Publications

PROJECT: Pleurogene

A nutrigenomic analysis of intestinal response to partial soybean meal replacement in diets for juvenile Atlantic halibut, Hippoglossus hippoglossus, L. (2009)

Murray, H. M., Lall, S. P., Rajaselvam, R., Boutilier, L. A., Blanchard, B., Flight, R. M., Mohindra, V. and Douglas, S. E.

Aquaculture DOI

Aquaculture feeds for carnivorous finfish species has been dependent upon the use of fish meal as the major source of dietary protein; however, the increasing demands upon the finite quantity of this high-quality protein source requires that feeds become increasingly comprised of alternative plant and/or animal protein. Soybean meal has been has been used to partially replace fish meal in the diets of several fish but it is known to cause enteritis in Atlantic salmon, Salmo salar. We have compared two groups of juvenile (207.2±6.6 g) Atlantic halibut, Hippoglossus hippoglossus, L., fed diets containing fish meal (FM; control) or 30% soybean meal (SBM; experimental) as a protein source for 3 weeks. No detectable difference in feed intake or palatability was evident with the SBM diet relative to the FM diet. Histological examination of the distal intestine was performed to examine leukocyte infiltration of the lamina propria and other changes in morphology commonly observed with soybean-induced enteritis of salmonids. No significant difference was found between fish fed the FM and SBM diets. Global gene expression profiling performed using a high-density oligonucleotide microarray containing 9260 unique features, printed in quadruplicate, from Atlantic halibut revealed subtle underlying changes in the expression of several immune genes and genes involved in muscle formation, lipid transport, xenobiotic detoxification, digestion and intermediary metabolism. These results indicate that SBM can be used successfully as a replacement for animal protein in diet for juvenile Atlantic halibut, although long-term effects on the immune system may ensue.

Effect of Early Introduction of Microencapsulated Diet to Larval Atlantic Halibut, Hippoglossus hippoglossus L. Assessed by Microarray Analysis (2009)

Murray, H. M., Lall, S. P., Rajaselvam, R., Boutilier, L. A., Flight, R. M., Blanchard, B. Colombo, S., Mohindra, V., Yufera, M. and Douglas, S. E.

Marine Biotechnology DOI

An experimental microdiet prepared using an internal gelation method was used to partially replace the traditional live feed (Artemia) for larval Atlantic halibut, Hippoglossus hippoglossus L. Three trials were conducted with microdiet introduced at 20, 32, and 43 days post first feeding and larvae were sampled at approximately 2, 13, 23, and 33 days after microdiet introduction in each trial. The success of feeding was assessed by morphometrics and histological analysis of gut contents. Microdiet particles were readily consumed after a period of adaptation and provided an adequate source of nutrients with no significant increase in mortality in the microdiet-fed group compared to the control group. However, growth was limited and there was an increased incidence of malpigmentation of the eye and skin. Subtle changes in underlying digestive and developmental physiology were revealed by microarray analysis of RNA from control and experimental fish given microdiet from day 20 post first feeding. Fifty-eight genes were differentially expressed over the four sampling times in the course of the trial and the 28 genes with annotated functions fell into five major categories: metabolism and biosynthesis, cell division and proliferation, protein trafficking, cell structure, and stress. Interestingly, several of these genes were involved in pigmentation and eye development, in agreement with the phenotypic abnormalities seen in the larvae.

A first generation Atlantic halibut Hippoglossus hippoglossus (L.) microarray: application to developmental studies (2008)

