Susan Douglas

Contact Information

Dr. Susan Douglas
Senior Research Officer, Molecular Biology and Functional Genomics
Telephone: (902) 426-4991
Fax: (902) 426-9413
E-mail: Susan.Douglas@nrc.ca
Résumé: Download

Interests

Molecular biology and functional genomics.

Education

Professional Affiliations

Selected Awards

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.
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.
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.
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.

PROJECT: Other

Algal evolution

Molecular chaperones encoded by a reduced nucleus: the cryptomonad nucleomorph (2001)
Archibald JM, Cavalier-Smith T, Maier U, Douglas S
J Mol Evol. 2001 Jun;52(6):490-501 DOI
The highly reduced genome of an enslaved algal nucleus (2001)
Douglas S, Zauner S, Fraunholz M, Beaton M, Penny S, Deng LT, Wu X, Reith M, Cavalier-Smith T, Maier UG
Nature. 2001 Apr 26;410(6832):1091-6 PubMed
A nucleomorph-encoded CbbX and the phylogeny of RuBisCo regulators (2000)
Maier UG, Fraunholz M, Zauner S, Penny S, Douglas S.
Mol Biol Evol. 2000 Apr;17(4):576-83 PubMed
Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph (2000)
Zauner S, Fraunholz M, Wastl J, Penny S, Beaton M, Cavalier-Smith T, Maier UG, Douglas S
Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):200-5. PubMed
The nucleomorph genomes of cryptophytes and chlorarachniophytes (2000)
Maier UG, Douglas SE, Cavalier-Smith T
Protist. 2000 Aug;151(2):103-9 PubMed
Ancient gene duplication and differential gene flow in plastid lineages: the GroEL/Cpn60 example (1999)
Wastl J, Fraunholz M, Zauner S, Douglas S, Maier UG
J Mol Evol. 1999 Jan;48(1):112-7 PubMed
The Atpa Gene Cluster of Guillardia Theta (Cryptophyta): A Piece in the Puzzle of Chloroplast Genome Evolution (1999)
Carola E. W. Leitsch, Klaus V. Kowallik, Susan Douglas
Journal of Phycology v35 p28 - February 1999 DOI
The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae (1999)
Douglas SE, Penny SL
J Mol Evol. 1999 Feb;48(2):236-44 PubMed
The secondary endosymbiont of the cryptomonad Guillardia theta contains alpha-, beta-, and gamma-tubulin genes (1999)
Keeling PJ, Deane JA, Hink-Schauer C, Douglas SE, Maier UG, McFadden GI
Mol Biol Evol. 1999 Sep;16(9):1308-13 PubMed
Bonsai genomics: sequencing the smallest eukaryotic genomes (1997)
McFadden GI, Gilson PR, Douglas SE, Cavalier-Smith T, Hofmann CJ, Maier UG
Trends Genet. 1997 Feb;13(2):46-9. DOI
The large ribosomal protein gene cluster of a cryptomonad plastid: gene organization, sequence and evolutionary implications (1997)
Wang SL, Liu XQ, Douglas SE
Biochem Mol Biol Int. 1997 Apr;41(5):1035-44 PubMed
DNA Strider. An inexpensive sequence analysis package for the Macintosh (1995)
Douglas SE
Mol Biotechnol. 1995 Feb;3(1):37-45 PubMed
Genetic relatedness of toxic and nontoxic isolates of the marine pennate diatom Pseudonitzschia (Bacillariophyceae): phylogenetic analysis of 18S rRNA sequences (1994)
Douglas DJ, Landry D, Douglas SE
Nat Toxins. 1994;2(4):166-74. PubMed
STRUCTURAL, TRANSCRIPTIONAL, AND PHYLOGENETIC ANALYSES OF THE atpB GENE CLUSTER FROM THE PLASTID OF CRYPTOMONASΨ (CRYPTOPHYCEAE) (1994)
Douglas, Susan E. & Murphy, Colleen A.
Journal of Phycology 30 (2), 329-340. DOI
The photosynthetic endosymbiont in cryptomonad cells produces both chloroplast and cytoplasmic-type ribosomes (1994)
McFadden GI, Gilson PR, Douglas SE
J Cell Sci. 1994 Feb;107 ( Pt 2):649-57 PubMed
A bchI homolog, encoding a subunit of Mg chelatase, is located on the plastid genomes of red and cryptomonad algae (1993)
Douglas, S. E. and Reith, M. R.
J. Marine Biotech. 1: 135-141 PubMed
A molecular and morphological analysis of the Gymnogongrus devoniensis (Rhodophyta, Phyllophoraceae) complex in the North Atlantic (1992)
Christine A Maggs, Susan E Douglas, John Fenety, Carolyn J Bird
Journal of Phycology, v 28, #2, pp214-232, Apr 1992 DOI
A secY homologue is found in the plastid genome of Cryptomonas Φ (1992)
Susan E. Douglas
FEBS Letters, v298, #1 pp 93-96 DOI
Eukaryote-eukaryote endosymbioses: insights from studies of a cryptomonad alga (1992)
Douglas SE
Biosystems. 1992;28(1-3):57-68 PubMed
Cryptomonad algae are evolutionary chimaeras of two phylogenetically distinct unicellular eukaryotes (1991)
Douglas SE, Murphy CA, Spencer DF, Gray MW
Nature. 1991 Mar 14;350(6314):148-51. PubMed
Plastid origins (1991)
Douglas SE, Gray MW
Nature. 1991 Jul 25;352(6333):290 PubMed

