team of Zhejiang Province, China (No. 2012R10026-07). Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.aquaculture.2016.11.001.
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Pathogen resistance and gene frequency stability of major histocompatibility complex class IIB alleles in the giant spiny frog Quasipaa spinosa Wenfang Hu a, Baojuan Dong a, Shenshen Kong a, Yuanyuan Mao a, Rongquan Zheng a,b,⁎ a b
Institute of Ecology, Zhejiang Normal University, Jinhua, Zhejiang 321004, China Key Lab of Wildlife Biotechnology and Conservation and Utilization of Zhejiang Province, Jinhua, Zhejiang 321004, China
a r t i c l e
i n f o
Article history: Received 29 December 2015 Received in revised form 28 October 2016 Accepted 1 November 2016 Available online 03 November 2016 Keywords: Quasipaa spinosa Major histocompatibility complex Pathogen resistance
a b s t r a c t The major histocompatibility complex (MHC) is a highly polymorphic genomic region, which widely exists in vertebrates and plays a pivotal role in immune response. As such, the association between MHC alleles and pathogen resistance has been investigated in many vertebrates. However, limited information is reported about the heredity of alleles associated with disease resistance, and the related research has not been reported in amphibians. In this study, PCR primers were developed for a portion of the second exon of the MHC class II B gene in the giant spiny frog Quasipaa spinosa, which is an important aquaculture species with high economical value in China. 31 MHC class II B alleles were identified from 100 individuals intraperitoneally injected with Aeromonas hydrophila. We selected six high-frequency alleles that exist in death and survival to investigate the association between MHC class II B alleles and disease resistance/susceptibility. Q. spinosa with qasp-DAB*h and qasp-DAB*l alleles exhibited a higher resistance to A. hydrophila than Q. spinosa with other alleles. By contrast, Q. spinosa with qasp-DAB*e alleles displayed a higher susceptibility to A. hydrophila than Q. spinosa with other alleles. We also determined an array of immunological and haematological parameters of Q. spinosa offspring after this organism was intraperitoneally injected with A. hydrophila, Acinetobacter baumannii and Citrobacter braakii. We found that the resistant offspring group showed higher immunity than the control offspring group. Twenty-six MHC class II B alleles were identified from 100 resistant offspring individuals. Two alleles qasp-DAB*0701 and qasp-DAB*1001 that is same to qasp-DAB*h and qaspDAB*l of parents were also determined in the resistant offspring. The constant transmission of the qasp-DAB*h and qasp-DAB*l alleles were observed. This study confirmed the association between MHC class II B gene alleles and disease resistance. This study also detected the two alleles correlated with high resistance to bacterial infection in Q. spinosa parents and offspring. Statement of relevance: Q. spinosa is highly valued in Chinese markets because of its medicinal and nutritional values. The paper plays an important role on the selective breeding of resistant and genetically stable Q. spinosa strains. © 2016 Published by Elsevier B.V.
1. Introduction The giant spiny frog (Quasipaa spinosa) is distributed in China and Vietnam, particularly in southern and southeast parts of China. It's an economically important frog aquaculture species in China which is highly valued in Chinese markets because of its nutritional and medicinal values (Ye et al., 2013; Yu et al., 2010; Chen, 2013). For example, 205 giant spiny frog farmers reach a certain scale and achieve an annual production value of 58.0276 million yuan in Zhejiang Province alone (Mei et al., 2015). However, frequent outbreaks of viral and bacterial diseases limit the profitability and development of cultured Q. spinosa. Various diseases, such as ⁎ Corresponding author at: Institute of Ecology, Zhejiang Normal University, Jinhua, Zhejiang 321004, China. E-mail address: [email protected] (R. Zheng).
http://dx.doi.org/10.1016/j.aquaculture.2016.11.001 0044-8486/© 2016 Published by Elsevier B.V.
