Huaping Zhu

Major histocompatibility complex class IIA and IIB genes of Nile tilapia Oreochromis niloticus: Genomic structure, molecular polymorphism and expression patterns

Major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism, and it plays an important role in the immune response of vertebrates. In the present study, we isolated MHC class II genes from Nile tilapia in order to investigate the immune mechanism in tilapia and develop better strategies for disease prevention. Moreover, we cloned the full-length cDNA sequences of MHC IIA and IIB from Nile tilapia by the RACE approach. In addition, the genomic structure, molecular polymorphism and expression patterns of MHC II genes in Nile tilapia were also examined. Compared with that of other teleosts, Nile tilapia MHC class IIA contained four exons and three introns. The deduced amino acid sequence of the MHC IIA molecule shared 25.4-64.5% similarity with those of other teleosts and mammals. Six exons and five introns were identified from Nile tilapia MHC IIB, and the deduced amino acid sequence shared 26.9-74.7% similarity with those of other teleosts and mammals. All the characteristic features of MHC class II chain structure could be identified in the deduced sequences of MHC IIA and IIB molecules, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. A total of 12 MHC IIA alleles were identified from six individuals. Four alleles originating from a single individual suggested that at least four MHC IIA loci existed. Moreover, 10 MHC IIB alleles were identified, among which four were detected in a single individual, suggesting that at least four MHC IIB loci existed. The expression of MHC IIA and IIB at the mRNA level in 10 types of normal tissues was determined using quantitative real-time PCR analysis. The highest expression level was detected in stomach and gill, whereas the lowest expression was detected in muscle and brain. Furthermore, MHC IIA and IIB were probably two candidate immune molecules involved in the resistance against streptococcosis, because their expression was significantly up-regulated in gill, kidney, intestine and spleen after the intraperitoneal injection of Streptococcus agalactiae.

Screening and identification of a microsatellite marker associated with sex in Wami tilapia, Oreochromis urolepis hornorum

In this study, primer pairs of 15 microsatellite markers associated with sex determination of tilapia were selected and amplified in Wami tilapia, Oreochromis urolepis hornorum. While one marker, UNH168, on linkage group 3 (LG3) was associated (P < 0.001) with the phenotypic sex in the experimental population, nine genotypes were detected in both sexes. Only 99-bp allele was detected in the female samples, while 141, 149 and 157-bp alleles were present in both male and female samples. UNH168 was localized by fluorescence in situ hybridization (FISH) on the long arm of the largest tilapia chromosome pair (chromosome 1, equivalent to LG3). This sex-linked microsatellite marker could potentially be used for marker-assisted selection in tilapia breeding programmes to produce monosex male tilapia.

Molecular characterization, expression and functional analysis of NOD1, NOD2 and NLRC3 in Nile tilapia (Oreochromis niloticus)

ABSTRACT The nucleotide‐binding oligomerization domain proteins NOD1, NOD2 and NLRC3 are cytoplasmic pattern recognition receptors (PRRs) of the Nod‐like receptor (NLR) family. In the present study, the Nile tilapia (Oreochromis niloticus) NOD1 (ntNOD1), NOD2 (ntNOD2) and NLRC3 (ntNLRC3) genes were cloned and characterized. The full‐length ntNOD1, ntNOD2 and ntNLRC3 genes were 3924, 3886 and 4574 bp, encoding 941, 986 and 1130 amino acids, respectively. The three Nod‐like receptors have a NACHT domain and a C‐terminal leucine‐rich repeat (LRR) domain. In addition, ntNOD1 and ntNOD2 have a N‐terminal CARD domain (ntNOD2 has two). Phylogenetic analysis showed that the three NLRs are highly conserved. Tissue expression analysis of the three receptors revealed that the highest mRNA and protein levels of ntNOD1, ntNOD2 and ntNLRC3 were in the spleen. The expression patterns of NLRs during embryonic development showed that the expression levels of ntNOD2 and ntNLRC3 significantly increased from 2 to 8 days post‐fertilization (dpf). The expression levels of ntNOD1 significantly increased from 2 to 6 dpf, decreased at 7 dpf and then increased at 8 dpf. Upon stimulation with an intraperitoneal injection of Streptococcus agalactiae, expression levels of the ntNOD1, ntNOD2 and ntNLRC3 mRNA and protein were clearly altered in the blood, spleen, kidney, intestine and gill. Furthermore, after cotransfection with an NF‐&kgr;B reporter plasmid, NF‐&kgr;B activation in ntNOD1‐overexpressing 293T cells significantly increased compared with that in control cells, before or after i‐EDPA‐stimulation. By contrast, compared with control, ntNOD2 and ntNLRC3 had no effect on NF‐&kgr;B activation in 293T cells, when their potential ligands were not stimulated. However, after MDP‐stimulation, ntNOD2 and ntNLRC3 overexpression increased NF‐&kgr;B activation in 293T cells. NOD1 and NLRC3 were uniformly distributed throughout the cytoplasm in 293T cells, whereas NOD2 was distributed throughout the cytoplasm and nucleus. Our results indicate that the three Nod‐like receptors are functionally conserved and may play pivotal roles in defense against pathogens such as Streptococcus agalactiae. HIGHLIGHTSNOD1, NOD2 and NLRC3 cDNAs were isolated from tilapia Oreochromis niloticus.NOD1, NOD2 and NLRC3 mRNAs and proteins expresses ubiquitously with predominant expression in spleen.Expressions of NOD1, NOD2 and NLRC3 mRNAs and proteins were up‐regulated after Streptococcus agalactiae challenge.The ntNOD1, ntNOD2, ntNLRC3 mRNAs were detected in various embryonic developmental stages.The ntNOD1, ntNOD2 and ntNLRC3 genes were positively involved in the NF‐&kgr;B‐mediated signaling pathway.

