Expression profile analysis of antioxidative stress and developmental pathway genes in the manganese-exposed intertidal copepod Tigriopus japonicus with 6K oligochip
Introduction
Manganese (Mn) is a trace metal, and is essential for enzymatic and metabolic processes of marine invertebrates. However, elevated levels of Mn would induce detrimental effects by accumulation of Mn on the early development, growth, metabolism, and physiology of animals (Sanchez et al., 1993, Colomina et al., 1996). Free Mn ions (Mn2+ and Mn4+) are being released into the aquatic environment by photochemical and chemical reduction of Mn oxides from diverse organic compounds (De Schamphelaire et al., 2007). The transport of Mn ions is modulated by several environmental factors such as temperature, pH, and oxygen concentration. Mn ions induce cytotoxicity and imbalance of homeostasis of organisms (Roth, 2006). However, to date, information on the effects of Mn in aquatic organisms is not well documented, compared to other heavy and trace metals.
Of all marine organisms, crustaceans and molluscs are known as the most manganese-sensitive invertebrates followed by echinoderms (Bryan and Ward, 1965). After Bryan and Ward (1965) firstly reported the absorption and effects of Mn in two common lobster species, Homarus gammarus and H. vulgaris, several researchers reported the effects of manganese. For example, Weinstein et al. (1992) showed a potential correlation between Mn accumulation and shell disease of the blue crab, Callinectes sapidus. Holmes et al. (1999) reported that Mn exposure inhibited neuromuscular transmission and muscle excitation as competitive inhibitors of calcium ion channels of the Norway lobster Nephrops norvegicus. Different accumulation patterns and the effects of Mn were associated with different seasonal and sex samples of mussels (Mytilus edulis and M. californianus) and oysters (Crassostrea gigas and C. virginica) (Nørum et al., 2005). In addition, Mn induced apoptosis of the circulating hemocytes and reduced granular hemocyte maturation in Nephrops norvegicus and M. edulis (Hernroth et al., 2004, Oweson and Hernroth, 2009). However, to date most studies on Mn have focused on the toxicities and physiological effects of Mn with marine invertebrates. Therefore, the information on gene/protein expressions of Mn-exposed marine invertebrates would be important for a better understanding of the molecular mechanisms of the Mn-induced mode of action.
To date, the potential of the copepod Tigriopus japonicus as a model organism has been recognized in environmental monitoring and ecotoxicogenomics (Raisuddin et al., 2007). The general genomic and physiological characters of T. japonicus provide several advantages for environmental studies, such as small size (≈1.0 mm in length), distinctive developmental stages, dimorphic sexes, short reproduction period, and extreme tolerance of diverse environmental conditions. Moreover, extensive genomic DNA information (10,894 unigenes) and RNASeq (59,983 assembled ESTs; total length 78.3 Mb; N50 = 2319 as of April 2, 2013) of T. japonicus have supported in vitro omics experiments (Lee et al., 2010; unpublished data), leading to gene expression-based environmental research with copepods (Kim et al., 2011, Kim et al., 2012). Supportingly, Ki et al. (2009) showed the usage of 6K oligomicroarray in quantifying and qualifying changes of genes modulated by copper (Cu) in T. japonicus. Therefore, in vivo and in vitro advantages with T. japonicus would provide many benefits to apply it in omics approaches towards biomonitoring and biomarker development. In this paper, we analyzed mRNA expression profiles of Mn-exposed organisms with a T. japonicus 6K oligochip to understand the molecular mechanisms of Mn toxicity.
Section snippets
Copepods
The intertidal copepod, T. japonicus was maintained and reared in 0.2 μm-filtered sea water adjusted to 25 °C temperature, a photoperiod of 12 h:12 h light/dark and a salinity of 30 ppt. Copepods were fed with the algae, Tetraselmis suecica (approximately 5 × 104 cells ml−1). Identification of the species was made by morphological characteristics and the sequence identity of the universal barcode marker, mitochondrial DNA COI.
Mn exposure
A large number (≈2000 adults of both sex) of copepods were exposed to Mn (as
T. japonicus 6K oligomicroarray
We used T. japonicus 6K oligomicroarray as developed previously (Ki et al., 2009). Briefly, 7219 unique expressed sequence tags (ESTs) of T. japonicus were identified by pyrosequencing method, and 6006 contigs were used for specific probes in the oligomicroarray. Each probe sequence, annotation information, and gene ontology (GO) functional compositions of 6006 contigs were represented as shown in Ki et al. (2009).
Analysis of differentially expressed genes in microarray
Exposure to 100 μg L of Mn for 24 h caused significant changes in transcript
Discussion
As one of the major environmental pollutants, trace heavy metals are of great concern in freshwater and marine ecosystems (Järup, 2003, Jenssen, 2003), and a lot of efforts are made to provide reliable and ecologically relevant invertebrate toxicity testing models for the monitoring of the aquatic environment. Of several testing species to Mn exposure, copepod species including Tigriopus spp. have shown sensitivity to metals (Kusk and Wollenberger, 2007, Raisuddin et al., 2007). Moreover, the
Acknowledgements
We thank Dr. Hans-U. Dahms for his comments on the manuscript. This research was a part of the project titled “Korea-Polar Ocean Development: K-POD” (Project No. PM12030)” funded by the Ministry of Oceans and Fisheries, Korea.
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