Evaluation of potential candidate genes involved in salinity tolerance in striped catfish (Pangasianodon hypophthalmus) using an RNA-Seq approach

Increasing salinity levels in freshwater and coastal environments caused by sea level rise linked to climate change is now recognized to be a major factor that can impact fish growth negatively, especially for freshwater teleost species. Striped catfish (Pangasianodon hypophthalmus) is an important freshwater teleost that is now widely farmed across the Mekong River Delta in Vietnam. Understanding the basis for tolerance and adaptation to raised environmental salinity conditions can assist the regional culture industry to mitigate predicted impacts of climate change across this region. Attempt of next generation sequencing using the ion proton platform results in more than 174 million raw reads from three tissue libraries (gill, kidney and intestine). Reads were filtered and de novo assembled using a variety of assemblers and then clustered together to generate a combined reference transcriptome. Downstream analysis resulted in a final reference transcriptome that contained 60,585 transcripts with an N50 of 683 bp. This resource was further annotated using a variety of bioinformatics databases, followed by differential gene expression analysis that resulted in 3062 transcripts that were differentially expressed in catfish samples raised under two experimental conditions (0 and 15 ppt). A number of transcripts with a potential role in salinity tolerance were then classified into six different functional gene categories based on their gene ontology assignments. These included; energy metabolism, ion transportation, detoxification, signal transduction, structural organization and detoxification. Finally, we combined the data on functional salinity tolerance genes into a hypothetical schematic model that attempted to describe potential relationships and interactions among target genes to explain the molecular pathways that control adaptive salinity responses in P. hypophthalmus. Our results indicate that P. hypophthalmus exhibit predictable plastic regulatory responses to elevated salinity by means of characteristic gene expression patterns, providing numerous candidate genes for future investigations.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg2+, Na+, K+ and NH4 (+) and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, . (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP ( (M) = 0.09 +/- A 0.01 mmol L-1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ ( (0.5) = 0.91 +/- A 0.04 mmol L-1) in decapodid III than in other stages; NH4 (+) had no modulatory effect. The affinity for Na+ ( (0.5) = 13.2 +/- A 0.6 mmol L-1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4 (+) obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval The NH4 (+)-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Sequence, circulating levels, and expression of C-type natriuretic peptide in a euryhaline elasmobranch, Carcharhinus leucas

The present study has examined expression and circulating levels of C-type natriuretic peptide (CNP) in the euryhaline bull shark, Carcharhinus leucas. Complementary DNA and deduced amino acid sequence for CNP in C leucas were determined by RACE methods. Homology of CNP amino acid sequence in C. leucas was high both for proCNP and for mature CNP when compared with previously identified elasmobranch CNPs. Mature CNP sequence in C. leucas was identical to that in Triakis seyllia and Seyliorhinus canicula. Levels of expression of CNP mRNA were significantly decreased in the atrium but did not change in either the brain or ventricle following acclimation to a SW environment. However, circulating levels of CNP significantly increased from 86.0 +/- 7.9 fmol ml(-1) in FW to 144.9 +/- 19.5 fmol ml(-1) in SW. The results presented demonstrate that changes in environmental salinity influences both synthesis of CNP from the heart and also circulating levels in C. leucas. Potential stimulus for release and modes of action are discussed. (c) 2005 Elsevier Inc. All rights reserved.

