New cytokines and TLR pathway signaling molecules in Chinese rare minnow (Gobiocypris rarus): Molecular characterization, basal expression, and their response to chlorpyrifos
Introduction
The immune system protects organisms from diseases by identifying and eliminating pathogens (Magnadottir, 2010). It plays an important role in the process of maintaining homeostasis during development, inflammatory reaction or tissue injury (Magnadottir, 2010, Tort, 2011). Fish have evolved to have innate immunity and an adaptive immune system (Flajnik and Kasahara, 2010). Pattern recognition receptors (PRRs), a part of the ancient innate immunity of the immune system, are evolutionarily conserved in invertebrate and vertebrate species (Akira et al., 2006). Until now, four classes of PRR families have been identified: toll-like receptors (TLRs), C-type lectin receptors, retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and nucleotide binding oligomerization domain (NOD)-like receptors (NLRs) (Kawai and Akira, 2010). Toll-like receptors (TLRs) are the earliest characterized and the most extensively studied family of type I transmembrane PRRs, which could sense pathogens outside of the cell and inside endosomes and lysosomes (Kawai and Akira, 2010). C-type lectin receptors were a diverse family of soluble and transmembrane proteins that share one or more carbohydrate-binding domains (Geijtenbeek and Gringhuis, 2009). The last two receptors (RLRs and NLRs) are cytosolic receptors that provide a second line of defense against invading pathogens and viruses (Takeuchi and Akira, 2010).
Over the past decade, many immune-related genes in teleosts have been discovered with the development of genomics research and genome sequencing. Myeloid differentiation primary-response protein 88 (Myd88) is the first identified adaptor protein and can be activated by all Tlrs, except Tlr3. The sequence of myd88 in Blunt Snout Bream (Megalobrama amblycephala) (Tran et al., 2015), spiny head croaker (Collichthys lucidus) (Sang et al., 2015), and turbot (Scophthalmus maximus) (Lin et al., 2015) have been reported. In addition, Interleukin-1 receptor-associated kinase-1 (Irak-1) is known for its intrinsic kinase activity and involved in the TLRs signaling pathway to activate downstream signal factors (Xu et al., 2017). The gene irak-1 had be studied in grass carp (Ctenopharyngodon idellus) (Huang et al., 2012) and mandar (Siniperca chuatsi) (Zhang et al., 2009). The interferon regulatory factors (Irfs) are initially identified as transcription factors and play vital roles in immune responses, including regulating inflammatory cytokine expression, controlling the cell-cycle and apoptosis, and mediating the development of lymphocytes (Honda and Taniguchi, 2006, Gabriele and Ozato, 2007, Paun and Pitha, 2007). The sequences of irf5 had been reported in rock bream (Oplegnathus fasciatus) (Wickramaarachchi et al., 2014), and half-smooth tongue sole (Cynoglossus semilaevis) (Zhang et al., 2015). Additionally, irf7 was found in Japanese flounder (Paralichthys olivaceus) (Hu et al., 2010), Pacific cod (Gadus macrocephalus) (Sun et al., 2016), and blunt snout bream (Zhan et al., 2017). However, Interleukin-8 (Il-8) belongs to the CXC chemokine family and plays a crucial role in acute inflammation (Mukaida et al., 1998). The il-8 in rainbow trout (Oncorhynchus mykiss) (Liang et al., 2017), and black rockfish (Sebastes schlegeli) (Herath et al., 2016) have been reported. Although the five immune-related genes have been documented in many researches, results showing data of them in freshwater fish are scarce.