Valsa sordida Nits, red leg disease and bacterial meningitis, influence the healthy development of the breeding industry of Q. spinosa. Consider to the negative impacts of antibiotic application, such as antibiotic residue accumulation in frogs, environmental pollution antibiotic resistance development and food safety problem (Xu et al., 2010; Zhou et al., 2013; Zhang et al., 2008). Therefore, frog strains with enhanced resistance to major viral and bacterial diseases should be cultured, disease resistance genes should be determined at a molecular level and molecular markerassisted selective breeding should be applied to cultivate resistant Q. spinosa varieties (Yu et al., 2014). The major histocompatibility complex (MHC) is a genomic region with high polymorphism, and it is implicated in innate and adaptive immune responses by presenting self and foreign peptides to T cells, and a specific immune response is initiated in vertebrates (Germain, 1994; Sheng et al., 2011). The major histocompatibility complex genes influence disease
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dynamics for encoding cell-surface glycoproteins, which regulate the acquired immune response in vertebrate organism and affect the occurrence and development of diseases (Bernatchez and Landry, 2003). The major histocompatibility complex genes are classified into three classes, namely, I, II and III, which are genomically linked among tetrapods (Savage and Zamudio, 2011). Antigen-presenting MHC molecules are encoded by classes I and II, which are two distinct gene families in structure and function (Klein and Horejsi, 1997; Marsh et al., 2000). High levels of polymorphism are found in antigen binding sites (ABS) of MHC classes I and II, and these sites are in the most dynamic coding regions of genome (Kelley et al., 2005). Specific MHC alleles of classes I and II genes have been discovered in some species and have been linked to some diseases (Briles et al., 1983; Medina and North, 1998; Paterson et al., 1998; Hill et al., 1991; Flores-Villanueva et al., 2003). The major histocompatibility complex class I genes and class II genes are predominantly implicated in immune responses to intracellular pathogens and extracellular pathogens respectively (Yu et al., 2014). The major histocompatibility complex class II loci are expressed on antigen-presenting cells and epithelial cells, and primarily present peptides derived from extracellular pathogens (Braciale et al., 1987; Kaufman et al., 1985). Therefore, the major histocompatibility complex class II genes are the most likely candidate in bacterial immunity because these genes are the main presenters of extracellular fungal pathogens (Braciale et al., 1987), and the dendritic and Langerhans lymphocytes of major histocompatibility complex class II-expression are also presented in amphibian skin (Du Pasquier and Flajnik, 1990; Carrillo-Farga et al., 1990). Moreover, the exon 2 sequence of the MHC class II B gene has been considered as a candidate molecular marker of the association between MHC class II B alleles and susceptibility/ resistance to diseases because the sequence has a high polymorphism (NikolichŽugich et al., 2004). The major histocompatibility complex genes are extensively investigated regions in vertebrate genomes (Horton et al., 2004) because of their important roles in immunity and fitness; the associations between MHC alleles and disease resistance or susceptibility in a large number of species have been documented (Sommer, 2005; Grimholt et al., 2003; Langefors et al., 2001; Paterson et al., 1998; Savage and Zamudio, 2011). However, research has focused on mammals, birds and fish. In amphibians, the whole MHC loci have been analysed and identified in model organisms, such as Xenopus laevis and Silurana (Xenopus) tropicalis (Kaufman et al., 1985; Sato et al., 1993; Flajnik et al., 1991; Liu et al., 2002). Attention for amphibian immunogenetics has been precipitated because of the emergence of amphibian infectious diseases (Berger et al., 1998; Gray et al., 2009). Studies have focused on the complete molecular structural analysis (Yu et al., 2014; Zeisset and Beebee, 2009), characterisation (Hauswaldt et al., 2007; Kiemnec-Tyburczy et al., 2010; May and Beebee, 2009; Shu et al., 2013; Zeisset and Beebee, 2009), disease association of MHC class II B in non-model amphibian species (Yu et al., 2014; Barribeau et al., 2008; Savage and Zamudio, 2011; Bataille et al., 2015). However, the disease association combine with genetic stability of MHC class II B in amphibians have been rarely investigated. In this study, the association between specific MHC class II B alleles and disease resistance to bacterial infections was detected in Q. spinosa. The immunity and disease resistance of Q. spinosa offspring were determined. The frequency of MHC class II B was also verified to evaluate the frequency stability of specific MHC class II B alleles between parent and next generation. This study helps elucidate MHC as a molecular marker of breeding. This study also provides a basis to improve disease resistance by applying molecular techniques and by selectively breeding resistant and genetically stable Q. spinosa strains. 2. Materials and methods 2.1. Animals A total of 100 individuals provided by an experimental aquaculture farm (Lanxi, China) were exposed to Aeromonas hydrophila to induce
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infection. After the infection experiment was completed, the surviving individuals were cultivated and reproduced in the farm. A total of about 5600 frog spawns were successfully obtained, 150 individuals were randomly selected from offspring (60 ± 10 g) and divide into three groups for A. hydrophila, Acinetobacter baumannii and Citrobacter braakii infection experiments. A. hydrophila strain QuSp1, Acinetobacter baumannii strain QuSp2 and Citrobacter braakii strain QuSp3 were isolated from an infected Q. spinosa individual collected from a farm in 2013.