The effects of freshwater to seawater transfer on circulating levels of angiotensin II, C-type natriuretic peptide and arginine vasotocin in the euryhaline elasmobranch, Carcharhinus leucas

This study examined the effect of transfer to increased environmental salinity on the circulating levels of angiotensin II (ANG II), C-type natriuretic peptide (CNP), and arginine vasotocin (AVT) in the euryhaline elasmobranch, Carcharhinus letteas. Plasma levels of ANG 11 and CNP were significantly increased in C. leucas chronically acclimated to seawater (SW) in comparison to freshwater (FW) acclimated fish. There was no difference in plasma AVT levels. Acute transfer of FW fish to 75% SW induced an increase in plasma ANG II levels within 12 h, and subsequent transfer from 75 to 100% SW further increased plasma ANG 11 levels at both 24 and 72 h. No change in plasma CNP was observed during acute transfer to increased salinity. However, a significant increase in plasma AVT levels was observed following 96 h in 75% SW and 24 h in 100% SW. In chronically SW acclimated C leucas plasma osmolality, sodium, chloride, and Urea were all significantly higher than FW acclimated fish but there was no difference in haematocrit. Acute transfer of C letteas to 75% SW induced a significant increase in plasma osmolality, sodium and urea concentrations within 96 h of transfer. Subsequent transfer from 75 to 100% SW induced a further increase in these variables within 24 h in addition to a significant increase in plasma chloride above control levels. Haematocrit did not differ between the experimental and control groups throughout the acute study. Circulating levels of ANG 11 were significantly correlated to plasma, sodium, chloride, and urea concentrations during acclimation to SW. Conversely, circulating levels of CNP and AVT did not correlate to plasma osmolytes, however, CNP was significantly correlated to haematocrit during acclimation to seawater. (c) 2005 Elsevier Inc. All rights reserved.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg2+, Na+, K+ and NH4 (+) and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, . (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP ( (M) = 0.09 +/- A 0.01 mmol L-1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ ( (0.5) = 0.91 +/- A 0.04 mmol L-1) in decapodid III than in other stages; NH4 (+) had no modulatory effect. The affinity for Na+ ( (0.5) = 13.2 +/- A 0.6 mmol L-1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4 (+) obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval The NH4 (+)-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Distribution of Diatoms Along Environmental Gradients in the Charlotte Harbor, Florida (USA), Estuary and Its Watershed: Implications for Bioassessment of Salinity and Nutrient Concentrations

The relative abundance of diatom species in different habitats can be used as a tool to infer prior environmental conditions and evaluate management decisions that influence habitat quality. Diatom distribution patterns were examined to characterize relationships between assemblage composition and environmental gradients in a subtropical estuarine watershed. We identified environmental correlates of diatom distribution patterns across the Charlotte Harbor, Florida, watershed; evaluated differences among three major river drainages; and determined how accurately local environmental conditions can be predicted using inference models based on diatom assemblages. Sampling locations ranged from freshwater to marine (0.1–37.2 ppt salinity) and spanned broad nutrient concentration gradients. Salinity was the predominant driver of difference among diatom assemblages across the watershed, but other environmental variables had stronger correlations with assemblages within the subregions of the three rivers and harbor. Eighteen indicator taxa were significantly affiliated with subregions. Relationships between diatom taxon distributions and salinity, distance from the harbor, total phosphorus (TP), and total nitrogen (TN) were evaluated to determine the utility of diatom assemblages to predict environmental values using a weighted averaging-regression approach. Diatom-based inferences of these variables were strong (salinity R 2 = 0.96; distance R 2 = 0.93; TN R 2 = 0.83; TP R 2 = 0.83). Diatom assemblages provide reliable estimates of environmental parameters on different spatial scales across the watershed. Because many coastal diatom taxa are ubiquitous, the diatom training sets provided here should enable diatom-based environmental reconstructions in subtropical estuaries that are being rapidly altered by land and water use changes and sea level rise.

