Molecular characterization of heat shock protein 70 genes in the liver of three warm freshwater fishes with differential tolerance to microcystin-LR

Heat shock protein 70 (HSP70) protect cell from oxidative stress by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR). Full-length cDNAs encoding the HSP70 were cloned from the livers of the three fishes. The HSP70 cDNAs of silver carp, grass carp, and Nile tilapia were 2356, 2348, and 2242 bp in length and contained an open-reading frame of 1950 bp (encoding a polypeptide of 649 amino acids), 1950 bp (649 amino acids), and 1917 bp (638 amino acids), respectively. Like mammalian HSP70, the HSP70 of the three fish was also composed of an ATPase domain from residues 1 to 383 (44 kDa), substrate peptide binding domain from residues 384 to 544 (18 kDa), and a C-terminus domain from residues 545 to 649 (10 kDa). The relatively high conservation of HSP70 sequences among different vertebrates is consistent with their important role in fundamental cellular processes. Using beta-actin as an external control, RT-PCR within the exponential phase was conducted to determine the constitutive and inducible expression level of HSP70 gene among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 μg kg(-1) body weight). Both constitutive and inducible liver mRNA levels of the fish HSP70 genes showed positive relationships with their tolerance to MC-LR: highest in Nile tilapia, followed by silver carp, and lowest in grass carp. The differential expression pattern of liver HSP70 genes in the three fish indicated a potential role of HSP70 in the detoxification process of MC-LR.     

Effects of acclimation salinity on the expression of selenoproteins in the tilapia,Oreochromis mossambicus

Selenoproteins are ubiquitously expressed, act on a variety of physiological redox-related processes, and are mostly regulated by selenium levels in animals. To date, the expression of most selenoproteins has not been verified in euryhaline fish models. The Mozambique tilapia, Oreochromis mossambicus, a euryhaline cichlid fish, has a high tolerance for changes in salinity and survives in fresh water (FW) and seawater (SW) environments which differ greatly in selenium availability. In the present study, we searched EST databases for cichlid selenoprotein mRNAs and screened for their differential expression in FW and SW-acclimated tilapia. The expression of mRNAs encoding iodothyronine deiodinases 1, 2 and 3 (Dio1, Dio2, Dio3), Fep15, glutathione peroxidase 2, selenoproteins J, K, L, M, P, S, and W, was measured in the brain, eye, gill, kidney, liver, pituitary, muscle, and intraperitoneal white adipose tissue. Gene expression of selenophosphate synthetase 1, Secp43, and selenocysteine lyase, factors involved in selenoprotein synthesis or in selenium metabolism, were also measured. The highest variation in selenoprotein and synthesis factor mRNA expression between FW- and SW-acclimated fish was found in gill and kidney. While the branchial expression of Dio3 was increased upon transferring tilapia from SW to FW, the inverse effect was observed when fish were transferred from FW to SW. Protein content of Dio3 was higher in fish acclimated to FW than in those acclimated to SW. Together, these results outline tissue distribution of selenoproteins in FW and SW-acclimated tilapia, and indicate that at least Dio3 expression is regulated by environmental salinity.     

Transcriptomic differences between euryhaline and stenohaline malaria vector sibling species in response to salinity stress

Evolution of osmoregulatory systems is a key factor in the transition of species between fresh‐ and saltwater habitats. Anopheles coluzzii and Anopheles merus are stenohaline and euryhaline malaria vector mosquitoes belonging to a larger group of sibling species, the Anopheles gambiae complex, which radiated in Africa within the last 2 million years. Comparative ecological genomics of these vector species can provide insight into the mechanisms that permitted the rapid radiation of this species complex into habitats of contrasting salinity. Here, we use RNA‐Seq to investigate gene expression differences between An. coluzzii and An. merus after briefly exposing both young and old larval instars of each species to either saltwater (SW) or freshwater (FW). Our study aims to identify candidate genes and pathways responsible for the greater SW tolerance of An. merus. Our results are congruent with the ability of gene induction to mediate salinity tolerance, with both species showing increasing amounts of differential gene expression between SW and FW as salt concentrations increase. Besides ion transporters such as AgAE2 that may serve as effectors for osmoregulation, we also find mitogen‐activated protein kinases that may serve in a phosphorylation signalling pathway responding to salinity, and report potential cross‐talk between the mosquito immune response and osmoregulation. This study provides a key step towards applying the growing molecular knowledge of these malaria vectors to improve understanding of their ecological tolerances and habitat occupancy.     

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.     