Chlorpyrifos (CPF), a well-known organophosphate pesticide, is widely used in agricultural practices to control pests throughout the world (Deb and Das, 2013). Previous reports showed that CPF in rainwater in Hisar, India was present at levels up to 3 μg/L (Kumari et al., 2007) and 4.3 μg/L in streams and lakes from agricultural runoff (Thoma and Nicholson, 1989). Previous studies have reported that CPF suppressed the mRNA transcription of il-1β and ifn-γ2b in the spleen and head kidney of common carp (Cyprinus carpio) (Wang et al., 2011), and increased the expression of the other 8 immune-response-related genes in the head kidney of common carp after exposure to CPF for 15 d (Zhang et al., 2017). In addition, phagocytosis capacity was significantly decreased in three-spined stickleback (Gasterosteus aculeatus) after exposure to 0.88 μg/L of CPF (Marchand et al., 2017). Moreover, Li et al. (2013) has been indicated CPF significantly change the mRNA and protein levels of C3 and IgM and inhibit the lysozyme activity in immune organs of common carp (Li et al., 2013). Thus, immune dysfunction is associated with fish exposure to CPF.
Although some genes for the cytokines and TLR pathway signaling molecules in the Chinese rare minnow (Gobiocypris rarus) were identified in previous studies (Su et al., 2008, Su et al., 2009, Tao et al., 2016), the mRNA expression of relative complete genes in pathway has not been reported. Therefore, in this study, new cytokines (il-8) and TLR pathway signaling molecules (irak-1, irf5, irf7, and myd88) in Chinese rare minnow were cloned and sequenced. Moreover, to better understand their potential roles in the response of external stimulus, the mRNA expression levels of those genes in the liver and spleen of Chinese rare minnow by CPF after 7 d and 28 d exposures were investigated. Chinese rare minnows, a Chinese fresh water cyprinid, are distributed mostly in the Yangtze River, Sichuan Province of China and considered as an appropriate species for aquatic toxicology studies due to their small size, rapid embryonic development, short life cycle, high fertilization and hatching rate and sensitivity to environmental contaminants (Yuan et al., 2013).
Section snippets
Chemicals
Chlorpyrifos (purity > 98%) and acetone (purity ≥ 99.5%) were purchased from Sigma (Chemical Co. USA). The stock solution of CPF was prepared by dissolving in acetone.
Experimental fish and culture conditions
The brood stock of Chinese rare minnows has been used in our laboratory as experimental materials for more than 15 years. The Chinese rare minnow was raised in a flow through recirculation system with dechlorinated filtered water (pH: 7.4 ± 0.2, temperature: 25 ± 1 °C, hardness: 44.0–61.0 mg of CaCO3/L, dissolved oxygen:
Identification and sequence analysis of new cytokines and TLR pathway signaling molecules in Chinese rare minnow
The cDNAs of il-8, irak-1, irf5, irf7, and myd88 were isolated and cloned from Chinese rare minnow. The partial fragments of these genes have been sequenced and submitted to GenBank. The individual accession numbers are MF426973, MF401442, MF426972, MF426971, and MF426974 for il-8, irak-1, irf5, irf7, and myd88, respectively. Phylogenetic analysis revealed that the predicted amino-acid sequences of il-8, irak-1, irf5, irf7, and myd88 in Chinese rare minnow were clustered together with
Discussion
The immune system of fish consists of a variety of defense mechanisms. Thus it plays an important role in maintaining the body's homeostasis and immune surveillance and protects organisms against damage from xenobiotics, such as pathogens and harmful environmental chemicals (Burns-Naas et al., 2001). The immune-related genes or proteins acting as indicators and indexes for early warning of environmental toxicant stress can respond rapidly by altering the contents or activities to protect organs
Conclusion
In summary, the partial cDNAs for immune-related genes in the rare minnow, including cytokines (il-8) and TLR pathway signaling molecules (irak-1, irf5, irf7, and myd88), were identified and cloned. Basal expression indicated tissue-specific regulation and different functions for these genes in the Chinese rare minnow. The results for the cytokines and TLR pathway signaling molecules of Chinese rare minnow following CPF exposure indicated that CPF could induce immunotoxicity in fish. Moreover,
Acknowledgements
The authors are grateful to the National Natural Science Foundation of China (21677165) for financial support. The financial support from the Major International Joint Research Project of the National Natural Science Foundation of China (51420105012) should also be highly appreciated.
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