2.2. Animal challenge The experimental frogs were temporarily placed in new rearing pond with a fresh water supply at 20 ± 0.5 °C (PH 6.7 ± 0.5) for a week to adapt to the surrounding environment before artificial infection was induced. In the infection experiment with A. hydrophila, the test individuals were inoculated with A. hydrophila were infected by intraperitoneal injection as described previously (Cosma et al., 2006) with 0.45 mm syringe needle. Control group trials were prepared under the same conditions. Different A. hydrophila concentrations were examined in a pre-challenge experiment with same-sized frogs as test organisms to determine the infective concentration. The median lethal concentration was determined according to Xu (Xu et al., 2008). The results revealed that the infective concentration was 4 × 107 cfu/ml. 100 individuals approximately weighing 200 ± 50 g were inoculated intraperitoneally with 0.2 ml of A. hydrophila colony forming units. The control group was inoculated intraperitoneally with the same dose of 0.65% sterile saline solution. The test lasted 18 days, and mortality and active state were recorded every 6 h for the succeeding test period. All of the dead individuals were stored at −20 °C. The dead individuals were considered to be susceptible organisms, and the surviving frogs were considered as disease-resistant individuals. Male and female surviving individuals were all selected as parental frogs by haphazard mating to produce offspring in an aquaculture farm. For the bacterial challenge of the offspring, the median lethal concentration was determined also according to Xu (Xu et al., 2008). Three bacteria, namely, A. hydrophila, Acinetobacter baumannii and Citrobacter braakii which isolated by our laboratory, were used for our challenge experiment. Bacteriums were inoculated on nutrient agar. Bacterial cells were obtained in sterile saline solution after overnight growth at 37 °C. The densities of A. hydrophila, Acinetobacter baumannii and Citrobacter braakin suspensions were respectively adjusted to 4 × 107, 1 × 108 and 1 × 109 cfu/ml, which were determined by colony counting. The 150 offsprings were divided into three groups and injected with 0.05 ml of each bacterial suspension (50 individuals per bacterium), and placed in different and separated rearing ponds. The control group comprised 150 ordinary and contemporary Q. spinosa, which were injected with same dose of bacterial suspension. In order to eliminate the wound affect individual normal breeding after injection, the blank control group was consisted of 150 ordinary and contemporary Q. spinosa, which were injected with the same dose of sterile saline solution. The test lasted 18 days.The survival rate was calculated at the end of experiment.
2.3. DNA extraction Genomic DNA was extracted from muscle or toe clip samples of 200 individuals (100 individuals of parent and 100 of offspring) through phenol-chloroform extraction in accordance with previously described methods (Ye et al., 2013). The quality and concentration of DNA were assessed through agarose gel electrophoresis. These parameters were determined by using a DNA spectrophotometer. The DNA was stored at − 20 °C for future use.
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2.4. Polymerase chain reaction (PCR) amplification and purification
3. Results
On the basis of the sequence primers of X. laevis (Hauswaldt et al., 2007), we used a degenerate MHC class II B forward primer MHC-F CCSCAGAKGATTWCGTGWMTCA and a reverse primer MHC-5R TGTCTGCAGACTGTYTCCACCHCAGCC to amplify exon 2. Polymerase chain reaction (PCR) was conducted to amplify exon 2 of the MHC class II B gene in a 25 μl reaction mixture under the following conditions: initial denaturation for 5 min at 95 °C; 34 cycles of denaturation for 30 s at 95 °C, annealing for 1 min at 58 °C and extension for 40s at 72 °C; and final extension for 10 min at 72 °C. PCR products were assessed through 1% agarose gel electrophoresis. The PCR products were then purified by using a Wizard PCR Preps DNA purification kit (Promega).The DNA data of 200 individuals (100 individuals of parent and 100 of offspring) were used for PCR and sequencing.
