Salmon Spawning Migration: Metabolic Shifts and Environmental Triggers
ABSTRACT: A large-scale functional genomics study revealed shifting energy generating processes in white muscle during the final 1,300 km migration of wild sockeye salmon to their spawning grounds in the Fraser River, British Columbia. In 2006, Lower Adams stock sockeye salmon ceased feeding after passing the Queen Charlotte Islands, 850 km from the Fraser River. Enhanced protein turnover and reduced transcription of actin, muscle contractile and heme-related proteins were early starvation responses in saltwater. Arrival to the estuarine environment triggered massive protein turnover through induction of proteasoma and lysosomal proteolysis and protein biosynthesis, and a shift from anaerobic glycolysis to oxidative phosphorylation. Response to entry into freshwater was modest, with up-regulation of heat shock proteins and nitric oxide biosynthesis. High river temperatures resulted in a strong defense/immune response and high mortalities in 50% of fish. Arrival to the spawning grounds triggered further up-regulation of oxidative phosphorylation and proteolysis, down-regulation of protein biosynthesis and helicase activity, and continued down-regulation of muscle proteins and most glycolytic enzymes. However, sharp up-regulation of PFK-I indicated induction of glycolytic potential at the spawning grounds. The identification of potential environmental cues triggering genome-wide transcriptional shifts in white muscle associated with migration and the strong activation of proteasomal proteolysis were both novel findings. Keywords: Functional genomics study on wild-caught fish The experiment was based on expression profiles of white muscle tissue collected from wild migrating adult sockeye salmon during their return spawning migration back to the Fraser River. Fish were collected from seven sites along the final 1,300 km migration path, and white muscle samples were quickly frozen in liquid nitrogen upon capture. Marine sampling sites included (from north to south) the Queen Charlotte Islands (QCI), Johnstone Strait (JS), Juan de Fuca Strait (JDFS), and the Strait of Georgia (SOG). Freshwater sampling sites included Whonnock (W), Savona (SV) and the Lower Adams Spawning Grounds. Genetically-based stock ID was used to identify the natal sites of fish collected from the wild. The experiment was designed to profile the transcriptional shifts associated with migration of the Adams River stock complex. The total experiment included 80 microarray slides, with a minimum biological replicate size per site of 6 (SV), and maximum of 18 (JS) (see supplemental table for details). Additional intra-site variables, which could only be addressed in some sites, included sex (female biased) and river entry timing (for JS, JDFS and W sites; identified through radio-tracking of marine collected fish). Total RNA was amplified (1 round) with MessageAmpTMII-96 kit (Ambion, TX, USA), and reverse transcribed to cDNA before labelling with ALEXA dyes using the Invitrogen Indirect Labelling Kit. The experiment was based on a reference design, with the reference containing the combined aRNA of all individuals used in the experiment. Individual samples were labelled with Alexa 555 and the reference control with Alexa 647, with no dye flips included. A single technical replicate of one SV fish (replicate 5) was included in the experimental design. This experiment is part of a larger white muscle experiment containing additional sockeye salmon stocks.
Project description:The transcriptional response of Atlantic salmon (Salmo salar) to infectious hematopoietic necrosis (IHN) virus was elucidated using 16,008 gene GRASP cDNA microarrays. S. salar were exposed to the IHN virus in a waterborne challenge, and kidney samples from five fish sampled on each of days 0, 1, 5 and 9 were analysed in the microarray study. Validation of nine of the significant genes was conducted on fish sampled on alternate days from 1 to 13 and compared to the unchallenged control sampled on day 0. The key pathways up-regulated in response to the virus included endosomal transport, type I and II interferon responses, the alternative complement pathway, apoptosis, phagocytosis and phagocytic cell oxidation, natural killer cell activity, antiviral replication via iron sequestering, virally-induced disruption of the cell cycle, retroviral DNA integration, T-cell activation and cellular immunity. Fish that did not contain viral titer on days 5 and 9 responded in a similar manner to those with titer, but further induced a number of genes involved in anti-viral replication (Glutathione peroxidase 1 and ferritin H), anti-proliferation (PTEN and B-cell receptor associated protein 32), lysosomal response (CD63 antigen), pro-inflammatory response (lipopolysaccharide binding protein, stress activated JNK1, thiol peroxidase), and T-cell activation (T-cell activation Rho-GTPase activating protein) that may increase resistance to the virus. Three genes that were potentially co-opted by the virus to enhance infectivity were also identified, including uPAR (angiogenesis), CypA (viral replication and infectivity), and BAF1 (viral protein biosynthesis). Perhaps the most notable finding was the up-regulation of uPAR which can function to increase the density of fibronectin, the receptor of the IHN virus, on the cell surface, hence facilitate viral entry into the cell. Keywords: Immunlogical response to IHN virus challenge A preliminary microarray experiment was run on the waterborne-challenged S. salar whereby the five individuals sampled on a given day were combined and run on a single slide, and we compared the expression profiles from each day (days 0 (unchallenged control) through 11). From this experiment, we noted that the most profound changes were occurring around days 1, 5, and 9, so we performed subsequent experiments at these time points, with five fish per day and 5 control fish analysed in total.