Improved salinity management for the Nebine / Mungallala / Wallam catchment

The low stream salinity naturally in the Nebine-Mungallala Catchment, extent of vegetation retention, relatively low rainfall and high evaporation indicates that there is a relatively low risk of rising shallow groundwater tables in the catchment. Scalding caused by wind and water erosion exposing highly saline sub-soils is a more important regional issue, such as in the Homeboin area. Local salinisation associated with evaporation of bore water from free flowing bore drains and bores is also an important land degradation issue particularly in the lower Nebine, Wallam and Mungallala Creeks. The replacement of free flowing artesian bores and bore drains with capped bores and piped water systems under the Great Artesian Basin bore rehabilitation program is addressing local salinisation and scalding in the vicinity of bore drains and preventing the discharge of saline bore water to streams. Three principles for the prevention and control of salinity in the Nebine Mungallala catchment have been identified in this review: • Avoid salinity through avoiding scalds – i.e. not exposing the near-surface salt in landscape through land degradation; • Riparian zone management: Scalding often occurs within 200m or so of watering lines. Natural drainage lines are most likely to be overstocked, and thus have potential for scalding. Scalding begins when vegetation is removed, and without that binding cover, wind and water erosion exposes the subsoil; and • Monitoring of exposed or grazed soil areas. Based on the findings of the study, we make the following recommendations: 1. Undertake a geotechnical study of existing maps and other data to help identify and target areas most at risk of rising water tables causing salinity. Selected monitoring should then be established using piezometers as an early warning system. 2. SW NRM should financially support scald reclamation activity through its various funding programs. However, for this to have any validity in the overall management of salinity risk, it is critical that such funding require the landholder to undertake a salinity hazard/risk assessment of his/her holding. 3. A staged approach to funding may be appropriate. In the first instance, it would be reasonable to commence funding some pilot scald reclamation work with a view to further developing and piloting the farm hazard/risk assessment tools, and exploring how subsequent grazing management strategies could be incorporated within other extension and management activities. Once the details of the necessary farm level activities have been more clearly defined, and following the outcomes of the geotechnical review recommended above, a more comprehensive funding package could be rolled out to priority areas. 4. We recommend that best-practice grazing management training currently on offer should be enhanced with information about salinity risk in scald-prone areas, and ways of minimising the likelihood of scald formation. 5. We recommend that course material be developed for local students in Years 6 and 7, and that arrangements be made with local schools to present this information. Given the constraints of existing syllabi, we envisage that negotiations may have to be undertaken with the Department of Education in order for this material to be permitted to be used. We have contact with key people who could help in this if required. 6. We recommend that SW NRM continue to support existing extension activities such as Grazing Land Management and the Monitoring Made Easy tools. These aids should be able to be easily expanding to incorporate techniques for monitoring, addressing and preventing salinity and scalding. At the time of writing staff of SW NRM were actively involved in this process. It is important that these activities are adequately resourced to facilitate the uptake by landholders of the perception that salinity is an issue that needs to be addressed as part of everyday management. 7. We recommend that SW NRM consider investing in the development and deployment of a scenario-modelling learning support tool as part of the awareness raising and education activities. Secondary salinity is a dynamic process that results from ongoing human activity which mobilises and/or exposes salt occurring naturally in the landscape. Time scales can be short to very long, and the benefits of management actions can similarly have immediate or very long time frames. One way to help explain the dynamics of these processes is through scenario modelling.

Coal seam gas waters - their geochemical signature and potential environmental implications