3.1. MHC II B exon 2 alleles in parents
2.5. Cloning and sequencing The PCR products were cloned into a pMD18-T vector (TaKaRa), which was subsequently used to transform Escherichia coli DH5α competent cells. Five to eight colonies from each transformed cell were selected to identify MHC class II B alleles in 100 individuals. Positive clones were screened through PCR with M13± primers. An average of 4 clones per individual was sequenced using an ABI 3730 automated sequencer with the M13 primer. 2.6. Sequence analysis We used translated amino acid queries in GenBank to confirm the MHC class II B homology. The alignment of the deduced amino acid sequence of the MHC class II B peptide was performed using Clustalx and DNAstar (http://www.dnastar.com). 2.7. Blood sampling and immune analysis After the offspring were subjected to bacterial challenge, 100 individuals were chosen randomly from surviving individuals (50 experimental group and 50 from control group) to collect blood sample. Afterward, 1.5–2 ml of blood sample was obtained from 100 individual frogs from each treatment via a puncture of the sciatic artery. The control group comprised ordinary and contemporary giant spiny frogs. Heparinised blood was used to determine haematological and immunological parameters. Haematological analyses included total and differential leucocyte counts and erythrocyte counts; immunological parameters consisted of the phagocytic capacity and phagocytic index of peritoneal phagocytes. 1.5–2.0 ml of blood were collected for the determination of several haematological parameters: the number of erythrocytes was calculated using a Neubauer chamber, and the erythrocyte were counted as described by Wintrobe (1933). Leucocyte counts were obtained from the smears stained with May-Grünwald and Giemsa in accordance with the methods described by Rosenfeld (1947). Phagocytes were calculated by a phase-contrast microscope. The phagocytic capacity was measured in accordance with the method described by Silva et al. (2002, 2005). The phagocytic index (PI) were calculated using the following equations (Zhang et al., 2008): PI = N1 / 100 × 100%, where N1 is the total number of bacteria engulfed by 100 random phagocytes. 2.8. Statistical analysis Data were expressed as means ± SE. Statistical analysis was performed in SPSS18.0. Different bacteria infected survival rate between experiment group and control group and allele frequency discrepancies were verified through Chi-square test. U test was conducted to analyze the trend in the changes in immune parameters. Differences were considered significant at P b 0.05.
After 100 individual parents were subjected to A. hydrophila challenge, 47 individuals survived, and the dead individuals appeared corresponding symptoms that infected with A. hydrophila disease. No mortality was observed in the control frogs. 27 females and 20 males of 47 surviving individuals used as parents by haphazard mating to produce offspring, and 20 females and 20 males contributed to offspring groups which could be ensured from the number and location of the frogs' spawns. The genomic DNA from 47 resistant frog and 53 susceptible individuals was amplified using a pair of primers, namely, MHC-F and MHC-5R. Afterward, 186 bp MHC II B exon 2 sequence was obtained. According to the accepted nomenclature rules, these alleles were designated as qasp-DAB*a to qasp-DAB*v. 31 distinct amino acid sequences of Q. spinosa MHC class II B is shown in Fig.1. Alleles that differ by less than five amino acid substitutions are considered as subtype within a single major type (Xu et al., 2007; Xu et al., 2005). Among these sequences, 16 were the same as those in a previous report (Yu et al., 2014). The 16 sequences include qasp-DAB*a, qasp-DAB*c, qaspDAB*e, qasp-DAB*g, qasp-DAB*h, qasp-DAB*i, qasp-DAB*l, qaspDAB*n1, qasp-DAB*n2, qasp-DAB*p1, qasp-DAB*p2, qasp-DAB*p3, qasp-DAB*p4, qasp-DAB*s, qasp-DAB*u and qasp-DAB*v. Furthermore, 15 sequences were newly discovered in this study. 