Genome-Wide QTL Analysis Identified Significant Associations Between Hypoxia Tolerance and Mutations in the GPR132 and ABCG4 Genes in Nile Tilapia

Exposure to hypoxia induces both acute and chronic stress responses, which plays an important role in health of cultured organisms including growth, reproduction, immunity, and other energy demanding activities. Application of advanced genomic technologies allows rapid identification of hypoxia trait-associated genes and precise selection of superior brood stocks with high tolerance in tilapia. By applying QTL-seq and double-digest restriction-site associated DNA sequencing (ddRAD-seq) techniques, we identified four genome-wide significant quantitative trait loci (QTLs) for hypoxia tolerance and many suggestive QTLs in Nile tilapia. These QTLs explained 6.6–14.7% of the phenotypic variance. Further analysis revealed that single nucleotide polymorphisms (SNPs) in exons of both GPR132 and ABCG4 genes located in genome-wide QTL intervals were significantly associated with hypoxia-tolerant traits. Expression analysis of both genes suggested that they were strong candidate genes involved into hypoxia tolerance in tilapia. Our findings suggest that both QTL-seq and ddRAD-seq techniques can be effectively utilized in QTL mapping of hypoxia traits in fish. Our data supply a basis for further marker-assisted selection of super lines with a high level of tolerance against low oxygen stress in the tilapia.     

Identifying a Major QTL Associated with Salinity Tolerance in Nile Tilapia Using QTL-Seq

Selection of new lines with high salinity tolerance allows for economically feasible production of tilapias in brackish water areas. Mapping QTLs and identifying the markers linked to salinity-tolerant traits are the first steps in the improvement of the tolerance in tilapia through marker-assisted selection techniques. By using QTL-seq strategy and linkage-based analysis, two significant QTL intervals (chrLG4 and chrLG18) on salinity-tolerant traits were firstly identified in the Nile tilapia. Fine mapping with microsatellite and SNP markers suggested a major QTL region that located at 23.0 Mb of chrLG18 and explained 79% of phenotypic variation with a LOD value of 95. Expression analysis indicated that at least 10 genes (e.g., LACTB2, KINH, NCOA2, DIP2C, LARP4B, PEX5R, and KCNJ9) near or within the QTL interval were significantly differentially expressed in intestines, brains, or gills under 10, 15, or 20 ppt challenges. Our findings suggest that QTL-seq can be effectively utilized in QTL mapping of salinity-tolerant traits in fish. The identified major QTL is a promising locus to improve our knowledge on the genetic mechanism of salinity tolerance in tilapia.     

淡咸水轮换浇灌抑制互花米草的克隆生长和繁殖

作为典型的盐沼植物,入侵种互花米草(Spartina alterniflora)具有较强的耐盐性,能否以淡水或淡咸水的轮换浇灌扰乱其耐盐机制,降低其生态入侵性,进而寻求其控制对策是一个重要命题。为此,对互花米草进行了6种浇灌处理:单一的淡水(D)或咸水(X)浇灌;1次浇灌转换,即先淡水后咸水(DX)或先咸水后淡水(XD);2次浇灌转换,即由淡水开始转而咸水,再转而淡水(DXD),或由咸水开始转而淡水,再转而咸水(XDX)。结果显示,D处理的总生物量最高,与其它各处理均差异显著(p<0.05),且是X处理的1.67倍;DXD和XDX处理的总生物量最低,与D和X处理的差异显著(p<0.05),两者均约为D处理的50%。浇灌处理对根冠比、茎重比、叶重比和根茎重比的影响不明显,对根重比有较大影响,X处理的根重比最高,与D处理的差异显著(p<0.05),但与DX、XD、DXD和XDX处理之间的差异不显著。D处理的克隆生长新生总株数(Propagule)最多,与其它处理均差异显著(p<0.05),且是X处理的1.34倍;DXD和XDX处理的新生总株数最少,与D和X处理的差异显著(p<0.05),仅为D处理的55%。浇灌处理对分蘖的影响明显,DX和DXD处理的分蘖数分别是D处理的62%和50%;XD和XDX处理的分蘖数分别是X处理的50%和47%。就开花株数而言,X处理显著高于其它处理(p<0.05);DXD和XDX处理的开花株数为0。因此,持续的淡水浇灌可能会促进互花米草的生物量积累,并且采取快速的克隆生长为主的繁殖策略;而在淡水和咸水交替的生境下,互花米草的生物量积累、无性和有性繁殖能力都会受到抑制。     

Flooding and salinity effects on growth and survival of four common forested wetland species