Antimicrobial peptides from fish

A C-terminal glycine suppresses production of pleurocidin as a fusion peptide in Escherichia coli (2006)
Bryksa, B.C., MacDonald, L.D., Patrzykat, A., Douglas, S.E., Mattatall, N.R.
Prot. Expr. Purif. Jan;45(1):88-98 PubMed
Effects of linear cationic a-helical antimicrobial peptides on immune-relevant genes in trout. (2006)
Chiou, P. P., Yang, B., Khoo, J., Bols, N. C., Douglas, S. and Chen, T. T.
Dev. Comp. Immunol. PubMed
There are increasing evidence of the potential role of antimicrobial peptides in the regulation of immune responses in mammalian species. However, the effects of these peptides in fish have yet to be investigated. In this study, we examined the transcriptional expression profile of representative immune-relevant genes in a trout macrophage cell line, RTS11, in response to three linear cationic ?-helical antimicrobial peptides (insect cecropin B, fish pleurocidin and a cecropin analogue CF17). The expression levels of two pro-inflammatory genes, interleukin-1? (IL-1?) and cyclo-oxygenase-2 (COX-2), increased in the peptide-treated RTS11 cells. The peptides did not appear to affect the expression levels of representative genes associated with antigen presentation, interferon response or JAK/STAT signal transduction. Furthermore, the induction of IL-1? and COX-2 in RTS11 by lipopolysaccharide was not adversely affected by these three antimicrobial peptides. Overall, the data indicate a pro-inflammatory effect of the three cationic antimicrobial peptides in the inflammatory response of salmonid species, suggesting a potential application of these peptides as immune adjuvant for fish vaccination.
Antimicrobial peptides: cooperative approaches to protection (2005)
Patrzykat A, Douglas SE
Protein Pept Lett. Jan;12(1):19-25 PubMed
Structural characterization of the antimicrobial peptide pleurocidin from winter flounder (2005)
Syvitski RT, Burton I, Mattatall NR, Douglas SE, Jakeman DL
Biochemistry 44(19): 7282-7293 PubMed
Cellular localization of pleurocidin gene expression and synthesis in winter flounder gill using immunohistochemistry and in situ hybridization (2003)
Murray HM, Gallant JW, Douglas SE
Cell Tissue Res. May;312(2):197-202. PubMed
Gone gene fishing: how to catch novel marine antimicrobials (2003)
Patrzykat A, Douglas SE
Trends Biotechnol. Aug;21(8):362-9. PubMed
Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish (2003)
Douglas SE, Gallant JW, Liebscher RS, Dacanay A, Tsoi SC
Dev Comp Immunol. Jun-Jul;27(6-7):589-601 PubMed
Identification, structure and differential expression of novel pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum) (2003)
Douglas SE, Patrzykat A, Pytyck J, Gallant JW
Eur J Biochem. Sep;270(18):3720-30. PubMed
Novel antimicrobial peptides derived from flatfish genes (2003)
Patrzykat A, Gallant JW, Seo JK, Pytyck J, Douglas SE
Antimicrob Agents Chemother. Aug;47(8):2464-70 PubMed
Cloning and developmental expression of a family of pleurocidin-like antimicrobial peptides from winter flounder, Pleuronectes americanus (Walbaum) (2001)
Douglas SE, Gallant JW, Gong Z, Hew C.
Dev Comp Immunol. 25(2):137-47. PubMed

