DNA methylation changes detected by methylation-sensitive amplified polymorphism in the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>) in response to salinity stress

Salinity is a considerable factor to the survival and distribution for a majority of marine organisms, the drawbacks of which are becoming a serious issue of aquaculture. DNA methylation, an extensively studied epigenetic modification in eukaryotes, plays a significant role in the regulation of gene expression in responding to environmental changes and triggering evolutionary consequences. The Pacific oyster Crassostrea gigas, as a eurythermal and euryhaline species, is considered to be tolerant to salinity fluctuation. In this study, fluorescent-labeled methylation-sensitive amplified polymorphism (F-MSAP) analysis was used to investigate the frequency and variation of DNA methylation in C. gigas under different salinity and time. The results showed that total methylation level was generally on a downward trend. At lower salinity, the total methylation level decreased at the earlier process and then increased during experiment process, but continued to shrink at the rest salinity. Fully methylation tended to better reflect the dynamics of total methylation. Recovery treatment showed that the extent and pattern of DNA methylation were difficult to return to the normal level in this research. The sequencing and BLAST analysis indicated that in salt stress most of the selected bands were closely related to the metabolism of nucleic acids and proteins, tropomyosin, and cellular transport, effecting on different biological processes of C. gigas. This work provides useful data to further elucidate the molecular mechanisms of salt stress response and tolerance in invertebrates.     

Climate change, precipitation and impacts on an estuarine refuge from disease

Background

Oysters play important roles in estuarine ecosystems but have suffered recently due to overfishing, pollution, and habitat loss. A tradeoff between growth rate and disease prevalence as a function of salinity makes the estuarine salinity transition of special concern for oyster survival and restoration. Estuarine salinity varies with discharge, so increases or decreases in precipitation with climate change may shift regions of low salinity and disease refuge away from optimal oyster bottom habitat, negatively impacting reproduction and survival. Temperature is an additional factor for oyster survival, and recent temperature increases have increased vulnerability to disease in higher salinity regions.

Methodology/Principal Findings

We examined growth, reproduction, and survival of oysters in the New York Harbor-Hudson River region, focusing on a low-salinity refuge in the estuary. Observations were during two years when rainfall was above average and comparable to projected future increases in precipitation in the region and a past period of about 15 years with high precipitation. We found a clear tradeoff between oyster growth and vulnerability to disease. Oysters survived well when exposed to intermediate salinities during two summers (2008, 2010) with moderate discharge conditions. However, increased precipitation and discharge in 2009 reduced salinities in the region with suitable benthic habitat, greatly increasing oyster mortality. To evaluate the estuarine conditions over longer periods, we applied a numerical model of the Hudson to simulate salinities over the past century. Model results suggest that much of the region with suitable benthic habitat that historically had been a low salinity refuge region may be vulnerable to higher mortality under projected increases in precipitation and discharge.

Conclusions/Significance

Predicted increases in precipitation in the northeastern United States due to climate change may lower salinities past important thresholds for oyster survival in estuarine regions with appropriate substrate, potentially disrupting metapopulation dynamics and impeding oyster restoration efforts, especially in the Hudson estuary where a large basin constitutes an excellent refuge from disease.     

Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation

Background

Studies of DNA methylomes in a wide range of eukaryotes have revealed both conserved and divergent characteristics of DNA methylation among phylogenetic groups. However, data on invertebrates particularly molluscs are limited, which hinders our understanding of the evolution of DNA methylation in metazoa. The sequencing of the Pacific oyster Crassostrea gigas genome provides an opportunity for genome-wide profiling of DNA methylation in this model mollusc.

Results

Homologous searches against the C. gigas genome identified functional orthologs for key genes involved in DNA methylation: DNMT1, DNMT2, DNMT3, MBD2/3 and UHRF1. Whole-genome bisulfite sequencing (BS-seq) of the oyster’s mantle tissues revealed that more than 99% methylation modification was restricted to cytosines in CpG context and methylated CpGs accumulated in the bodies of genes that were moderately expressed. Young repeat elements were another major targets of CpG methylation in oysters. Comparison with other invertebrate methylomes suggested that the 5’-end bias of gene body methylation and the negative correlation between gene body methylation and gene length were the derived features probably limited to the insect lineage. Interestingly, phylostratigraphic analysis showed that CpG methylation preferentially targeted genes originating in the common ancestor of eukaryotes rather than the oldest genes originating in the common ancestor of cellular organisms.

Conclusions

Comparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these ''relatively young’ genes was critical for the origin and radiation of eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1119) contains supplementary material, which is available to authorized users.     

Ecophysiological response of Crambe maritima to airborne and soil-borne salinity

Background and Aims

There is a need to evaluate the salt tolerance of plant species that can be cultivated as crops under saline conditions. Crambe maritima is a coastal plant, usually occurring on the driftline, with potential use as a vegetable crop. The aim of this experiment was to determine the growth response of Crambe maritima to various levels of airborne and soil-borne salinity and the ecophysiological mechanisms underlying these responses.