Coal Seam Gas (CSG) is a form of natural gas (mainly methane) sorbed in underground coal beds. To mine this gas, wells are drilled directly into an underground coal seam and groundwater (CSG water) is pumped out to the surface. This lowers the downhole piezometric pressure and enables gas desporption from the coal matrix. In the United States, this gas has been extracted commercially since the 1980s. The economic success of US CSG projects has inspired exploration and development in Australia and New Zealand. In Australia, Queensland’s Bowen and Surat basins have been the subject of increased CSG development over the last decade. CSG growth in other Australian basins has not matured to the same level but exploration and development are taking place at an accelerated pace in the Sydney Basin (Illawarra and the Hunter Valley, NSW) and in the Gunnedah Basin. Similarly, CSG exploration in New Zealand has focused in the Waikato region (Maramarua and Huntly), in the West Coast region (Buller, Reefton, and Greymouth), and in Southland (Kaitangata, Mataura, and Ohai). Figure 1 shows a Shcoeller diagram with CSG samples from selected basins in Australia, New Zealand, and the USA. CSG water from all of these basins exhibit the same geochemical signature – low calcium, low magnesium, high bicarbonate, low sulphate and, sometimes, high chloride. This water quality is a direct result of specific biological and geological processes that have taken part in the formation of CSG. In general, these processes include the weathering of rocks (carbonates, dolomite, and halite), cation exchange with clays (responsible for enhanced sodium and depleted calcium and magnesium), and biogenic processes (accounting for the presence of high bicarbonate concentrations). The salinity of CSG waters tends to be brackish (TDS < 30000 mg/l) with a fairly neutral pH. These particular characteristics need to be taken into consideration when assessing water management and disposal alternatives. Environmental issues associated with CSG water disposal have been prominent in developed basins such as the Powder River Basin (PRB) in the United States. When disposed on the land or used for irrigation, water having a high dissolved salts content may reduce water availability to crops thus affecting crop yield. In addition, the high sodium, low calcium and low magnesium concentrations increase the potential to disperse soils and significantly reduce the water infiltration rate. Therefore, CSG waters need to be properly characterised, treated, and disposed to safeguard the environment without compromising other natural resources.

The environmental enigma : why do producers professing stewardship continue to practice poor natural resource management?

Despite a wide acceptance that primary producers in Australia subscribe to a stewardship ethic, land and water degradation remains an ongoing problem. Recent calculations suggest that the economic cost of Australia's environmental degradation is amounting to more than $A3.5 billion a year with an estimated cost of managing (not overcoming) problems of salinity, acidification, soil erosion totalling $A60 billion over the next decade. This paper argues that stewardship itself is an unsatisfactory concept when looking to landholders to respond to environmental problems, for rarely does the attitude of stewardship translate into behaviours of improving natural resource management practices on private land. Whilst there is some acceptance of the environmental problem among primary producers, a number of external constraints may also impede the uptake of conservation-orientated practices. In light of the prevailing accounts of poor adoption of sustainable practices a number of policy options are reviewed in this paper, including formal regional partnerships, regulatory frameworks and market-based measures. It is concluded that the contentious nature of some of these new opportunities for change will mean that any moves aimed at reversing environmental degradation in Australia will be slow.

A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery

The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9 ppt which showed best growth performance. Total sequence data generated was 467.8 Mbp, consisting of 4,116,424 reads with an average length of 112 bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

Engineering salinity tolerance in rice by exogenous expression of cell death regulators

Rice, an important crop that feeds more than half of the world's population is very sensitive to salinity stress – a growing problem affecting crop production globally. This PhD study addressed this problem by manipulating the programmed cell death pathways in rice resulting in significant enhancement of salinity stress tolerance. The impact of this work is that farmers would be in a position to grow rice containing such a trait in environments where salinisation of the soil exists, thereby addressing food security needs.

An evaluation of potential candidate genes involved in salinity tolerance in striped catfish (Pangasianodon hypopthalmus) using an RNA-SEQ approach

The project investigated the molecular response of Tra catfish (Pangasianodon hypophthalmus) to elevated salinity conditions. We employed Next generation sequencing platform to evaluate differential gene expression profiles of key genes under two salinity conditions. Results of the current project can form the basis for further studies to confirm the functional roles of specific genes that influence salinity tolerance in the target species and more broadly in other freshwater teleost fishes. Ultimately, the approach can contribute to developing superior culture stocks of the target species.

Cyclic Salinity Model for deposition of large-scale coal deposits

Existing field data for Rangal coals (Late Permian) of the Bowen Basin, Queensland, Australia, are inconsistent with the depositional model generally accepted in the current geological literature to explain coal deposition. Given the apparent unsuitability of the current depositional model to the Bowen Basin coal data, a new depositional model, here named the Cyclic Salinity Model, is proposed and tested in this study.