Fish

Small scale cDNA microarray analysis of expression of genes for lipid metabolism in liver of Atlantic salmon (Salmo salar L.) – effect of dietary rapeseed oil replacement. (2005)
O.-Jordal, A.-E., Torstensen, B. E., Tsoi, S. C., Tocher, D. R., Lall, S. and Douglas, S. E.
J. Nutr. 135(10): 2355-61. PubMed
Supplies of marine fish oils (FO) are limited, and sustainable production in aquaculture dictates that alternatives that do not compromise fish health and product quality, such as vegetable oils, must be found. Nutrigenomics will increase our understanding of how nutrition influences metabolic pathways and homeostatic control, and may be used to measure and validate subtle changes in organ-specific, metabolic gene expression signatures. We compared 2 groups of Atlantic salmon fed diets containing 100% FO or 75% rapeseed oil (RO) for 42 wk. A small-scale cDNA microarray was constructed to screen for changes in the expression of lipid metabolism genes in the liver resulting from this partial substitution of RO for FO. 5 fatty acid desaturase gene expression was significantly greater in fish fed 75% RO than in fish fed the control diet; this was confirmed by quantitative real time PCR analysis. In addition, several genes, among these mitochondrial proteins, peroxisome proliferator-activated receptor , as well as other transcription factors, coactivators, and signal transducers, showed significant differential regulation. This partially validated microarray may be used for further gene expression profiling using other dietary comparisons, and for further characterization of selected genes.
Use of parasite and genetic markers in delineating populations of winter flounder from the central and southwest Scotian Shelf and northeast Gulf of Maine (2005)
G. McClelland, J. Melendy, J. Osborne, D. Reid and S. Douglas
Journal of Fish Biology Volume 66 Page 1082 - April 2005 DOI
Genetic characterization, morphometrics and gonad development of induced interspecific hybrids between yellowtail flounder, Pleuronectes ferrugineus (Storer) and winter flounder, Pleuronectes americanus (Walbaum) (2003)
I-S Park, YK Nam, SE Douglas, SC Johnson3 & DS Kim
Aquaculture Research Volume 34 Page 389 - April 2003 DOI

Functional genomics of the salmonid response to infection

Identification of a transcript encoding a soluble form of toll-like receptor 5 (TLR5) in the liver of Atlantic salmon infected with Aeromonas salmonicida. (2006)
Tsoi, S. C., Park, K., Kay, H. H., O’Brien,T. J., Podor, E., Sun, G., Douglas, S. E., Brown, L. L. and Johnson, S. C.
Vet. Immunol. Immunopathol. 109:183-7. PubMed
Toll-like receptors (TLRs) are involved in the innate immune response against microbial pathogens in vertebrates and insects. The extracellular region of a TLR recognizes pathogen-associated molecules, while the intracellular region initiates the signaling pathway leading to immune response. Membrane-bound TLRs have been found in most vertebrates, but few soluble forms have been reported. A novel transcript corresponding to a portion of a soluble TLR was identified in liver of infected Atlantic salmon. The complete coding sequence of this TLR was obtained and BLASTN analysis showed the highest sequence identity to a recently described full-length cDNA sequence of a soluble TLR5 from rainbow trout (GenBank Accession No.: AB062504). The deduced protein is 40% identical to the mammalian counterpart of the leucine-rich repeat (LRR)/LRR-like motifs of TLR5. Based on the structure of human TLRs, it contains 21 LRRs with conserved LxxLxLxxNx*xx*xxxxFxxL pattern. Since TLR5 is essential for the recognition of bacterial flagellins, we hypothesize that flagellin and perhaps some other pathogen-derived factors from Aeromonas salmonicida bind to this soluble TLR through an unknown binding domain within the LRR.
Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cDNA microarray technology (2005)
Ewart KV, Belanger JC, Williams J, Karakach T, Penny S, Tsoi SC, Richards RC, Douglas SE
Dev Comp Immunol. 2005;29(4):333-47 PubMed
Identification of immune-relevant genes from atlantic salmon using suppression subtractive hybridization (2004)
Tsoi SC, Ewart KV, Penny S, Melville K, Liebscher RS, Brown LL, Douglas SE
Mar Biotechnol (NY). 2004 May-Jun;6(3):199-214 PubMed
Use of human cDNA microarrays for identification of differentially expressed genes in Atlantic salmon liver during Aeromonas salmonicida infection (2003)
Tsoi SC, Cale JM, Bird IM, Ewart V, Brown LL, Douglas S
Mar Biotechnol (NY). 2003 Nov-Dec;5(6):545-54 PubMed

