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Oreochromis Mossambicus Lam et al. BMC Genomics 2014, 15:921 http://www.biomedcentral.com/1471-2164/15/921 RESEARCH ARTICLE Open Access Differential transcriptomic analyses revealed genes and signaling pathways involved in iono-osmoregulation and cellular remodeling in the gills of euryhaline Mozambique tilapia, Oreochromis mossambicus Siew Hong Lam1,2*, Eei Yin Lui1, Zhengjun Li1, Shaojiang Cai3, Wing-Kin Sung3,4, Sinnakaruppan Mathavan4, Toong Jin Lam2 and Yuen Kwong Ip2 Abstract Background: The Mozambique tilapia Oreochromis mossambicus has the ability to adapt to a broad range of environmental salinities and has long been used for investigating iono-osmoregulation. However, to date most studies have focused mainly on several key molecules or parameters hence yielding a limited perspective of the versatile iono-osmoregulation in the euryhaline fish. This study aimed to capture transcriptome-wide differences between the freshwater- and seawater-acclimated gills of the Mozambique tilapia. Results: We have identified over 5000 annotated gene transcripts with high homology (E-value <1.0E-50) to human genes that were differentially expressed in freshwater- and seawater-acclimated gills of the Mozambique tilapia. These putative human homologs were found to be significantly associated with over 50 canonical signaling pathways that are operating in at least 23 biological processes in relation to branchial iono-osmoregulation and cellular remodeling. The analysis revealed multiple signaling pathways in freshwater-acclimated gills acting in concert to maintain cellular homeostasis under hypo-osmotic environment while seawater-acclimated gills abounded with molecular signals to cope with the higher cellular turn-over rate, energetics and iono-regulatory demands under hyper-osmostic stress. Additionally, over 100 transcripts encoding putative inorganic ion transporters/channels were identified, of which several are well established in gill iono-regulation while the remainder are lesser known. We have also validated the expression profiles of 47 representative genes in freshwater- and seawater-acclimated gills, as well as in hypersaline-acclimated (two-fold salinity of seawater) gills. The findings confirmed that many of these responsive genes retained their expression profiles in hypersaline-acclimated gills as in seawater-acclimated gills, although several genes had changed significantly in their expression level/direction in hypersaline-acclimated gills. (Continued on next page) * Correspondence: [email protected] 1NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive, 117411 Singapore, Singapore 2Department of Biological Science, National University of Singapore, S3-Level 5, 14 Science Drive 4, 117543 Singapore, Singapore Full list of author information is available at the end of the article © 2014 Lam et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lam et al. BMC Genomics 2014, 15:921 Page 2 of 23 http://www.biomedcentral.com/1471-2164/15/921 (Continued from previous page) Conclusions: This is the first study that has provided an unprecedented transcriptomic-wide perspective of gill iono-osmoregulation since such studies were initiated more than 80 years ago. It has expanded our molecular perspective from a relatively few well-studied molecules to a plethora of gene transcripts and a myriad of canonical signaling pathways driving various biological processes that are operating in gills under hypo-osmotic and hyper-osmotic stresses. These findings would provide insights and resources to fuel future studies on gill iono-osmoregulation and cellular remodeling in response to salinity challenge and acclimation. Keywords: Transcriptomic analysis, RNA sequencing, Gene expression, Signaling pathways, Iono-osmoregulation, Cellular remodeling, Euryhaline fish gills, Mozambique tilapia Oreochromis mossambicus Background remodel the cellular landscape of the gill by changing cell The ability of the euryhaline fish to adapt to a broad types along with their transporters/channels and intercel- range of aquatic environmental salinities is an interesting lular organization in order to cope with the reversed phenomenon of adaptive homeostatic response that is iono-osmoregulatory challenges. It is this versatility in crucial for survival and therefore provides an excellent cellular remodeling along with the transporters and model for investigating iono-osmoregulation and its mo- channels within the gills in response to salinity challenge lecular plasticity. Understanding the euryhalinity of fish that is