Project description:Although studies have established that exogenous growth hormone (GH) treatment stimulates growth in fish, its effects on target tissue gene expression are not well characterized. We assessed the effects Posilac® (Monsanto Co., St. Louis, MO), a recombinant bovine somatotropin, on tissue transcript levels. Transcript abundance was measure in liver and muscle using the GRASP 16 K cDNA microarray. A selection of the genes identified as altered with the microarray, and also transcripts for insulin-like growth factors, growth hormone receptors (GHR) and myostatins were measured by realtime PCR in the liver, muscle, brain, kidney, intestine, stomach, gill and heart. In general, transcripts identified as differentially regulated in the muscle on the microarray showed similar direction of expression in the other non-hepatic tissues. Rainbow trout were selected from two high growth rate and two low growth rate families. A total of 113 and 67 transcripts were identified by microarray as differentially expressed with GH treatment across growth rate for muscle and liver respectively. The largest proportion of the transcripts represented novel transcripts, followed by immune and metabolism related genes. The immune related genes were primarily modulated in the liver and indicate activation of a non-specific immune response. The metabolic genes include lipid metabolism, oxidative phosphorylation and one carbon metabolism pathway transcripts. Most notable among the growth axis genes measured by realtime PCR were increases in GHR1 and-2 transcript in liver and muscle. Our results indicate that short-term GH treatment activates the immune system, shifts the metabolic sectors and modulates growth regulating genes. Keywords: Growth Hormone Injection Muscle and Liver Gene Expression Rainbow trout (hatched March 2005) selected for extreme growth rate were obtained from NCCCWA brood stock. Families were selected based on body weight at 7 months of age and thermal growth coefficient for the final month of growth. The two high growth families used in the study were in the top 2% in terms of growth rate, and the low growth families were in the lowest 10% for growth rate. Fish acclimated to the new tanks for two weeks prior to initiation of the treatments. Fish from each family were randomly selected to receive one of three treatments: 1) Posilac® injection (120 mg/kg BW, n = 4 per family); 2) vehicle injection (n = 4 per family); or 3) untouched controls (n = 2 per family). We had determined there was no effect to growth or the GH/IGF-I axis in the vehicle treated fish, and therefore, all of the microarray hybridizations were made between the GH and vehicle injected groups. This study included a total of 16 two-channel arrays designed for the direct comparison of GH treatment levels. That is, for each of the four groups, 1) High Growth Rate Liver; 2) Low Growth Rate Liver; 3) High Growth Rate Muscle; and 4) Low Growth Rate Muscle, four slide were hybridizes using individual RNA samples from individual fish. RNA isolation from each tissue/organ sample was handled separately (without pooling) with the purpose of using biological replications. We hybridized two slides with the GH cDNA labeled with Alexa 555 and vehicle cDNA labeled with Alexa 647; and two slides, using unique RNA samples, for the with GH cDNA labeled with Alexa 647 and tissue from the vehicle injected group labeled with Alexa 647 within each tissue and growth rate. Sixteen slides were used in the current study representing 32 individual tissue samples, meaning a total of four biological replicates for each treatment group.
Project description:A comparison of "maturing" and "prespawn" ovarian and testicular transciptomes was performed to determine the genes that are involved in regulating gametic and accessory cell function during maturation and development of the rainbow trout gonad. To identify some fo the genes involved in these processee, total RNA was compared between three-year-old normal vs two-year-old normal (maturing) and three-year-old normal vs two-year-old precocious (prespawn) gonadal tissue. Three biological replicates for both "maturing" and "prespawn" rainbow trout ovary and testis with corresponding dye flips.
Project description:Comparison of gene expression of exposed versus non-exposed Oncorhynchus mykiss hepatocytes to four model chemicals and a synthetic mixture. Hepatocytes were exposed for 24 hours to a single chemical and a synthetic mixture of 10 nM 17 alpha-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 μM paraquat and 0.75 μM 4-nitroquinoline-1-oxide (NQO). Four biological replicates for both exposed and non-exposed Oncorhynchus mykiss hepatocytes with corresponding dye flips.