Molecular biological approaches to digestion in larval marine fish

Characterization of a partial a-amylase clone from red porgy (Pagrus pagrus): Expression during larval development. (2006)
Darias, M. J., Murray, H. M., Gallant, J. W., Astola, A., Douglas, S. E., Yufera, M. and Martinez-Rodriguez, G.
Dev. Comp. Immunol. PubMed
A partial ?-amylase cDNA was isolated from red porgy (Pagrus pagrus, Teleostei: Sparidae) and its tissue specific expression during larval development was examined. The cDNA was 949 bp long and showed 90% identity with other fish amylases. A 545 bp fragment was used to study amylase expression using in situ hybridization and RT-PCR techniques. Both methods showed a similar pattern: high and relatively constant expression for the first 30 days after hatching (dah), subsequently decreasing until the end of the experiment at 60 dah. The goal of this work was to extend the existing knowledge of the functionality of larval fish digestive systems and to provide new information about ?-amylase gene expression.
Cloning and expression analysis of three digestive enzymes from Atlantic halibut (Hippoglossus hippoglossus) during early development: Predicting gastrointestinal functionality (2006)
H.M. Murray, J.W. Gallant, S.C. Johnson and S.E. Douglas
Aquaculture (in press) DOI
Cloning and expression analysis of three digestive enzymes from Atlantic halibut during early development: predicting gastrointestinal functionality. (2006)
Murray, H.M., Gallant, J.W., Johnson, S.C., and Douglas, S.E.
Dev. Comp. Immunol. PubMed
The objective of the present study was to describe the histological and physiological development of the gastrointestinal system in Atlantic halibut from the time of first-feeding until metamorphosis. At first-feeding, the gastrointestinal (GI) tract is divided into anterior, mid and hindgut regions. The liver is present, as is the pancreas. During development the pancreas changes from a compact organ to a diffuse tissue interspersed through much of the mesentery surrounding the GI tract. Functional gastric glands are not present until approximately 66 days post-hatch (dph). Using primers based upon winter flounder digestive enzyme gene sequences, we were able to amplify partial sequences for bile salt-activated lipase (BAL), trypsinogen (TRP), and pepsinogen (PEP) from RNA extracted from whole larvae and juveniles using Reverse Transcription-PCR (RT-PCR). PCR products were sequenced and the sequences used to design halibut gene-specific primers for BAL and PEP. RT-PCR analysis revealed that BAL and TRP gene expression was evident at least from the time of first-feeding but PEP gene expression was not detectable until 80 dph. In situ hybridization using molecular probes from winter flounder sequences localized expression of BAL and TRP to the exocrine pancreas. PEP expression was only localized to the glandular regions of the stomach. These data provide a first step toward understanding the molecular events governing the ontogeny of digestive capacity in Atlantic halibut.
Development of digestive capacity in larvae of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua) (2006)
Perez-Casanova, J.C., Murray, H.M., Gallant, J.W., Ross, N.W., Douglas, S. E. and Johnson, S.C.
Aquaculture 251:377-401 PubMed
Using biochemical and molecular biological techniques, we describe the expression of several key digestive enzymes throughout the ontogeny of larval haddock and Atlantic cod. The pattern of activity of general proteases (GP), trypsin-like enzymes (TLE), pepsin-like enzymes (PLE), general lipase (GL), bile salt-activated lipase (BAL), alkaline phosphatase (AP) and ?-amylase were studied in larvae of both species using colourometric techniques. All enzymes were detected as early as hatch, except for ?