partly responsible for euryhalinity in fish. could provide evolutionary perspective into the adapta- The Mozambique tilapia Oreochromis mossambicus tion of fish in various aquatic environments including has versatile iono-osmoregulatory ability that enables it the great divide between the freshwater and marine to live in both FW (salinity at 0 ppt) and SW (salinity at species [1] while probing deeper into its molecular 30–35 ppt), and can tolerate extreme hypersalinity of up plasticity would yield insight into the versatile iono- to 80–120 ppt, i.e. about 3–4 fold the salinity of SW. osmoregulatory mechanism and control [2,3]. In turn, it This makes Mozambique tilapia among the few remark- would enhance our understanding of iono-osmoregulation able successful teleost species inhabiting extreme hyper- from the molecular, cellular, tissue-organ, systems to saline environment [9,10]. The versatility of the tilapia to whole-organism levels, which may contribute to fishery, iono-osmoregulate across a broad range of extreme sal- environmental and even biomedical research. To be eury- inity has been the focus of many studies ranging from haline, the fish must be able to cope with salt depletion physiological, morphological, biochemical to molecular and water gain when it is in freshwater (FW), and deal levels. Recent reviews had documented studies investi- with osmotic water loss and salt gain when it is in sea- gating iono-osmoregulatory changes in, although not water (SW) or hypersaline water (HSW). Euryhalinity limited to, tilapia gills and interested readers are advised therefore requires extreme versatility in the switching of to refer to them for details [7,8,11]. While many of the iono-osmoregulatory mechanisms and cellular remodel- studies had been elegantly executed, most of them had ing in iono-osmoregulatory tissue-organ in response to focused mainly on several key molecules or parameters changes in environmental salinity [4]. hence yielding important, albeit limited, perspectives of The fish gill is a major iono-osmoregulatory organ and the iono-osmoregulation changes in response to different is therefore a principal site for entry and departure environmental salinities. An earlier attempt to capture a of ion and water molecules between its internal fluid broader molecular perspective employing suppression and its external environment [5]. As part of the iono- subtractive hybridization approach reported 20 genes as- osmoregulatory mechanism, the gills are actively se- sociated with 6 molecular processes with immediate re- questering salt and minimizing water gain when in sponse to salinity challenge in O. mossambicus gills [12]. FW environment, while in SW the gills are actively ex- The study was focused on immediate response to salinity truding excess salt and minimizing water loss into the ex- challenge that occurred few hours after transfer to SW ternal environment. The ability to sequester or extrude hence detecting only early gene responses and not those salt against concentration gradient in FW and SW, re- involved in gill salt transport in acclimated state. More- spectively, are performed by specialized distinct cell types over, the suppression subtractive hybridization approach is which occupy the epithelial layer of the gill [5,6]. Embed- low-throughput and is only sensitive to detect high abun- ded in the plasma membrane of these cells are specific dant transcript changes, and the challenges of annotating transporters and channels that worked collaboratively to partial sequences in non-model organism at the time of ensure selective movement of ions across the epithelial the study may have limited the identification of more layer of the gill [4-8]. For a euryhaline fish to move from genes. This was similarly reflected in two other osmo- FW to SW environment or vice versa, it would have to regulation studies on non-model euryhaline fish species Lam et al. BMC Genomics 2014, 15:921 Page 3 of 23 http://www.biomedcentral.com/1471-2164/15/921 subjected to salinity challenge; one study on the European gill tissues from Mozambique tilapias that had acclimated eel Anguilla anguilla, employed a combination of sub- in their respective FW and SW environments for 3 weeks. tracted libraries and cDNA arrays, identified 95 known We extracted total RNA from the gill tissues of the FW genes differentially expressed in various tissues of eel accli- and SW groups and pooled four of the highest quality mated to SW [13], while another early response study on RNA samples from each group for preparation
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