Seminar Details
Donald Mykles and David Towle (organizers), SICB 2006 Annual Meeting
Genomic and Proteomic Approaches in Crustacean Biology
January 7, 2006 to
January 8, 2006
Orlando, FL
Abstract:
EcoGenomics: analysis of complex systems
Robert W. Chapman. South Carolina Department of Natural Resources, Hollings Marine Laboratory
Ecogenomics is a convenient descriptor for the application of advanced molecular technologies to studies of organismal responses to environmental challenges in their natural settings. The development of molecular tools to survey changes in the transcript profile of thousands of genes has presented scientist with enormous analytical challenges. In the main, these center about the reduction of massively paralleled data to statistics or indices comprehensible to the human mind. Historically, scientists have used linear statistics such as ANOVA to accomplish this task, but the sheer volume of information available from microarrays severely limits this approach. In addition, important information in microarrays may not reside solely in the up or down regulation of individual genes, but rather in their dynamic, and probably non-linear, interactions. In this presentation we will explore alternative approaches to extracting of these signals using artificial neural networks and fractal geometry. The goal is to produce predictive models of gene dynamics in individuals and populations under environmental stress and reduce the number of genes that must be surveyed in order to recover transcript profile patterns of environmental challenges.
Gene discovery in Daphnia by expressed sequence tag sequencing
John K. Colbourne, Erika Lindquist, Darren Bauer, Peter Brokstein, Kelley Thomas, and Justen Andrews
Center for Genomics and Bioinformatics, Indiana University
Department of Energy, Joint Genome Institute
Hubbard Center for Genome Studies, University of New Hampshire
The Daphnia genome project is a consortium based endeavor to characterize the genome and transcriptome of a crustacean and to generate resources for genomic studies in the fields of ecology, toxicology and evolution. To facilitate the annotation of the genome sequence and to accelerate the production of a full transcriptome microarray, we created unidirectional cDNA libraries from Daphnia exposed to 12 distinct ecological stressors ranging from toxic metals and UV radiation to hypoxia, starvation and predation. We are sequencing 5’ and 3’ EST sequence reads from randomly selected clones from each library. At the mid-point in the project, we identified over 12,600 unique genes from 71,000 high quality ESTs. These gene sequences were queried, using a variety of homology searches, against those from other arthropod genomes to discover their evolutionary conservation with respect to putative gene functions. This genome-wide comparative analysis of Daphnia genes with the functionally well characterized genes of model insects provides important findings into similarities / differences between these two classes of Arthropoda and provides clues into the biology of a fascinating sentinel species within freshwater habitats.
Functional genomics of environmental adaptations in marine crustaceans
David W. Towle, Mount Desert Island Biological Laboratory
The expression of genes in response to changes in salinity and temperature is being examined in our laboratory through the generation of expressed sequence tags and subsequent microarray analysis. Using the traditional gene-by-gene approach and quantitative PCR, the expression of ten different genes has been monitored in gills and other tissues of crabs and lobsters challenged with an environmental stimulus. For example, in the European shore crab Pachygrapsus marmoratus acclimated to normal seawater, salinity dilution leads to rapid up-regulation of transporter and heat-shock protein mRNA levels in all gills tested. In contrast, salinity increase produces an up-regulation of transporter gene expression only in gill number 7 (of 9). Current expressed sequence tag (EST) and microarray programs are developing tools for the global analysis of changes in gene expression. Starting with mixed tissue cDNA libraries normalized to reduce the abundance of highly expressed messages, we have produced 4,604 ESTs for the American lobster Homarus americanus and 5,362 ESTs for the green shore crab Carcinus maenas, available for searching at www.ncbi.nlm.nih.gov. Clustering has identified 3,773 unique sequences for H. americanus and 1,928 unique sequences for C. maenas. Oligonucleotides based on these ESTs are being arrayed for studies of transcriptome changes following environmental perturbation. In addition, the ESTs are being used directly to identify specific targets for further study, including the recent characterization of a zinc transporter in gills of C. maenas. Supported by NSF (DBI-0100394 and IBN-0340622) and NCRR (1-P20-RR16463).
Transcriptome changes during thermal acclimation, acclimatization, and stress in porcelain crabs
Jonathon H Stillman and Kristen S Teranishi
The thermal phenotype of an organism (i.e., heat tolerance, cold tolerance, eurythermality) is an essential determinant of performance across thermal environments and in response to thermal stress. We are working to understand the mechanistic bases of thermal phenotype by analysis of correlated changes between transcriptome profiles and cardiac thermal performance of thermally acclimated porcelain crabs, Petrolisthes cinctipes. Crabs were acclimated for 28 days to 8°C or 18°C, at which time cardiac upper and lower thermal performance limits significantly differ. On day 29, crabs were swapped between 8°C and 18°C. Transcriptome profiles were generated from crabs sampled during days 27-31 of the experiment using cDNA microarrays of 13,824 cloned ESTs from P. cinctipes. R/maanova was used to identify ESTs that had significantly different expression profiles between pre-swap and 12h intervals post-swap. Following transfer of 8°C-acclimated crabs to 18°C, 4957 ESTs had significant changes in expression. In contrast, 1400 ESTs had significant changes in gene expression following transfer of 18°C acclimated crabs to 8°C. Only 300 ESTs were identified as having significant changes in expression in both groups of crabs, thus we conclude that largely different sets of genes are involved in warm and cold acclimation states. Functional characterization of genes expressed in both groups of crabs as well as those expressed only during warm or cold acclimation is underway. I will also present an overview of our studies to examine variation in thermal phenotype in an ecological context by examination of acclimatization across natural thermal gradients as well as responses to thermal stresses common in intertidal zone habitats. This work is funded by NSF IOB 0533920 to JHS and by the JGI Community Sequencing Program.