Project description:Hybridization of one kidney of cortisol treated fish vs. one kidney of control fish. Kidneys were collected from untreated juvenile sea bream (n=4) and from fish, which received for 72h a coconut-oil implant containing 10mg/Kg (fish wet weight) (n=4) cortisol. Experiments were carried out at the University of the Algarve, Portugal in accordance with National legislation for the welfare of animals. Experiments were conducted in two 125 l cylindriconical tanks supplied with a continuous through-flow of oxygenated seawater at 20+1 °C using juvenile sea bream (25 g + 3 g) adapted for 1 week to the experimental conditions. One tank contained 8 untreated fish (control) and the other tank 8 cortisol treated fish and the end of experiments fish were removed form tanks, decapitated and the kidneys rapidly removed and place in RNAlater (Qiagen) at –20 °C. No mortality occurred in the control tank but 2 fish died in the cortisol treated tank.
Project description:All above ground organs of higher plants are ultimately derived from specialized organogenic structures called shoot apical meristems (SAMs). It is comprised of pluripotent stem cells, which divide to regenerate themselves as well as to provide cells to form other organs such as leaves and stems. To study global gene expression in maize SAM and very young primordia (P0 and P1), RNA was extracted from both SAMs (SAMs per se plus P0 and P1) and above-ground portions of seedlings collected from 14-day old B73 seedlings. These RNA samples were labeled and hybridized with cDNA microarrays that have 14,400 informative spots from maize. Statistical analyses showed that approximately 6.3% and 6.5% of the tested genes were significantly (p <0.0001) up- and down-regulated, respectively. Several control genes, whose expressions were confirmed in the maize SAM in the previous studies, were also up-regulated (p <0.01). The significantly up-regulated genes involved many novel transcription factor genes and regulatory genes as well as some kinds of enzymes, suggesting these genes play important roles in meristem maintenance and the early stage of leaf development in maize. Interestingly, several retrotransposon-related genes were greatly up-regulated in the SAM. This finding raised the possibility that retrotransposons are involved in regulatory mechanisms of maize SAM. An experimental aim is to identify genes preferentially expressed in maize SAM by comparing the transcript abundant between SAMs and seedlings using cDNA microarrays that have 14,400 informative spots from maize.
Project description:Vegetative phase change is the developmental transition from the juvenile phase to the adult phase during which a plant becomes competent for sexual reproduction. Gain of ability to flower is often accompanied by changes in patterns of differentiation in newly forming vegetative organs. In maize, juvenile leaves differ from adult leaves in morphology, anatomy, and cell wall composition. Whereas the normal sequence of juvenile followed by adult is repeated with every sexual generation, this sequence can be altered in maize by the isolation and culture of the shoot apex from an adult phase plant; an “adult” meristem so treated reverts to forming juvenile vegetative organs. To investigate the molecular differences between the juvenile and adult phases in maize comparisons among two juvenile samples, leaf 4 and culture-derived leaf 3 or 4, and an adult sample (leaf 9) were made using cDNA microarrays. All samples were leaf primordia at plastochron 6. A gene was scored as “phase specific” if it was up- (or down-) regulated in both juvenile samples compared to the adult sample with at least a twofold-change in gene expression at P-value less than or equal to 0.005. Some 221 ESTs up-regulated in juvenile and 28 ESTs up-regulated in adult were identified. Altered patterns of expression of selected ESTs in the phase change mutants Tp2, d1 and gl15 further confirmed these genes as being phase-specific and allowed us to position these genes in the known genetic hierarchy regulating phase change. Keywords: Transcript profiling among seed-derived juvenile leaf 4 and adult leaf 9 and culture-rejuvenated leaf 3 or 4 in maize To identify juvenile or adult specific ESTs, total RNA from leaf primordia at plastochron 6 (P6) was isolated from leaves 4 (L4) and 9 (L9) from seed-derived plants and leaf 3 or 4 (RL3/4) from culture-derived plants. For each of six biological replications, each of the three pairwise comparisons of P6-staged leaf primordia from L4, L9 and RL3/4 was made on one slide. With six biological replications and three slides per replication (L4 vs. L9, L9 vs. RL3/4, RL3/4 vs. L4), this replicated loop design used a total of 18 slides. To ensure dye balance, each of the 18 target samples was measured once with Cy3 labeling and once with Cy5 labeling.