3.2. Association between allele frequency and resistance/susceptibility to A. hydrophila A total of 31 different MHC II exon 2 alleles were identified from the sampled individuals. The frequency of alleles was not distributed equally. Six high-frequency alleles were selected to investigate the association between MHC class II B allele polymorphism and disease resistance/susceptibility. The frequencies of qasp-DAB*e (χ2 = 8.999, df = 1, P = 0.025), qasp-DAB*h (χ2 = 15.468, df = 1, P = 0.001) and qasp-DAB*l (χ2 = 16.329, df = 1, P = 0.001) were significantly different between the dead individuals (Dd) and the surviving individuals (Fig. 2). qasp-DAB*h and qasp-DAB*l may be associated with the increased resistance to A. hydrophila of Q. spinosa (Sv). qasp-DAB*e may be associated with the susceptibility to A. hydrophila of Q. spinosa (Dd). 3.3. Disease resistance and immunity of the next generation After the pathogenic bacterial challenge was conducted in offsprings, the control Q. spinosa manifested various symptoms, such as poor activity and appetite loss. The experimental Q. spinosa also displayed these symptoms, but these symptoms appeared at a later time than those of the control group. The survival rate of the resistant group was higher than that of the control group (Table 1). The survival rate of the blank control group was 100%. There is a significantly difference between the resistant groups and control group which were injected with A. hydrophila (χ2 = 3.841 , df = 1, P = 0.035), The groups are also with significantly difference which were injected with Citrobacter braakii (χ2 = 4.233, df = 1, P = 0.023). The survival rate of the blank control group was 100%. The blood samples obtained from the resistant group and the control group were evaluated to determine the immune status of the next generation. Haematological and immunological parameters were detected in the resistant offspring and the control Q. spinosa (Table 2). The numbers of leucocytes in blood of bacterium-injected offspring were observed to be significantly higher than those in the control group (u = 11.913, df = 1, P = 0.002), and a sharp increase in the number of erythrocyte in bacterium-injected offspring was observed compared with control group (u = 34.474, df = 1, P = 0.001).There was a slight increase in the phagocytosis activity and phagocytic index of bacteriuminjected offspring, so the phagocytosis of phagocytes of Q. spinosa in
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Fig. 1. Putative amino acid sequences for MHCIIB exon2 alleles of Q. spinosa. Dots indicate identity with the top sequences; the putative peptide binding region is indicated with asterisks.
the resistant offspring group was stronger than those in the control group. 3.4. Inheritance of the alleles in the next generation In this study, 300 MHC class II nucleotide sequences and 24 distinct MHC II exon 2 alleles were identified from the 100 randomly selected offspring of the disease-resistant individuals (Fig.3). The full alignment of 186 bp exon 2 of the MHC II B gene did not show any gap with the
aligned MHC II exon 2 alleles of their parent (Fig. 1). A total of 24 distinct MHC II exon 2 alleles were found in the parents. qasp-DAB*h and qaspDAB*l were associated with the disease resistance allele of the parents; these alleles also corresponded to qasp-DAB*0701 and qasp-DAB*1001, respectively. This finding revealed that the MHC II B alleles were transmitted to the progeny and the frequency of the alleles was not distributed equally. To confirm the hereditary stability of the two disease resistance alleles, we calculated the frequency of the alleles. We found that the frequencies of qasp-DAB*l and qasp-DAB*h were 0.19 and 0.15 in the parents. The frequencies of qasp-DAB*1001 and qaspDAB*0701 were 0.57 and 0.46 in the offspring. After Chi-square test, we found frequencies of two alleles were exist highly significant difference (p = 0.001). 4. Discussion
Fig. 2. Distribution of MHC class IIB alleles in dead individuals (Dd) and survival individuals (Sv) of the giant spiny frog. Note: * for p b 0.05; ** for p b 0.01.