-amylase. Activity of GP generally increased from 0 to 144 degree-days (DD), decreased until 333 DD and then increased again in the oldest larvae of both species. Activity of TLE in haddock was high at hatch and generally showed an inverse pattern to that of Atlantic cod. Using zymography, we detected serine proteases, particularly trypsin, in the protein digestion of both species. Using RT-PCR, trypsinogen transcripts were detected as early as hatch. For both species, in situ hybridization analysis showed localization of trypsinogen expression to the pancreas during larval development. Although activity of PLE was detected as early as hatch in both species, pepsinogen transcripts were detected by RT-PCR only in the oldest larvae sampled, and after the gastric glands were identified morphologically. Atlantic cod BAL activity showed no significant differences over time. Activity of GL remained constant over time in larvae of both species. Levels of AP activity in haddock larvae showed no significant differences from hatch to 333 DD, except at 66 and 401 DD when there was a significant increase compared to other time points. In Atlantic cod, AP activity remained relatively constant until 476 DD, when a significant increase was detected. ?-Amylase activity could not be detected in either species, except at 43 and 60 DD for Atlantic cod and 144 DD for haddock when large numbers of live prey were present in the gut. The estimated contribution of Pavlova lutheri-enriched rotifers to the total larval digestion was negligible, except for ?-amylase, for which their contribution was estimated to be 100%. Our results suggest that both Atlantic cod and haddock larvae are capable of digesting protein and lipids at the time of mouth opening and that they have a limited capacity to digest carbohydrates.
Ontogeny of lipase expression in winter flounder (2006)
H. M. Murray, J. W. Gallant, J. C. Perez-Casanova, S. C. Johnson and S. E. Douglas
Journal of Fish Biology Volume 62 Page 816 - April 2003 DOI
Dietary rapeseed oil affects the expression of genes involved in hepatic lipid metabolism in Atlantic salmon (Salmo salar L.) (2005)
Jordal AE, Torstensen BE, Tsoi S, Tocher DR, Lall SP, Douglas SE
J Nutr. 2005 Oct;135(10):2355-61 PubMed
Bile salt-activated lipase expression during larval development in the haddock (Melanogrammus aeglefinus) (2004)
J.C. Perez-Casanova, H.M. Murray, J.W. Gallant, N.W. Ross, S.E. Douglas and S.C. Johnson
Aquaculture Volume 235, Issues 1-4 , 1 June 2004, Pages 601-617 DOI
Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus) (2004)
Murray HM, Perez-Casanova JC, Gallant JW, Johnson SC, Douglas SE
Comp Biochem Physiol A Mol Integr Physiol. 2004 May;138(1):53-9. PubMed
Cellular expression of the pepsinogen and gastric proton pump genes in the stomach of winter flounder as determined by in situ hybridization (2001)
A. Gawlicka, C.T. Leggiadro, J.W. Gallant and S.E. Douglas
Journal of Fish Biology Volume 58 Page 529 DOI
Molecular analysis of the amylase gene and its expression during development in the winter flounder, Pleuronectes americanus (2000)
Susan E. Douglas, Suzan Mandla and Jeffrey W. Gallant
Aquaculture v190, # 3-4 , 1 Nov 2000, pp 247-260 DOI
Molecular investigation of aminopeptidase N expression in the winter flounder, Pleuronectes americanus (1999)
S. E. Douglas, J. W. Gallant and C. E. Bullerwell
Journal of Applied Ichthyology, Volume 15 Page 80 - May 1999 DOI
Winter Flounder Expressed Sequence Tags: Establishment of an EST Database and Identification of Novel Fish Genes (1999)
Douglas SE, Gallant JW, Bullerwell CE, Wolff C, Munholland J, Reith ME
Mar Biotechnol (NY). 1999 Sep;1(5):458-0464 PubMed
Isolation of cDNAs for trypsinogen from the winter flounder, Pleuronectes americanus. (1998)
Douglas SE, Gallant JW
J. Mar. Biotechnol.. 1998 Dec;6(4):214-219. PubMed

Production of transgenic fish expressing the enzyme for vitamin C biosynthesis

Isolation and transient expression of a cDNA encoding -gulono-γ-lactone oxidase, a key enzyme for -ascorbic acid biosynthesis, from the tiger shark Scyliorhinus torazame (2002)
Yoon Kwon Nam, Young Sun Cho, Susan E. Douglas, Jeffrey W. Gallant, Michael E. Reith and Dong Soo Kim
Aquaculture Volume 209, Issues 1-4 DOI