S. E. Douglas, L. C. Knickle, J. Williams, R. M. Flight M. E. Reith

Journal of Fish Biology Volume 72 Issue 9, Pages 2391 DOI

An oligonucleotide microarray containing 50-mer oligonucleotides representing 9277 unique Atlantic halibut Hippoglossus hippoglossus genes has been designed, printed and is currently being used for the study of gene expression in developing halibut. The oligonucleotides are based on all the Atlantic halibut data available at the time of printing, these included expressed sequence tags (ESTs) and complete cDNAs derived from the Pleurogene sequencing project as well as sequences deposited in GenBank by other groups as of September 2006. Of the Pleurogene ESTs, 5040 are functionally annotated; the remainder are unknown (1016) or are similar to unannotated sequences in GenBank (1626). In addition to Atlantic halibut features, several control features have been incorporated, including an oligonucleotide representing a heterologous plant gene (92 spots) and empty spots containing buffer only (1344). The array contains 48 subgrids, each comprised of 32 columns and 26 rows. Every feature is printed at least four times as side-by-side quadruplicates, resulting in a microarray containing 39 936 features. This microarray has been utilized to identify genes differentially expressed in larval Atlantic halibut during the developmental period from post-hatch to post-metamorphosis. Early in development, transcription of the gene for hatching enzyme was down-regulated, whereas a gene involved in eye development was up-regulated. Midway to metamorphosis, transcription of genes encoding several key digestive enzymes was up-regulated, and in pre-metamorphic larvae, transcription of genes encoding muscle proteins was prominent.

Ontogenetic and tissue-specific expression of preproghrelin in the Atlantic halibut, Hippoglossus hippoglossus L. (2008)

Manning, T., Murray, H., Gallant, J. W., Matsuoka, M., Radford, E. and Douglas, S. E.

J. Endocrinol. 196: 181-192 DOI

Ghrelin is a conserved vertebrate hormone that affects both GH release and appetite. We have cloned and characterized Atlantic halibut preproghrelin cDNA and examined for the first time preproghrelin expression during fish larval development using quantitative real-time PCR. In addition, cellular sites of expression in larvae and tissue-specific expression in 3-year-old halibut were studied. A full-length cDNA for preproghrelin was isolated from halibut stomach tissue. The 899 bp cDNA encodes an open reading frame of 105 amino acids that is comprised of a signal peptide and two peptides with high similarity to ghrelin and obestatin. The deduced amino acid sequence of halibut ghrelin peptide (GSSFLSPSHKPPKGKPPRA) shows significant conservation relative to other teleostean sequences and is identical to human ghrelin for the first seven amino acids of the sequence. The putative obestatin peptide is well-conserved among fishes but shares limited similarity with its human counterpart. Expression of ghrelin was localized to two different cell types in the stomach of larval halibut by in situ hybridization. However, sensitive PCR assays on tissues collected from 3-year-old fish additionally identified ghrelin transcripts in pyloric caecae, intestine, and in immature ovary and testis. Ontogenetic studies detected ghrelin expression prior to exogenous feeding during larval development (hatching and mouth-opening stages) with increased expression occurring through metamorphosis. This increase was pronounced during climax metamorphosis and coincided with stomach differentiation. Patterns of preproghrelin expression suggest that ghrelin has important roles during and after larval development in halibut, and that ghrelin is associated with digestive and gonadal tissues in this teleost.

Translational machinery of Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.): comparative sequence analysis of the complete set of 60S ribosomal proteins and their expression. (2008)

Matsuoka, M., Infante, C., Cañavate, J. P., Reith, M. E., Douglas, S. E. and Manchado, M.

Mar. Biotechnol. 10:676–691 DOI

Ribosomal proteins (RPs) comprise a large set of highly evolutionarily conserved proteins that are often over-represented in complementary DNA libraries. They have become very useful markers in comparative genomics, genome evolution, and phylogenetic studies across taxa. In this study, we report the sequences of the complete set of 60S RPs in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species. Amino-acid sequence comparisons of the encoded proteins showed a high similarity both between these two flatfish species and with respect to other fish and human counterparts. Expressed sequence tag analysis revealed the existence of paralogous genes for RPL3, RPL7, RPL41, and RPLP2 in Atlantic halibut and RPL13a in Senegalese sole as well as RPL19 and RPL22 in both species. Phylogenetic analysis of paralogs revealed distinct evolutionary histories for each RP in agreement with three rounds of genome duplications and lineage-specific duplications during flatfish evolution. Steady-state transcript levels for RPL19 and RPL22 RPs were quantitated during larval development and in different tissues of sole and halibut using a real-time polymerase chain reaction approach. All paralogs were expressed ubiquitously although at different levels in different tissues. Most RP transcripts increased coordinately after larval first-feeding in both species but decreased progressively during the metamorphic process. In all cases, expression profiles and transcript levels of orthologous genes in Senegalese sole and Atlantic halibut were highly congruent. The genomic resources and knowledge developed in this survey will be useful for the study of Pleuronectiformes evolution.

