Kelli Anderson

Meta-Analysis of Studies Using Suppression Subtractive Hybridization and Microarrays to Investigate the Effects of Environmental Stress on Gene Transcription in Oysters

Many microarray and suppression subtractive hybridization (SSH) studies have analyzed the effects of environmental stress on gene transcription in marine species. However, there have been no unifying analyses of these data to identify common stress response pathways. To address this shortfall, we conducted a meta-analysis of 14 studies that investigated the effects of different environmental stressors on gene expression in oysters. The stressors tested included chemical contamination, hypoxia and infection, as well as extremes of temperature, pH and turbidity. We found that the expression of over 400 genes in a range of oyster species changed significantly after exposure to environmental stress. A repeating pattern was evident in these transcriptional responses, regardless of the type of stress applied. Many of the genes that responded to environmental stress encoded proteins involved in translation and protein processing (including molecular chaperones), the mitochondrial electron transport chain, anti-oxidant activity and the cytoskeleton. In light of these findings, we put forward a consensus model of sub-cellular stress responses in oysters.

Rapid transcriptional acclimation following transgenerational exposure of oysters to ocean acidification.

Marine organisms need to adapt in order to cope with the adverse effects of ocean acidification and warming. Transgenerational exposure to CO2 stress has been shown to enhance resilience to ocean acidification in offspring from a number of species. However, the molecular basis underlying such adaptive responses is currently unknown. Here, we compared the transcriptional profiles of two genetically distinct oyster breeding lines following transgenerational exposure to elevated CO2 in order to explore the molecular basis of acclimation or adaptation to ocean acidification in these organisms. The expression of key target genes associated with antioxidant defence, metabolism and the cytoskeleton was assessed in oysters exposed to elevated CO2 over three consecutive generations. This set of target genes was chosen specifically to test whether altered responsiveness of intracellular stress mechanisms contributes to the differential acclimation of oyster populations to climate stressors. Transgenerational exposure to elevated CO2 resulted in changes to both basal and inducible expression of those key target genes (e.g. ecSOD, catalase and peroxiredoxin 6), particularly in oysters derived from the disease‐resistant, fast‐growing B2 line. Exposure to CO2 stress over consecutive generations produced opposite and less evident effects on transcription in a second population that was derived from wild‐type (nonselected) oysters. The analysis of key target genes revealed that the acute responses of oysters to CO2 stress appear to be affected by population‐specific genetic and/or phenotypic traits and by the CO2 conditions to which their parents had been exposed. This supports the contention that the capacity for heritable change in response to ocean acidification varies between oyster breeding lines and is mediated by parental conditioning.

Hepatic reference gene selection in adult and juvenile female Atlantic salmon at normal and elevated temperatures

BackgroundThe use of quantitative real-time polymerase chain reaction (qPCR) has become widespread due to its specificity, sensitivity and apparent ease of use. However, experimental error can be introduced at many stages during sample processing and analysis, and for this reason qPCR data are often normalised to an internal reference gene. The present study used three freely available algorithms (GeNorm, NormFinder and BestKeeper) to assess the stability of hepatically expressed candidate reference genes (Hprt1, Tbp, Ef1α and β-tubulin) in two experiments. In the first, female Atlantic salmon (Salmo salar) broodstock of different ages were reared at either 14 or 22°C for an entire reproductive season, therefore a reference gene that does not respond to thermal challenge or reproductive condition was sought. In the second, estrogen treated juvenile salmon were maintained at the same temperatures for 14 days and a reference gene that does not respond to temperature or estrogen was required. Additionally, we performed independent statistic analysis to validate the outputs obtained from the program based analysis.ResultsBased on the independent statistical analysis performed the stability of the genes tested was Tbp > Ef1α > Hprt1 > β-tubulin for the temperature/reproductive development experiment and Ef1α > Hprt1 > Tbp for the estrogen administration experiment (β-tubulin was not analysed). Results from the algorithms tested were quite ambiguous for both experiments; however all programs consistently identified the least stable candidate gene. BestKeeper provided rankings that were consistent with the independent analysis for both experiments. When an inappropriate candidate reference gene was used to normalise the expression of a hepatically expressed target gene, the ability to detect treatment-dependent changes in target gene expression was lost for multiple groups in both experiments.ConclusionsWe have highlighted the need to independently validate the results of reference gene selection programs. In addition, we have provided a reference point for those wishing to study the effects of thermal challenge and/or hormonal treatment on gene stability in Atlantic salmon and other teleost species.