The major histocompatibility complex genes have been extensively investigated because these genes are significant elements of the vertebrate immune system. In this study, 31 and 24 different MHC II exon 2 alleles were identified from the 100 parental individuals of Q. spinosa and 100 offspring, respectively. The associations between MHC class II B alleles and A. hydrophila resistance or susceptibility were presented. We also determined some immune parameters and the inheritance of the alleles associated with the disease resistance of their next generation. The major histocompatibility complex allele diversity is critical for resistance against parasites (Wegner et al., 2006). The high polymorphism of MHC genes in animals provides high disease resistance (Xu
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Table 1 The comparison of survival rate in resistant and control group of Q. spinosa. Method
Aeromonas hydrophila injection
group
Before /number
After /number
Survival rate
Before/number
Acinetobacter baumannii injection After/number
Survival rate
Citrobacter braakii injection Before/number
After /number
survival rate
E C
50 50
45 24
90% 48%
50 50
42 26
84% 52%
50 50
40 20
80% 40%
Note: E: Experiment group. C: Control group.
et al., 2008). In our study, sequence analysis revealed the high polymorphism of MHC II B gene in Q. spinosa parents. At least three loci of MHC II B gene were found in each individual. This result is similar to that reported by Chen et al. (2006) and Zhang and Chen (2006). Diseases are major factors seriously influencing the development of aquatic agriculture. Diseases pose a significant threat to commercial production in freshwater aquaculture in terms of the presence of ubiquitous pathogens and their frequent occurrence. A. hydrophila is considered as a secondary pathogen that is likely to infect immunocompromised animals (Barribeau et al., 2008; Taylor et al., 1999). A. hydrophila affects the growth and survival of tadpoles, and MHC mediates these responses (Barribeau et al., 2008; Song et al., 2014). A. hydrophila can also lead to the high mortality of farmed Q. spinosa with epidemic diseases (Ma et al., 2013). In this study, the different allele distributions indicated the association between MHC class II B alleles and resistance to A. hydrophila of Q. spinosa. qasp-DAB*h (χ2 = 15.468, df = 1, P = 0.001) and qasp-DAB*l (χ2 = 16.329, df = 1, P = 0.001) yielded a significantly higher frequency in the Sv individuals than in the Dd individuals. This result possibly demonstrated the association of the allele with the resistance to A. hydrophila of Q. spinosa parents. After sequence alignment was performed, the resistant alleles detected in our study are consistent with those described in a previous study (Yu et al., 2014). qasp-DAB*e alleles (χ2 = 8.999, df = 1, P = 0.025) were also associated with the susceptibility to A. hydrophila. The experimental result of the three bacterial challenges of the disease-resistant Q. spinosa offspring revealed that the immunity to bacterial pathogens of the disease-resistant offspring was significantly higher than that of the control group. The immunity of the resistance groups against different pathogens was not significantly different. Zhao (2010) confirmed that resistance to different pathogenic bacteria is controlled by different alleles. Nevertheless, a specific allele may exhibit resistance to various pathogens. Therefore, the survival of the individuals which was infected with A. hydrophila could be contributed to the resistance to other bacterial pathogens. Non-specific immunity is a natural trait of animals. This trait has evolved during the development of defense mechanisms to resist pathogenic microorganism infection. This trait can also remain stable and be transmitted to progenies. In amphibians, frogs have developed specific immunity (Marchalonis and Edelman, 1966), but specific immune responses are triggered several days or weeks after bacterial invasion occurs. Non-specific immunity plays a major role in the anti-infection immune response in the initial stage. Several immunological and haematological parameters have been introduced to evaluate the non-specific immune response of some aquatic animals to bacterial infection (Zhang et al., 2008; Dias et al., 2009; Yildiz, 1998; Benli and Yildiz,
Table 2 Values of leucocyte counts, erythrocytes counts, phagocytosis activity and phagocytic index of peritoneal phagocytes in the resistant offspring and control group of Q. spinosa. Category leucocyte counts erythrocytes counts phagocytosis activity phagocytic index
n 50 50 50 50
Disease-resistant offspring 3
−3
11.82 ± 0.16 × 10 mm 4.68 ± 0.03 × 104 mm−3 85 ± 0.32% 1.71 ± 0.02%
Note: Mean values and standard error for result.