Methods

In the greenhouse, plants were exposed to salt spray (400 mm NaCl) as well as to various levels of root-zone salinity (RZS) of 0, 50, 100, 200 and 300 mm NaCl during 40 d. The salt tolerance of Crambe maritima was assessed by the relative growth rate (RGR) and its components. To study possible salinity effects on the tissue and cellular level, the leaf succulence, tissue Na⁺ concentrations, Na⁺ : K⁺ ratio, net K⁺/Na⁺ selectivity, N, P, K⁺, Ca²⁺, Mg²⁺, proline, soluble sugar concentrations, osmotic potential, total phenolics and antioxidant capacity were measured.

Key Results

Salt spray did not affect the RGR of Crambe maritima. However, leaf thickness and leaf succulence increased with salt spray. Root zone salinities up to 100 mm NaCl did not affect growth. However, at 200 mm NaCl RZS the RGR was reduced by 41 % compared with the control and by 56 % at 300 mm NaCl RZS. The reduced RGR with increasing RZS was largely due to the reduced specific leaf area, which was caused by increased leaf succulence as well as by increased leaf dry matter content. No changes in unit leaf rate were observed but increased RZS resulted in increased Na⁺ and proline concentrations, reduced K⁺, Ca²⁺ and Mg²⁺ concentrations, lower osmotic potential and increased antioxidant capacity. Proline concentrations of the leaves correlated strongly (r = 0·95) with RZS concentrations and not with plant growth.

Conclusions

Based on its growth response, Crambe maritima can be classified as a salt spray tolerant plant that is sensitive to root zone salinities exceeding 100 mm NaCl.     

Carry-over of differential salt tolerance in plants grown from dimorphic seeds of Suaeda splendens

Background and Aims

Halophytic species often show seed dimorphism, where seed morphs produced by a single individual may differ in germination characteristics. Particular morphs are adapted to different windows of opportunity for germination in the seasonally fluctuating and heterogeneous salt-marsh environment. The possibility that plants derived from the two morphs may also differ physiologically has not been investigated previously.

Methods

Experiments were designed to investigate the germination characteristics of black and brown seed morphs of Suaeda splendens, an annual, C₄ shrub of non-tidal, saline steppes. The resulting seedlings were transferred to hydroponic culture to investigate their growth and photosynthetic (PSII photochemistry and gas exchange) responses to salinity.

Key Results

Black seeds germinated at low salinity but were particularly sensitive to increasing salt concentrations, and strongly inhibited by light. Brown seeds were unaffected by light, able to germinate at higher salinities and generally germinated more rapidly. Ungerminated black seeds maintained viability for longer than brown ones, particularly at high salinity. Seedlings derived from both seed morphs grew well at high salinity (400 mol m⁻³ NaCl). However, seedlings derived from brown seeds performed poorly at low salinity, as reflected in relative growth rate, numbers of branches produced, F_v/F_m and net rate of CO₂ assimilation.

Conclusions

The seeds most likely to germinate at high salinity in the Mediterranean summer (brown ones) retain a requirement for higher salinity as seedlings that might be of adaptive value. On the other hand, black seeds, which are likely to delay germination until lower salinity prevails, produce seedlings that are less sensitive to salinity. It is not clear why performance at low salinity, later in the life cycle, might have been sacrificed by the brown seeds, to achieve higher fitness at the germination stage under high salinity. Analyses of adaptive syndromes associated with seed dimorphism may need to take account of differences over the entire life cycle, rather than just at the germination stage.Key words: Chlorophyll fluorescence, germination, growth rate, halophyte, photosynthesis, photosystem II, salt tolerance, seed dimorphism, seed viability, Suaeda splendens     

Metabolic and osmoregulatory changes in response to reduced salinities in the red grouper, Epinephelus akaara (Temminck & Schlegel), and the black sea bream, Mylio macrocephalus (Basilewsky)

Red groupers (Epinephelus akaara Temminck & Schlegel) and black sea breams (Mylio macrocephalus Basilewsky) were transferred from 30‰ into 3, 7, 12, 20, and 30‰ salinity. Fish were sampled at 0, 6, 24, 96, 168 and 336h after transfer. Serum osmolality, glucose, protein, Na⁺, K⁺, Ca²⁺, liver glycogen, liver protein, muscle water and haematocrit were determined. In general, transient disturbances in these variables were observed after transfer. For both species, no tissue hydration was observed upon acclimation to different salinities, whereas a progressive increase in haematocrit value was found as salinity decreased. Liver glycogen of both species, however, was higher in hypo-osmotic salinities. Serum Na⁺ of the red groupers declined upon acclimation to 7‰ salinity but the opposite was found for the black sea breams. The results indicate that both species are extremely euryhaline, and physiological stress is unlikely to occur within the salinity regime of 7 to 30‰ Comparatively, the black sea bream appears to be a more efficient osmoregulator.     