Functional genomics approaches to understanding stress responses and disease in shrimp
Paul S. Gross
Pathogens in the marine environment have caused catastrophic losses to shrimp aquaculture. The manner in which marine crustaceans respond to pathogens is complex and largely unknown. In addition, the role of anthropogenic and natural factors in the etiology of shrimp disease and their effects on immunity is, at best, poorly understood. This is particularly true in the case of antiviral immunity for which there is virtually nothing known about host resistance. We have taken a functional genomics approach to gain an understanding of the underlying genetics of disease response and immunity in crustaceans, using the Pacific white shrimp, Litopenaeus vannamei and White Spot Syndrome Virus (WSSV) as models. The Pacific white shrimp is a commonly aquacultured species in many parts of the world and WSSV has had a devastating effect on farms growing the Pacific white shrimp. The strategy employed relies on the discovery of novel genes with putative roles in immune response using of ESTs derived from standard and redundancy depleted cDNA libraries and to a lesser extent enriched disease-responsive libraries generated by suppression subtractive hybridization (SSH). In addition, these cDNA clones are being used to generate microarrays to examine disease response in detail. We hypothesize that infection triggers changes in gene expression that are at least partially reflective of an immune response. By characterizing these disease-responsive genes, we aim to gain an understanding about the kinds of pathways involved in the invertebrate antiviral defense.
Lobster olfactory genomics
Tim McClintock
Several fundamental observations about olfaction were made first using lobsters, largely due to advantages that stem from their anatomy and a wealth of background knowledge. Investigating the molecular basis for the function of the lobster organ has also proved fruitful. Reasoning that gene products specific to, or enriched in, the olfactory organ will help identity its unique properties, we have used expression profiling strategies such as differential amplification by PCR to identify molecular markers of the olfactory sensory neurons, their auxiliary cells, secretory cells of the associated aesthetasc tegumental glands, and reactive epithelial cells critical for the remarkable ability of the organ to regenerate itself. More importantly, the identify of these markers make predictions about novel physiological processes such as glutamatergic input to the olfactory sensory neurons and the production of neuroactive biogenic amine hormones by the auxiliary cells that surround the inner dendrites of the olfactory sensory neurons. More recently, bioinformatics of ESTs from more than 5,500 cDNAs confirms the preponderance of neurons in the mature zone of the olfactory organ. To allow testing of hypotheses about which mRNAs are most closely associated with the olfactory organ and its functions, we generated a cDNA microarray. As a first test of the array, we compared olfactory organ with dactyl, a taste organ, and identified 115 gene products that discriminate between these two chemosensory tissues. We are now in position to use mRNA abundance to investigate the olfactory organ as a molecular system by observing the responses of thousands of mRNAs to experimental manipulation.
Limb regeneration and expressed sequence tag sequencing in fiddler crab
Durica, D.S., Kupfer, D., Najar, F., So, S. Tang, Y., Griffin, K., Hopkins, P.M. and Roe, B. University of Oklahoma
We have constructed several directional and randomly-primed cDNA libraries from mRNAs isolated during progressive stages of fiddler crab limb regeneration. Data from these libraries is being assembled into an on-line database (http://www.genome.ou.edu/crab) that is both BLAST and keyword searchable; the data set will also be available through GenBank. The first characterized library was made from mRNA isolated fours days post-autotomy, when the first sign of morphological differentiation, cuticle secretion, is observed. Analysis of approximately 1500 cDNA clones led to assignment of 485 contigs and 429 singlets, for a total of 914 sequences. Of these, approximately 63% showed no homology on database searching. Analysis of these unclassified sequences relative to sequencing with 5’ or 3’ specific primers showed approximately equal numbers in each category, suggesting that the ‘no hit’ clones may contain a large number of new genes. Biological assignments for the approximately 37% of ESTs based on KEGG (Kyoto Encyclopedia of Genes and Genomes) are broken down into metabolism or regulatory categories, and a detailed list of assignments based on the NCBI COG (Clusters of Othologous Groups) outline format is available. These ESTs include several genes that may be potentially ecdysteroid-responsive, such as homologs to chaperone proteins and cuticle protein genes. Homologs to arthropod proteins involved in retinoid/terpenoid metabolism have also been identified. To investigate potential ecdysteroid-responsive candidate genes, hormone induction experiments are currently in progress, using primer sets designed from database sequence information and quantitative real-time PCR.