Control group 9.408 ± 80.128 × 8103 mm−3 3.208 ± 80.038 × 8104 mm−3 71 ± 0.48% 1.56 ± 0.03%
2004). Bacteria-injected promote the transfer and accumulation of leukocytes to the infection site, and the continual proliferation of leucocytes can eliminate invasive bacteria (Lydyard et al., 2000). Increase in the numbers of leukocytes during fish defense against the invasive pathogenic bacteria was well known (Yildiz, 1998; Caruso et al., 2002) and many types of frog leucocytes have pseudopodia to ingest particles (phagocytosis) (Pan, 1999). Though the enhancement in the phagocytosis activity of fish peripheral blood neutrophil during bacterial infection has been reported (Park and Wakabayashi, 1992), erythrocyte, a type of immunocyte, has a great number of receptors of complement fragment (C3b),which can mediate the bonding of erythrocyte with pathogenic granule, and facilitate the bacteria clearing by phagocyte (Passantino et al., 2002). Phagocytes can excrete many types of molecules into blood which kill bacteria directly or indirectly during phagocytosis (Lydyard et al., 2000). So we can determine the animal's immune status by calculating the numbers of related immunocyte. In a previous study on the correlation of MHC genes with immune response, animals with different MHC alleles exhibit various immune responses (Liu, 2003). In our study, the number of erythrocytes, the number of leucocytes and the phagocytosis of phagocytes were verified between the resistant offspring group and the control group. The numbers of leucocytes and erythrocytes in blood of bacterium- injected offspring were observed to be higher than those in the control group, and the phagocytosis activity and phagocytic index of bacterium-injected offspring was increased in various degrees. These parameters significantly differed (P b 0.05) between the treatment groups and the control group. Major histocompatibility complex (MHC) class II antigens can activate different cellular functions in immune cells (Altomonte et al., 1999). We found larger numbers of two kinds of blood cells in injected offspring, but a hypothesis that whether the MHC-related resistance genes influenced the differentiation and expression of the blood cells which needed further study. In this study, the associations between MHC class II B alleles and resistance to bacterial infections were detected in the MHC II exon 2 alleles of the resistant offspring. The qasp-DAB*0701 and qaspDAB*1001 alleles were associated with the resistance to the three pathogens; these alleles corresponded to the qasp-DAB*h and qasp-DAB*l of the parents, respectively. The allele frequencies qasp-DAB*0701and qasp-DAB*1001 in the resistant offspring were 0.46 and 0.57 respectively. No new resistant alleles appeared. The constant heredity of the alleles observed in our study is similar to that reported by Xu et al. (2008). The frequencies of the two disease resistance alleles in the offspring were higher than those of the parents and no new resistant alleles were observed possibly because of the following. (i) The methods of data statistics and analysis. The allele frequency of individual which is lower than 1% of the sequences were eliminated because such alleles are generally consider to be almost impossible to be continued survival in the next generation, but maybe some of those alleles can survival in the next generation. (ii) Considering the condition of that we only determined the first generation and without any new genotype. The high frequency may be a result of sexual selection during mating, that is, individuals may prefer mates with “better alleles.” For instance, female tuco-tucos prefer males with special MHC loci considered as “good genes” (Ana et al., 2012). Therefore, Q. spinosa females may be more attracted to males with a disease-resistant gene than to males without this gene.
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Fig. 3. Putative amino acid sequences for MHC IIB exon2 alleles of Q. spinosa. Dots indicate identity with the top sequences.
The association between genes and diseases can be attributed to the effects of the gene or the linkage disequilibrium of the studied gene with a resistance gene (Langefors et al., 2001; Kjøglum et al., 2005). Nevertheless, another linked gene possibly causes the observed association; in our study, qasp-DAB*h and qasp-DAB*l alleles exhibited a higher resistance to A. hydrophila than other alleles. The qasp-DAB*h and qaspDAB*l alleles were continuously passed on from the parents to the offspring. Thus, our research may be used as a basis to develop disease resistance-related MHC markers for the molecular marker-assisted selective breeding of Q. spinosa with enhanced resistance to diseases caused by bacterial infections. This marker will be used in our future research to select individuals for mating. This study also helps improve the selective breeding of Q. spinosa with enhanced disease resistance. Acknowledgments The research was supported by the National Natural Science Foundation of China (Nos. 31172116 and 31472015), the Major Science and Technology Specific Projects Zhejiang Province, China (No. 2012C12907-9), the Agricultural Science and technology achievement transformation project of China (No. 2014GB2C200199), and Science and Technology Innovation Team of Zhejiang Province, China (No. 2012R10026-07). Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.aquaculture.2016.11.001.
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