The survival, growth, and biomass of baldcypress (Taxodium distichum (L.) Rich.), water tupelo (Nyssa aquatica L.), Chinese tallow (Sapium sebiferum (L.) Roxb.), and green ash (Fraxinus pennsylvanica Marsh.) seedlings were examined in an experiment varying water levels (watered, flooded) and salinity levels (0, 2, and 10 ppt, plus a simulated storm surge with 32 ppt saltwater). All seedlings, except for those flooded with 10 ppt saltwater, survived to the end of the experiment. In 10 ppt saltwater, flooded baldcypress, water tupelo, and green ash survived two weeks whereas Chinese tallow survived for 6 weeks. However, a second set of slightly older baldcypress, water tupelo, and Chinese tallow seedlings survived eight weeks of flooding with 10 ppt saltwater. When carried through the winter to the beginning of the second growing season, flooded baldcypress and Chinese tallow seedlings from the 0 and 2 ppt treatments leafed out, but only Chinese tallow recovered from the saltwater surge treatment. The diameter and growth (height) of each species was not affected when watered with 2 ppt saltwater, except for the effects of the height growth of baldcypress. Growth was reduced for all species when watered with 10 ppt saltwater. The diameter growth of green ash was reduced by freshwater flooding. The diameter growth of baldcypress and water tupelo was greater when flooded with fresh water. Flooding with 2 ppt saltwater caused a significant reduction in diameter growth in water tupelo, green ash, and Chinese tallow, but not in baldcypress. Root and stem biomass values were not significantly different for any species between the 0 and 2 ppt salinity watering treatments. However, seedlings watered with 10 ppt saltwater had significantly lower root and stem biomass values, except for baldcypress roots and green ash stems. Baldcypress was least affected by flooding with 0 and 2 ppt saltwater, although there were slight reductions in root biomass with increasing salinity. Because of the susceptibility of the seedlings of these four species to increases in flooding and salinity, their regeneration may be limited in the future, thereby causing shifts in species composition.     

3种不同食性淡水鱼类谷胱甘肽S-转移酶Pi、Mu、Theta型cDNA序列的克隆分析及表达

鱼类谷胱甘肽S-转移酶(glutathione S-transferase,GST)是鱼类一种重要的Ⅱ相去毒酶,在催化毒素与还原谷胱甘肽(GSH)加合去毒代谢过程中具有关键作用。采用RT-PCR及RACE法,分离、克隆得到草鱼、尼罗罗非鱼pi、mu、theta型GST(GSTpi、GSTmu、GSTtheta)基因、鲢鱼GSTmu、GSTtheta基因的cDNA部分序列并推测各自对应的氨基酸序列。氨基酸序列同源性比较和系统进化分析均表明,鲢鱼、草鱼、尼罗罗非鱼与鱼类GST同源性较高,与哺乳类、鸟类、两栖类GST同源性较低,可能与鱼类GST基因在水环境毒素去毒代谢中承担的特殊功能有关。而不同种鱼类GSTtheta的同源性明显要较GSTpi、GSTmu的同源性低,可能与不同淡水鱼类食性及对毒素耐受性不同有关。用实时荧光定量PCR(RT-PCR)检测三种鱼肝脏中三型GST基因组成型表达水平,发现三种鱼各型之间皆有一定差异,尼罗罗非鱼肝脏整体GSTs基因表达很低,GSTtheta显著低于草鱼(P<0.05),GSTmu显著低于鲢鱼(P<0.05)。本研究为从分子水平上研究不同型谷胱甘肽S-转移酶基因在不同食性淡水鱼类体内代谢去毒过程中的作用提供了基础。     

A Morphometric Study of Euryhalinity in Marine Populations of the Ciliate Genus Euplotes

ABSTRACT. Twenty different clonal strains of marine and brackish Euplotes , representing four morphotypes, were tested for hyposalinity tolerance by a method which gradually acclimated the cells to lower salinity medium. The lowest salinities in which the strains could thrive ranged from 60% of normal seawater to complete freshwater. The morphological effects of culture medium salinity were also examined for two strains of a small "Euplotes charon" morphotype, as well as for two mating compatible "Euplotes vannus" strains and several of their exconjugates. There were no differences between the euryhaline strains grown in fresh or saltwater, except for a slight increase in overall cell size in one strain when cultured in freshwater medium. E. vannus strains increased in overall cell size with decreased salinity; also, the dorsal surface of the cells can become disorganized when the cells are cultured in 30% normal seawater.     

Variation in gene expression along a salinity gradient in wild populations of the euryhaline black-chinned tilapia Sarotherodon melanotheron

This study evaluated variation in expression of 11 genes within and among six wild populations of the black-chinned tilapia Sarotherodon melanotheron distributed along a salinity gradient from 0 to 100. Previous laboratory studies had shown that expression of these genes was sensitive to water salinity; the current study confirmed that a number of them also varied in expression in wild populations along the salinity gradient. Principal component analysis (PCA) first distinguished two, not mutually exclusive, sets of genes: trade-off genes that were highly expressed at one or other extreme of the salinity gradient and stress genes that were up-regulated at the two salinity extremes (i.e. a U-shaped expression pattern). The PCA clearly partitioned the populations into three groups based on their gene expression patterns and their position along the salinity gradient: a freshwater (GL; 0) population, four brackish and seawater (GB, HB, SM, SF; ranging from 20 to 50) populations and a hypersaline (SK, 100) population. Individual variation in gene expression was significantly greater within the populations at the extreme compared to intermediate salinities. These results reveal phenotypically plastic regulation of gene expression in S. melanotheron, and greater osmoregulatory and plasticity costs at extreme salinities, where fitness-related traits are known to be altered.