Comparative sequence analysis of the complete set of 40S ribosomal proteins in the Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.) (Teleostei: Pleuronectiformes): phylogeny and tissue- and development-specific (2007)

Manuel Manchado, Carlos Infante, Esther Asensio, Jose Pedro Canavate and Susan E Douglas

BMC Evolutionary Biology 7:107 DOI

Background Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. Their levels can vary as a function of organism growth and development; however, some RPs have been associated with other cellular processes or extraribosomal functions. Their high representation in cDNA libraries has resulted in the increase of RP sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis. Results The development of large-scale genomics of Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of RP sequences for the small (40S) ribosome subunit. Amino acid sequence comparisons showed a high similarity both between these two flatfish species and with respect to other fish and human. EST analysis revealed the existence of two and four RPS27 genes in Senegalese sole and Atlantic halibut, respectively. Phylogenetic analysis clustered RPS27 in two separate clades with their fish and mammalian counterparts. Steady-state transcript levels for eight RPs (RPS2, RPS3a, RPS15, RPS27-1, RPS27-2, RPS27a, RPS28, and RPS29) in sole were quantitated during larval development and in tissues, using a real-time PCR approach. All eight RPs exhibited different expression patterns in tissues with the lowest levels in brain. On the contrary, RP transcripts increased co-ordinately after first larval feeding reducing progressively during the metamorphic process. Conclusion The genomic resources and knowledge developed in this survey will provide new insights into the evolution of Pleuronectiformes. Expression data will contribute to a better understanding of RP functions in fish, especially the mechanisms that govern growth and development in larvae, with implications in aquaculture.

Comprehensive EST analysis of Atlantic halibut (Hippoglossus hippoglossus), a commercially relevant aquaculture species (2007)

Susan E Douglas, Leah C Knickle, Jennifer Kimball and Michael E Reith

BMC Genomics 2007, 8:144 DOI

Background An essential first step in the genomic characterisation of a new species, in this case Atlantic halibut (Hippoglossus hippoglossus), is the generation of EST information. This forms the basis for subsequent microarray design, SNP detection and the placement of novel markers on genetic linkage maps. Results Normalised directional cDNA libraries were constructed from five different larval stages (hatching, mouth-opening, midway to metamorphosis, premetamorphosis, and post-metamorphosis) and eight different adult tissues (testis, ovary, liver, head kidney, spleen, skin, gill, and intestine). Recombination efficiency of the libraries ranged from 91–98% and insert size averaged 1.4 kb. Approximately 1000 clones were sequenced from the 5'-end of each library and after trimming, 12675 good sequences were obtained. Redundancy within each library was very low and assembly of the entire EST collection into contigs resulted in 7738 unique sequences of which 6722 (87%) had matches in Genbank. Removal of ESTs and contigs that originated from bacteria or food organisms resulted in a total of 7710 unique halibut sequences. Conclusion A Unigene collection of 7710 functionally annotated ESTs has been assembled from Atlantic halibut. These have been incorporated into a publicly available, searchable database and form the basis for an oligonucleotide microarray that can be used as a tool to study gene expression in this economically important aquacultured fish.

Gynogenesis and sex determination in Atlantic Halibut (Hippoglossus hippoglossus) (2006)