Project description:Natural Antisense Transcripts (NATs) can regulate gene expression by virtue of their ability to form double-stranded RNA duplexes. To investigate NATs in the maize transcriptome, cDNAs from seedling of two inbred lines (B73 and Mo17) were hybridized to an oligonucleotide microarray designed to validate the expression of in silico detected NATs and to screen for NATs that can anneal to a random set of 3’ UTRs and selected repeats found in 3’ UTRs regions. Quantitative Real-Time PCR experiments were conducted to determine the minimum detection threshold of microarray experiment and to thereby identify genes for which both sense and antisense transcripts accumulate to detectable levels. Two independent approaches, strand-specific RT-PCR and S1 nuclease assays were conducted to validate the results of the microarray experiment. Based on these conservative assays, NATs accumulate in seedlings that can anneal to over 70% of a random set of maize genes. In addition, both sense and antisense transcripts anneal to more than 80% of a set of maize repeats. Significantly, sense and antisense transcripts exhibit significant different expression patterns between the two genotypes. Based on these findings we hypothesize that interactions between sense and antisense transcripts may contribute to the differential patterns of gene expression in maize hybrids and to heterosis. Keywords: Global antisense transcripts profiling between two maize inbreds To systematically identify NATs in maize, we employed multiple strategies to computationally identify putative antisense transcripts from our partial genome assembly (MAGIs) and a collection of ESTs we sequenced with known sequence orientation. Additionally, we randomly sampled and surveyed maize UTRs which often harbor transposons. Strand-specific oligonucleotides which can hybridize to the antisense strand were designed and spotted with together with the oligonucleotides hybridizing sense strands on a custom, strand-specific oligoarray, providing the first global expression profiling study of NATs in maize UTRs. In total, 10 biological replication were conducted to compare the global gene expression profiles between two maize genotypes (B73 and Mo17).
Project description:All above ground organs of higher plants are ultimately derived from specialized organogenic structures termed shoot apical meristems (SAMs). The SAM exhibits distinctive structural organization, marked by cell layering. Maize SAMs are comprised of two cell layers, L1 (single cell layered tunica) and L2 (corpus). To identify genes required for layer-specific functions intact maize SAMs were fixed, embedded in paraffin and sectioned. L1 and L2 cells were isolated from these sections via laser capture microdissection (LCM). RNA was isolated from six biological replications of L1 and L2, amplified and hybridized to microarrays spotted with ~37,000 maize cDNA clones. This experiment identified ~700 ESTs that are preferentially expressed in the L1 or the L2 (P <0.001). The L1-up-regulated ESTs included ZmOCL1 and ZmOCL4, which are known to exhibit L1-specific expression in the maize SAM. The L2-up-regulated ESTs included KNOTTED1, whose transcripts are known to accumulate in the L2 but not in the L1 of the maize SAM. Differentially expressed ESTs included genes involved in transcription, signal transduction, transport and metabolism, many of which are novel candidates that are required for layer-specific functions in the maize SAM. Several L1-up-regulated ESTs were annotated as yabby family genes or basic helix-loop-helix transcription factor-like genes, which have not previously been reported as having layer-specific expression in the SAM. Novel WW domain-containing genes (WW genes) were identified in this study. The WW domain mediates protein-protein interactions, often with signal transduction components. These WW genes were substantially up-regulated in the L1 relative to the L2. Keywords: Cell Type Comparison An experimental aim is to identify genes that are differentially expressed in distinct histological cell layers of maize SAM by comparing the transcript accumulation between L1 (single cell layered tunica) and L2 (corpus) using cDNA microarrays that have over 37,000 informative spots from maize.
Project description:All above ground organs of higher plants are ultimately derived from specialized organogenic structures called shoot apical meristems (SAMs). It is comprised of pluripotent stem cells, which divide to regenerate themselves as well as to provide cells to form other organs such as leaves and stems. To study global gene expression in maize SAM and very young primordia (P0 and P1), RNA was extracted from both whole SAMs (SAMs, P0 and P1) and whole seedlings (above ground part of seedlings) collected from 14-day old B73 seedlings. These RNA samples were labeled and hybridized with cDNA microarrays that have about 30,000 maize cDNA clones. Statistical analyses showed that approximately 7% and 8% of the tested genes were significantly up-regulated and down-regulated, respectively. Several control genes, whose expressions were confirmed in the maize SAM in the previous studies, were significantly up-regulated. Many histone genes and cell cycle related genes were also significantly up-regulated. Those observations validated our microarray results. The significantly up-regulated genes involved many novel transcription factor genes and regulatory genes as well as some kind of enzymes, suggesting those genes play important roles in meristem maintenance and the early stage of leaf development in maize. Surprisingly, several retrotransposon-related genes were greatly up-regulated with the statistical significance. This finding raised the possibility that retrotransposons are involved in regulatory mechanism of maize SAM. An experimental aim is to identify genes preferentially expressed in maize SAM by comparing the transcript abundant between whole SAMs and whole seedlings using cDNA microarrays that have about 30,000 maize cDNA clones.