Proteomics and signal transduction in the crustacean molting gland
Mykles, D.L. Biology, Colorado State University
Molting in decapod crustaceans is controlled by the X-organ/sinus gland complex, a neurosecretory center in the eyestalks. The complex secretes a neuropeptide, molt-inhibiting hormone (MIH), that suppresses production of molting hormone (ecdysone), an ecdysteroid secreted by a pair of molting glands (Y-organs or YOs) located in the anterior of the body. Binding of MIH to a YO membrane receptor results in a cyclic nucleotide-dependent inhibition of ecdysteroidogenesis. Eyestalk ablation (ESA) induces a rapid increase of hemolymph ecdysteroid titers. Proteomics has become a powerful tool to identify proteins involved with specific physiological or disease states. We are using expression and cell-map proteomics to determine which proteins are involved in YO signaling, YOs from intact and ES-ablated land crabs (Gecarcinus lateralis) were analyzed by 2-D gel electrophoresis and mass spectrometry. ESA causes dramatic changes in the levels of proteins between 12 kDa and 27 kDa. These proteins were selected as putative candidates for molt-regulating factors from both silver and phosphoprotein-stained gels. Nitric oxide synthase (NOS) was identified by immunoblotting and peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry as one of the proteins transiently phosphorylated in response to ESA. In addition, MIH interacting proteins were detected using immunoprecipitation with an anti-MIH antibody. These putative MIHBPs (MIH binding proteins) were observed only in intact YO and not in YOs from ES-ablated animals. This suggests that these proteins could be involved in the binding of MIH to its receptor in the YO membrane. We are currently characterizing the MIHBPs by PMF and internal peptide sequencing using ion-trap tandem mass spectrometry. Supported by NSF (IBN-0342982).
Expressed sequence tag sequencing to identify genes involved in exoskeleton calcification in the blue crab
Shafer, TH University of North Carolina
A blue crab (Callinectes sapidus) expressed sequence tag project was designed for multiple purposes including the discovery of genes for cuticle proteins regulating calcification. One of the expression libraries sequenced was from hypodermal tissue, the epithelium that deposits the cuticle. The RNA used for cDNA synthesis was pooled from arthrodial and dorsal hypodermis of both pre-ecdysis (D2) and 3-h postecdysis crabs. This ensured representation from both calcifying and non-calcifying cuticle regions and from cuticle layers deposited both before and after exuviation. The EST data have been mined for cuticle protein sequences. First, we searched for known cuticle-specific motifs, the Rebers-Riddiford chitin-binding sequence and a mineralized-tissue motif described by Andersen. Second, we checked the BLAST annotations and the GO analysis of the entire EST project for evidence of similarity to known (often insect) cuticle proteins. Finally, all crustacean cuticle protein sequences in NCBI were placed into an alignment-based dendrogram resulting in broad similarity groups. Then BLAST was used to search the EST data for significant homologies to each group. In all, the database appears to contain approximately 70 contigs or singlets representing transcripts of cuticle proteins. Forty-five distribute among ten clusters of very similar transcripts, possibly representing alternate splicing or recent gene duplications, while the rest share less similarity. We have obtained complete sequences for about 25 of the transcripts, and we are determining gene expression patterns across the molt cycle in calcifying versus non-calcifying cuticle regions. The combination of homology analysis and gene expression analysis allows us to infer possible functions in cuticle synthesis and calcification.
Crustacean hemocyanin gene family: oxygen probes of the global gene scene
Terwilliger, N. amd Ryan, M. University of Oregon
Global expression profiling simultaneously monitors vast numbers of genes in an organism, and it identifies groups of co-regulated genes. The principles of molecular recognition at the genomic and proteomic levels were developed for single gene analysis, and data analysis of global expression profiling requires knowledge of gene families and products. Oxygen delivery in decapod crustaceans is provided by hemocyanin (Hc). The Hc gene family, like hemoglobin, has undergone a process of gene duplication and divergence. These events have resulted in a Hc molecule composed of multiple subunits with a functional spectrum of homeotropic and heterotropic responses to allosteric effectors, metabolites and environmental factors. Other duplication events have resulted in new proteins with marked changes in function, as evidenced by phenoloxidase, cryptocyanin, and insect hexamerins. Continuing investigations at the gene family level in concert with genomics and proteomics will provide new insights into crustacean physiology. We have determined the cDNA sequences of all six subunits that compose the Hc molecule in the adult Dungeness crab, Cancer magister. The subunits are the products of six separate genes. Analyses of protein and genomic structures of these Hc subunits confirm the clustering into two motifs. Hc genes 1 and 2 are tandemly linked on the same chromosome and have one intron. Genes 3,4,5 and 6 have an additional intron. The exon-intron pattern in C. magister will facilitate Hc gene structure comparisons in the Arthropoda and other phyla. Analyses of upstream regulatory regions of the Hc genes in conjunction with ongoing development and construction of a C. magister microarray will yield insights into transcriptome changes during development, molting and environmental stress. Supported by NSF 9984202.