Tvedt HB, Benfey TJ, Martin-Robichaud DJ, McGowan C, Reith M

Aquaculture Volume 252, Issues 2-4, Pages 573-583 DOI

Gynogenesis refers to a process of uniparental inheritance whereby the resulting offspring retain only maternal DNA. It has been used to identify sex-determining mechanisms in fish and to produce all-female populations for aquaculture. The objective of this study was to develop a protocol for the production of gynogenetic Atlantic halibut for both these purposes. Various milt concentrations (1:20, 1:40 and 1:80 dilutions in seminal plasma) and UV doses (0–1382 mJ/cm2) were tested for providing genetically inactivated, yet motile, spermatozoa for the production of gynogenetic haploids. Spermatozoon motility, assessed both as duration of swimming and percentage of motile spermatozoa, declined with increasing UV dose. Haploidy in developing embryos was determined either visually (‘haploid syndrome’) or by genetic analysis using microsatellite DNA. The optimum milt treatment for the production of haploids was 1:80 dilution followed by a UV exposure of 65 mJ/cm2. Retention of the second polar body for the production of gynogenetic diploids was achieved by exposing newly activated eggs (15 min post-activation at 5–6 °C) to hydrostatic pressure of 8500 psi for 5 min. These treatments were combined to produce a population of gynogenetic diploids that was subsequently demonstrated to be comprised solely of females, thereby demonstrating that female is the homogametic sex in this species. It should therefore be possible to produce all-female populations of Atlantic halibut for commercial culture through gynogenesis or by crossing hormonally masculinized genotypic females to normal females.

Real-time PCR analysis of ovary- and brain-type aromatase gene expression during Atlantic halibut (Hippoglossus hippoglossus) development (2006)

Matsuoka MP, van Nes S, Andersen O, Benfey T, Reith M

Comparative Biochemistry and Physiology Part B, Volume 144, Issue 1, Pages 128-135 DOI

Two forms of cytochrome P450 aromatase, acting in both the brain and the ovary, have been implicated in controlling ovarian development in fish. To better understand the expression of these two enzymes during sexual differentiation in Atlantic halibut (Hippoglossus hippoglossus), real-time PCR was used to quantify the mRNA levels of ovary- (cyp19a) and brain-type cytochrome P450 aromatase (cyp19b) genes in the gonad and brain during gonadal development. Both enzymes showed high levels of expression in both tissues in developmental stages prior to histologically detectable ovarian differentiation (38 mm fork length), with increased expression occurring slightly earlier in the brain than the gonad. Cyp19a showed a second peak of expression in later stages (> 48 mm) in the gonad, but not the brain. Cyp19b expression was generally higher in the brain than the gonad. These results suggest that sexual differentiation may begin in the brain prior to gonadal differentiation, supporting the idea that steroid hormone expression in the brain is a key determinant of phenotypic sex in fish. In an examination of sexually immature adults, cyp19a was highly expressed in female gonad while cyp19b was very highly expressed in the pituitary of both sexes. The ratio of cyp19a to cyp19b expression was much higher in ovaries than in testes in the adult fish, so this ratio was analyzed in the developing gonads of juvenile halibut in an attempt to infer their sex. This was only partially successful, with about half the fish in later developmental stages showing apparently sex-specific differences in aromatase expression.

Characterization of steroid enzyme genes and real-time PCR analysis of their expression during gender differentiation in Atlantic halibut (Hippoglossus hippoglossus), summer flounder (Paralichthys dentatus), and black sea bass (Centropristis striata) (2005)

M.P. Matsuoka, S. van Nes, Ø. Andersen, D.L. Berlinsky, T.J. Benfey, and M. Reith

International Marine Biotechnology Conference, June 7-12, 2005

We have been characterizing steroid enzyme genes, including ovary- and brain-type aromatase and 11β-hydroxylase, which catalyze key steps in the synthesis of 17β-estradiol and 11-ketotestosterone, respectively. We also have been investigating their expression during gender differentiation period in juveniles, as well as in various adult tissues using quantitative real-time PCR. Determining the period when gonad development occurs would define the critical time when hormonal or environmental treatments should be applied to influence gender ratios. We characterized complete cDNA sequences of three genes in Atlantic halibut, and partial gene sequences in summer flounder and black sea bass. The results of gene expression analysis from Atlantic halibut suggest that treatments to manipulate gender ratios should be started in fish as small as 16 mm, when both of the aromatase gene expression levels are elevated in the brain. For all three genes, elevation of the expression levels was observed in both brain and gonad regions in fish prior to histological gender differentiation (approximately 32 mm). The elevation in the brain occurred slightly earlier than in the gonad, supporting the idea that steroid hormone expression in the brain is an early determinant of phenotypic gender in fish. However, we were unable to detect a consistent pattern of gene expression that would be diagnostic of gender.