Nostoc, Microcoleus and Leptolyngbya inoculums are detrimental to the growth of wheat (Triticum aestivum L.) under salt stress

Background and aims

This study investigated the effect of cyanobacterial inoculants on salt tolerance in wheat.

Methods

Unicyanobacterial crusts of Nostoc, Leptolyngbya and Microcoleus were established in sand pots. Salt stress was targeted at 6 and 13 dS m^?1, corresponding to the wheat salt tolerance and 50 % yield reduction thresholds, respectively. Germinated wheat seeds were planted and grown for 14 (0 and 6 dS m^?1) and 21 (13 dS m^?1) days by which time seedlings had five emergent leaves. The effects of cyanobacterial inoculation and salinity on wheat growth were quantified using chlorophyll fluorescence, inductively coupled plasma-optical emission spectrometry and biomass measurements.

Results

Chlorophyll fluorescence was negatively affected by soil salinity and no change was observed in inoculated wheat. Effective photochemical efficiency correlated with a large range of plant nutrient concentrations primarily in plant roots. Inoculation negatively affected wheat biomass and nutrient concentrations at all salinities, though the effects were fewer as salinity increased.

Conclusions

The most likely explanation of these results is the sorption of nutrients to cyanobacterial extracellular polymeric substances, making them unavailable for plant uptake. These results suggest that cyanobacterial inoculation may not be appropriate for establishing wheat in saline soils but that cyanobacteria could be very useful for stabilising soils.     

A native Glomus intraradices strain from a Mediterranean saline area exhibits salt tolerance and enhanced symbiotic efficiency with maize plants under salt stress conditions

Aims

Arbuscular mycorrhizal (AM) fungi have been shown to occur naturally in saline environments and it has been suggested that differences in fungal behaviour and efficiency can be due to the origin and adaptation of the AM fungus. These findings invite to look out for AM fungal species isolated in saline environments and compare their salt-tolerance mechanisms with those of species living in non-saline areas.

Methods

A fungal strain of G. intraradices (Gi CdG) isolated from a region with serious problems of salinity and affected by desertification, has been compared with a collection strain of the same species, used as a model fungus. An in vitro experiment tested the ability of both AM fungi to grow under increasing salinity and an in vivo experiment compared their symbiotic efficiency with maize plants grown under salt stress conditions.

Results

The isolate Gi CdG developed better under saline conditions and induced considerably the expression of GintBIP, Gint14-3-3 and GintAQP1 genes, while it showed a lower induction of GintSOD1 gene than the collection G. intraradices strain. The isolate Gi CdG also stimulated the growth of maize plants under two levels of salinity more than the collection strain. The higher symbiotic efficiency of Gi CdG was corroborated by the enhanced efficiency of photosystem II and stomatal conductance and the lower electrolyte leakage exhibited by maize plants under the different conditions assayed.

Conclusions

The higher tolerance to salinity and symbiotic efficiency exhibited by strain Gi CdG as compared to the collection G. intraradices strain may be due to a fungal adaptation to saline environments. Such adaptation may be related to the significant up-regulation of genes encoding chaperones or genes encoding aquaporins. The present study remarks that AM fungi isolated from areas affected by salinity can be a powerful tool to enhance the tolerance of crops to saline stress conditions.     

A preliminary integrated genetic map distinguishes every chromosome pair and locates essential genes related to abiotic adaptation of <Emphasis Type="Italic">Crassostrea angulata</Emphasis>/<Emphasis Type="Italic">gigas</Emphasis>

Background

The re-sequencing of C. angulata has revealed many polymorphisms in candidate genes related to adaptation to abiotic stress that are not present in C. gigas; these genes, therefore, are probably related to the ability of this oyster to retain high concentrations of toxic heavy metals. There is, in addition, an unresolved controversy as to whether or not C. angulata and C. gigas are the same species or subspecies. Both oysters have 20 metacentric chromosomes of similar size that are morphologically indistinguishable. From a genomic perspective, as a result of the great variation and selection for heterozygotes in C. gigas, the assembly of its draft genome was difficult: it is fragmented in more than seven thousand scaffolds.

Results

In this work sixty BAC sequences of C. gigas downloaded from NCBI were assembled in BAC-contigs and assigned to BACs that were used as probes for mFISH in C. angulata and C. gigas. In addition, probes of H3, H4 histone, 18S and 5S rDNA genes were also used. Hence we obtained markers identifying 8 out the 10 chromosomes constituting the karyotype. Chromosomes 1 and 9 can be distinguished morphologically. The bioinformatic analysis carried out with the BAC-contigs annotated 88 genes. As a result, genes associated with abiotic adaptation, such as metallothioneins, have been positioned in the genome. The gene ontology analysis has also shown many molecular functions related to metal ion binding, a phenomenon associated with detoxification processes that are characteristic in oysters. Hence the provisional integrated map obtained in this study is a useful complementary tool for the study of oyster genomes.