Effect of elevated temperature on estrogenic induction of vitellogenesis and zonagenesis in juvenile Atlantic salmon (Salmo salar)

Fertility and embryo survival rates are often low in eggs from thermally challenged Tasmanian Atlantic salmon, partly due to a reduction in plasma 17β-estradiol (E2) levels. We used juvenile Atlantic salmon to assess whether hepatic tissue remains responsive to stimulation by E2 at the higher temperatures sometimes encountered by Tasmanian salmon during summer. E2 administration stimulated vitellogenin (Vtg) and estrogen receptor alpha transcription at 14°C and 22°C, although induction of Vtg occurred more rapidly at 22°C. Consequently, plasma Vtg levels increased and reached a plateau more quickly at 22°C. Zona pellucida (Zp) B and C transcription was significantly lower in E2-treated fish at 22°C relative to 14°C. This shows that the Vtg gene is E2-responsive at high temperature unlike Zp B and C genes that displayed traits of thermal inhibition. Therefore, estrogen replacement therapy in adult salmon may offset some, but not all thermal inhibition of reproductive function.

A transcriptomic investigation of digestive processes in orange-spotted grouper, Epinephelus coioides, before, during, and after metamorphic development

The orange-spotted grouper (OG), Epinephelus coioides, is an ecologically and economically important species with strong market demand. However, larval rearing for this species is especially difficult, with mass mortality occurring at multiple stages including the period coinciding with metamorphic development. The aim of the present study was to characterise the molecular ontogenesis of genes that influence appetite, feeding, and digestion in OG larvae head and body tissue at 12, 18, and 50 days post hatch (dph), which coincides with the beginning and end of metamorphic development. The sequences of many transcripts involved in the regulation of appetite, feeding and digestive processes were detected from 12 dph in OG larvae, including those that were differentially expressed in body tissue in fish at different stages of development such as cholecystokinin, peptide Y, and meprin A. Of the transcripts encoding digestive enzymes, only the expression level of bile salt-activated lipase decreased as development progressed. In contrast, a dramatic increase in expression for other body-expressed transcripts encoding digestive enzymes and a proton pump subunit was observed at 50 dph, which is indicative of an increase in digestive capacity. In addition, we have provided evidence suggesting that various trypsinogen isoforms are present, and have differing expression patterns throughout larval development in whole body tissue. We also report on the presence of a prey-specific transcript encoding α-amylase that was present in the body-transcriptome. Taken together, these results give insight into the processes underpinning attainment of digestive capacity, and form the basis of a new transcriptomic database that will aid further study into the digestive development and dietary requirements of orange-spotted grouper larvae.

Effects of GnRHa treatment during vitellogenesis on the reproductive physiology of thermally challenged female Atlantic salmon (Salmo salar)

Tasmanian Atlantic salmon (S. salar) broodstock can experience temperatures above 20 °C, which impairs reproductive development and inhibits ovulation. The present study investigated the prolonged use of gonadotropin releasing hormone analogue (GnRHa) during vitellogenesis as a means of maintaining endocrine function and promoting egg quality at elevated temperature in maiden and repeat spawning S. salar. GnRHa-treatment during vitellogenesis did not compensate for the negative effects of thermal challenge on the timing of ovulation, egg size, egg fertility or embryo survival in any fish maintained at 22 °C relative to 14 °C. The lack of effectiveness was reflected by the endocrine data, as plasma follicle stimulating hormone and luteinising hormone levels were not different between treated and untreated groups at 22 °C. Furthermore, plasma testosterone and E2 levels were unchanged in GnRHa-treated fish at 22 °C, and plasma levels were generally lower in both groups maintained at 22 °C relative to 14 °C. Transcription of vitellogenin, and zona pellucida B and C was not enhanced in GnRHa-treated fish relative to untreated fish at 22 °C, presumably due to observed suppression of plasma E2. These results indicate that thermal impairment of reproduction is likely to occur on multiple levels, and is difficult to overcome via hormonal manipulation.

Estrogen therapy offsets thermal impairment of vitellogenesis, but not zonagenesis, in maiden spawning female Atlantic salmon (Salmo salar)

In female Atlantic salmon (Salmo salar), exposure to warm summer temperatures causes a reduction in plasma 17β-estradiol (E2), which impairs downstream vitellogenesis and zonagenesis, and reduces egg fertility and embryo survival. The aim of the present study was to determine whether E2-treatment could offset thermal impairment of endocrine function and maintain egg quality in maiden (first-time-spawning) S. salar reared at 22 °C. Treatment with E2 at 22 °C stimulated vitellogenin (vtg) gene expression and subsequent protein synthesis which promoted oocyte growth and increased egg size relative to untreated fish at 14 and 22 °C. However, E2-treatment at 22 °C was not associated with an increase in egg fertility and embryo survival relative to untreated fish at 22 °C, despite the positive effects of E2-treatment on vitellogenesis and oocyte growth. As there was no evidence to suggest that the estrogen receptor alpha expression was suppressed by high temperature, this could be due to the lack of stimulation on zonagenesis by E2-treatment observed at high temperature during oocyte development. Our results demonstrate that treatment with E2 is not able to maintain zonagenesis or egg quality in maiden S. salar at high temperature, even when vtg gene expression, protein synthesis and subsequent oocyte growth is promoted. This implies that the mechanisms regulating zonagenesis, but not vitellogenesis are impaired at elevated temperature in female S. salar broodstock, and highlights the remarkable complexity of thermally induced endocrine disruption in fish.