Flatfish Genomics at IMB: Past Results and Future Plans (2005)

Douglas, S. E., Matsuoka, M., Murray, H. M., Cerdà, J., Martin-Robichaud, D., Reid, D., Blanchard, B. and Reith, M.

International Marine Biotechnology Conference, St. John’s, Nfld. June 7-12, 2005.

Flatfish such as Atlantic halibut (Hippoglossus hippoglossus), winter flounder (Pseudopleuronectes americanus) and yellowtail flounder (Pleuronectes ferrugineus) have good potential for aquaculture in Atlantic Canada. Production-related problems in these species may be addressed with improved knowledge of basic biological processes such as reproduction, development, nutrition, and immunity obtained through genomic approaches. Initially, EST surveys of six winter flounder tissues were conducted, generating information on genes expressed in adult tissues and providing molecular probes for developmental and other studies. Subsequent sequencing of several genomic regions revealed basic characteristics of the flatfish genome. Analysis of wild winter flounder using microsatellites enabled their discrimination into genetically distinct populations and provided tags for following interspecific crosses between winter and yellowtail flounders. Similar analysis of Atlantic halibut broodstock and their progeny has allowed the creation of a preliminary linkage map. PLEUROGENE is a new initiative funded through Genome Canada-Genome Spain (2004-2007) that will extend these genomic studies in Atlantic halibut and provide complementary information on Senegal sole (Solea senegalensis), a species under aquaculture development in Spain. There are two main goals: construction of genetic linkage maps for use in marker-assisted selection of superior broodstock, and design, construction and use of a flatfish microarray for gene expression studies in these two species. High-throughput genome- and proteome-based technologies will be used for the identification, characterization and mapping of genes important for reproduction, larval development, immunity and nutrition, ultimately leading to the establishment of new technologies for the control of reproduction and optimization of larval health and nutrition in the Senegal sole, Atlantic halibut, and other related flatfish species under intensive culture conditions.

Microarray Research at IMB: Past, Present and Future (2005)

Douglas, S. E.

Atlantic Microarray Symposium, Moncton, New Brunswick. Aug. 25, 2005

Acquisition of a ScanArrayTM 5000XL Microarray Acquisition scanner (Packard BioChip Technologies) in 1999 allowed the initiation of microarray experiments at the Institute for Marine Biosciences. Following a ramp-up period during which we adapted various labeling and hybridization protocols from other genomics institutes, we embarked on a series of experiments aimed at investigating the host-pathogen relationship in Atlantic salmon following infection with the bacterial pathogen, Aeromonas salmonicida. We designed a cDNA microarray comprised of 4104 different features, predominantly selected from 4 non-normalized salmonid cDNA libraries from liver, head kidney, macrophage and spleen as well as cDNA libraries constructed by suppression subtraction hybridization from the same tissues infected by A. salmonicida. Features were printed as side-by-side duplicates and the entire array of 8208 features was duplicated on the bottom portion of the slide, resulting in a microarray containing over 16,000 features. We routinely prepare amplified cDNA from 100 ng of mRNA from control and experimental samples using the SuperSmart PCR cDNA Synthesis Kit and Advantage 2 PCR kit (both from BD Biosciences) and label with Cy3- and Cy5-dCTP (Amersham). Spot intensities are quantified using the QuantArrayTM software, and all normalization and data analysis are carried out using the GeneTrafficTM software (Iobion Informatics) and Significance Analysis of Microarrays (SAM). An overview of the experiments conducted using the IMB salmon microarray will be presented.

Microsatellite analysis indicates an absence of population structure among Atlantic halibut (Hippoglossus hippoglossus) in the northwest Atlantic Ocean (2005)

Reid D, Pongsomboon S, Jackson T, McGowan C, Murphy C, Martin-Robichaud D, Reith M

J. Fish Biology 67, 570-576 DOI

To explore the population structure of Atlantic halibut Hippoglossus hippoglossus, 160 fish from four locations in the north‐west Atlantic (Bay of Fundy, Scotian Shelf, Gulf of St Lawrence and Iceland) were examined for evidence of population structure using 18 microsatellite markers. Pair‐wise FST and a model‐based cluster analysis revealed no significant differentiation between samples, although uncertainties surrounding Atlantic halibut reproductive behaviour made it difficult to ascertain that only a single breeding population had been sampled at each location.

