Gene Expression Profiles as Molecular Indicators of Dissolved Oxygen Stress in Grass Shrimp Palaemonetes pugio Holthuis 1949
Occurrence and severity of hypoxia is increasing in coastal and estuarine environments, and recovery of impacted habitats and living resources is slow. Detection of early biological effects of hypoxia is needed for timely remedial action to be taken. The overall objectives of this research was to develop molecular indicators of dissolved oxygen stress to assess the biological impact of hypoxia in coastal estuaries and validate their use through a combination of laboratory and field studies. To achieve these goals, grass shrimp, Palaemonetes pugio, oxygen-sensitive and hypoxia-tolerant species abundant in estuarine systems, were exposed to hypoxia under controlled laboratory conditions, and significant changes in gene expression were identified. Grass shrimp were collected from hypoxic field sites to evaluate if these hypoxia-responsive genes can be used as indicators of dissolved oxygen (DO) stress in the aquatic environment. Hypoxia inducible factor 1α (HIF-1α), a key transcription factor that controls a variety of cellular and systemic homeostatic responses to hypoxic stress, was successfully cloned and characterized in crustaceans using RT-PCR and RACE. Grass shrimp HIF-1α protein shows a high level of conservation with other HIF-1α proteins in the bHLH, PAS, ODD, and TAD domains. Phylogenetic analysis indicates that grass shrimp and vertebrate HIFs belong to distinct clades within the HIF protein family. HIF mRNA levels were not responsive to chronic or cyclic hypoxia. Six libraries of expressed sequence tags (ESTs) were constructed by suppression subtractive hybridization (SSH) from the grass shrimp exposed to environmental stress: moderate (DO 2.5 mg/L) and severe (1.5 mg/L) hypoxia, cyclic hypoxia (1.5 [arrow right]7 mg/L), contaminant-induced stress (pyrene and copper), and biological stress (molt). Gene Ontology (GO) analysis of libraries showed several genes that were present in only one library suggesting that their expression may be stressor specific. The molting process was accompanied by changes in expression of many genes not found in the hypoxia/copper/pyrene libraries. The resulting annotated transcripts were used to design and construct a cDNA microarray to measure the expression changes in response to hypoxia conditions. The microarrays were used to examine differentially expressed genes in hypoxic vs. normoxic groups at 6 (H6), 12 (H12), 24 (H24), 48 (H48), 120 (H120), and 240 (H240) hours exposure to chronic hypoxia. Cluster analysis showed two response patterns, composed of an up- (including H6, H24, and H120) and down-regulated (including H12, H48, and H240) dominated cluster. Changes in gene expression are dynamic and transient. There is no differentially expressed gene up- or down-regulated common to all six groups. Differentially expressed genes were determined in hypoxic vs. normoxic groups after 1, 2, 5 and 10 days exposure to cyclic hypoxia. Sampling on each day was conducted at two different time series, one in the morning (representing low DO, CA) and one in the afternoon (representing high DO, CP). There are distinct differences between the number and identity of specific genes that are significantly down- or up-regulated in shrimp collected at the low DO and high DO points of the cyclic DO cycle. Only a few genes are differentially expressed in grass shrimp exposed to cyclic hypoxia in the field relative to those collected from a normoxic reference site. In conclusion, grass shrimp HIF is constitutively expressed and not induced by chronic and cyclic hypoxia exposures in both laboratory and field studies. Some differentially expressed genes appear unique at certain time points during laboratory and field exposures. However, changes of significant genes are too dynamic to serve as biomarkers of hypoxia stress in grass shrimp. Gene expression changes of grass shrimp in response to cyclic hypoxia conditions are not only dependent on the duration of exposure but also on the time of day.