Conclusions

In this study 8 out of 10 chromosome pairs of Crassostrea angulata/gigas were identified using BAC clones as probes. As a result all chromosomes can now be distinguished. Moreover, FISH showed that H3 and H4 co-localized in two pairs of chromosomes different that those previously escribed. 88 genes were annotated in the BAC-contigs most of them related with Molecular Functions of protein binding, related to the resistance of the species to abiotic stress. An integrated genetic map anchored to the genome has been obtained in which the BAC-contigs structure were not concordant with the gene structure of the C. gigas scaffolds displayed in the Genomicus database.

     

High Tolerance to Salinity and Herbivory Stresses May Explain the Expansion of Ipomoea Cairica to Salt Marshes

Background

Invasive plants are often confronted with heterogeneous environments and various stress factors during their secondary phase of invasion into more stressful habitats. A high tolerance to stress factors may allow exotics to successfully invade stressful environments. Ipomoea cairica, a vigorous invader in South China, has recently been expanding into salt marshes.

Methodology/Principal Findings

To examine why this liana species is able to invade a stressful saline environment, we utilized I. cairica and 3 non-invasive species for a greenhouse experiment. The plants were subjected to three levels of salinity (i.e., watered with 0, 4 and 8 g L⁻¹ NaCl solutions) and simulated herbivory (0, 25 and 50% of the leaf area excised) treatments. The relative growth rate (RGR) of I. cairica was significantly higher than the RGR of non-invasive species under both stress treatments. The growth performance of I. cairica was not significantly affected by either stress factor, while that of the non-invasive species was significantly inhibited. The leaf condensed tannin content was generally lower in I. cairica than in the non-invasive I. triloba and Paederia foetida. Ipomoea cairica exhibited a relatively low resistance to herbivory, however, its tolerance to stress factors was significantly higher than either of the non-invasive species.

Conclusions/Significance

This is the first study examining the expansion of I. cairica to salt marshes in its introduced range. Our results suggest that the high tolerance of I. cairica to key stress factors (e.g., salinity and herbivory) contributes to its invasion into salt marshes. For I. cairica, a trade-off in resource reallocation may allow increased resources to be allocated to tolerance and growth. This may contribute to a secondary invasion into stressful habitats. Finally, we suggest that I. cairica could spread further and successfully occupy salt marshes, and countermeasures based on herbivory could be ineffective for controlling this invasion.     

Change in uptake, transport and accumulation of ions in Nerium oleander (rosebay) as affected by different nitrogen sources and salinity

Background and Aims

The source of nitrogen plays an important role in salt tolerance of plants. In this study, the effects of NaCl on net uptake, accumulation and transport of ions were investigated in Nerium oleander with ammonium or nitrate as the nitrogen source in order to analyse differences in uptake and cycling of ions within plants.

Methods

Plants were grown in a greenhouse in hydroponics under different salt treatments (control vs. 100 mm NaCl) with ammonium or nitrate as the nitrogen source, and changes in ion concentration in plants, xylem sap exuded from roots and stems, and phloem sap were determined.

Key Results

Plant weight, leaf area and photosynthetic rate showed a higher salt tolerance of nitrate-fed plants compared with that of ammonium-fed plants. The total amount of Na⁺ transported in the xylem in roots, accumulated in the shoot and retranslocated in the phloem of ammonium-fed plants under salt treatment was 1·8, 1·9 and 2·7 times more, respectively, than that of nitrate-treated plants. However, the amount of Na⁺ accumulated in roots in nitrate-fed plants was about 1·5 times higher than that in ammonium-fed plants. Similarly, Cl⁻ transport via the xylem to the shoot and its retranslocation via the phloem (Cl⁻ cycling) were far greater with ammonium treatment than with nitrate treatment under conditions of salinity. The uptake and accumulation of K⁺ in shoots decreased more due to salinity in ammonium-fed plants compared with nitrate-fed plants. In contrast, K⁺ cycling in shoots increased due to salinity, with higher rates in the ammonium-treated plants.

Conclusions

The faster growth of nitrate-fed plants under conditions of salinity was associated with a lower transport and accumulation of Na⁺ and Cl⁻ in the shoot, whereas in ammonium-fed plants accumulation and cycling of Na⁺ and Cl⁻ in shoots probably caused harmful effects and reduced growth of plants.Key words: Mineral cycling, Nerium oleander, nitrogen source, salinity, xylem and phloem transport