PLEUROGENE: Flatfish Genomics and Proteomics for Aquaculture (2005)

Cerdà, J., Douglas, S. E., Reith, M., Buesa, C., Canavate, P. Lopez-Barea, J., Manchado, M., Martinez, G., Navas, J. I., Piferrer, F., Planas, J. V., Prat, F., Ruiz-Rejoin, M., Yufera, M.

Society for Experimental Biology, Barcelona, Spain. July 11-15, 2005.

Senegal sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus) are two flatfish yielding high value market products with good potential for aquaculture in Mediterranean Europe and eastern North America, respectively. Production-related problems in these two evolutionary-related species may be addressed with improved knowledge of important basic biological processes such as reproduction, development, nutrition, genetics and immunity. The use of genomic and proteomic approaches to thoroughly characterize these processes will translate into knowledge that can be used to overcome the production obstacles and create (for Senegal sole) or expand (for Atlantic halibut) solid aquaculture industries. PLEUROGENE is a new research programme funded by Genome Spain and Genome Canada with two main goals: the analysis of global gene expression during sex differentiation, reproduction, larval development, immunity and nutrition, and the construction of genetic linkage maps for use in the selection of improved broodstock. High-throughput genome- and proteome-based technologies will be applied to establish gene expression profiling during these processes and to discover novel genes. All genetic, molecular and morphological information obtained in this project will be integrated into an interactive bioinformatics platform specifically developed, the Solea-mold. The knowledge generated by the PLEUROGENE project will ultimately lead to the establishment of new technologies for the control of reproduction and optimization of larval health and nutrition in the Senegal sole, Atlantic halibut, and other related flatfish species under intensive culture conditions.

Avoiding genetic bottlenecks in broodstock selection (2004)

Reith M, Jackson T, and Martin-Robichaud, D.

Global Aquaculture Alliance Advocate, February 2004

link to article: http://pdf.gaalliance.org/pdf/gaa-reith-feb04.pdf

PLEUROGENE: Genomics for the enhancement of commercial production of Atlantic halibut and Senegal sole (2004)

Matsuoka, M. Reith, M., Cerdà, J., Murray, H. M., Martin-Robichaud, D., Blanchard B. and Douglas, S. E.

Flatfish Biology Conference, Westbrook, CT, Dec 1-2, 2004.

Atlantic halibut (Hippoglossus hippoglossus) and Senegal sole (Solea senegalensis) are two flatfish yielding high value market products with good potential for aquaculture in eastern North America and Mediterranean Europe, respectively. Production-related problems in these two evolutionarily-related species may be addressed with improved knowledge of important basic biological processes such as reproduction, development, nutrition, genetics and immunity. The use of genomic approaches to thoroughly characterize these processes will translate into knowledge that can be used to overcome the production obstacles and create (for Senegal sole) or expand (for Atlantic halibut) solid aquaculture industries for these fish. PLEUROGENE is a new initiative funded through Genome Canada-Genome Spain for three years (2004-2007). There are two main goals: the construction of genetic linkage maps for Atlantic halibut and Senegal sole for use in the selection of improved broodstock based on molecular markers, and the design, construction and use of a flatfish microarray for studies of gene expression in these two species. High-throughput genome- and proteome-based technologies will be used for the identification, characterization and mapping of genes important for reproduction, larval development, immunity and nutrition. All the genetic and molecular information obtained in this project will be integrated into an interactive bioinformatic platform specifically developed for the project. The knowledge generated by the PLEUROGENE project will ultimately lead to the establishment of new technologies for the control of reproduction and optimization of larval health and nutrition in the Senegal sole, Atlantic halibut, and other related flatfish species under intensive culture conditions.

Use of molecular genetic markers in Atlantic halibut broodstock management (2004)

Martin-Robichaud, D.J., Reid D., Jackson T., Reith M.

Bull. Aquacult. Assoc. Canada 104-2, 19-25