Characterization and expression of Na+/K+-ATPase in gills and kidneys of the Teleost fish Oreochromis mossambicus, Oreochromis urolepis hornorum and their hybrids in response to salinity challenge

Tilapia (Oreochromis mossambicus, O. urolepis hornorum, their hybrids O. mossambicus♀ × O. hornorum♂ and O. hornorum♀ × O. mossambicus♂) were exposed to a high salinity environment to evaluate their osmoregulatory responses. The plasma osmolality of all the tilapia species were elevated with the salinity challenge. The activities of Na+/K+-ATPase (NKA) in both the gill and kidney showed a similar increased change tendency compared with the control. The distribution of NKA α1 mRNA in all the examined tissues suggested that NKA α1 has a possible housekeeping role for this isoform. The amount of NKA α1 mRNA in the gill and kidney was elevated in the four fishes with similar expression patterns after transfer from freshwater to seawater. The NKAα1 mRNA expression levels in the gill reached their peak level at 24 h after transfer (P < 0.01) compared to the freshwater group, following decreases in the pretreatment level at 48 h (P > 0.05). However, the NKAα1 mRNA expression levels in the kidney were not significantly affected with increasing environmental salinity (P > 0.05). The differences in the responses to saltwater challenge may be associated with differences in saltwater tolerance between the four tilapia. The drastic increase in the plasma osmolality, NKA activities and mRNA expression suggested that the hybrids (O. mossambicus♀ × O. hornorum♂) possess heterosis in salinity responsiveness compared to that of both the parents, indicating a maternal effect on the salinity tolerance of the tilapia hybrids. This study provides a theoretical basis to further study the mechanism of fish osmoregulation response to salinity challenge.

Effect of temperature on the virulence of Streptococcus agalactiae from Nile tilapia( Oreochromis niloticus )

In order to understand the influence and mechanism of temperature on the virulence of Streptococcus agalactiae,the effects of culture temperature on the virulence related parameters of S. agalactiae including bacteria grow th,adhesion,invasion,expression of virulence genes and mortality rates of tilapia w ere studied. The results show ed that the grow th speeds of S. agalactiae at different temperatures( 25-40 ℃) were different; 37 ℃ was the optimum growth temperature and the slowest growth speed appeared at 25 ℃. The adhesive capacity of S. agalactiae reached the peak at 37 ℃ and decreased at 40 ℃. There w as no significant difference betw een the absorbance( OD 590 nm) of adhesive S. agalactiae incubated in the range of 25- 34 ℃. The number of colony forming units in the brain of tilapia infected artificially w ith S. agalactiae show ed a temperature-dependent grow th pattern. The number of colony forming units increased along w ith the increase of culture temperature and reached the peak at 37 ℃,and then decreased at 40 ℃. The number of colony forming units w as positively associated w ith its mortality rate. The expression of virulence genes w as also influenced by culture temperature. The expression level of hly and cfb genes show ed a temperature-dependent expression pattern similar to the number of colony forming units. The expression of hly and cfb genes significantly increased and reached the peak at 34 ℃ and 37 ℃,respectively,and then dropped rapidly w hile the culture temperature continued to rise. The expression of sip gene w as minimally influenced by culture temperature w hile the expression of scpB gene decreased along w ith the increase of the culture temperature. The mortality rates of tilapia infected w ith S. agalactiae cultured at different temperatures show ed temperature-dependence. The mortality rate increased along w ith the increase of culture temperature and peaked 66. 67% ± 6. 67% at 37 ℃,and then slightly decreased at 40 ℃. In conclusion,the culture temperature influenced the grow th,adhesion,invasion and expression of some virulence genes of S. agalactiae,w hich may contribute to its virulence.