The fish has to preserve the biological structures and sustain a slow rate of waste production to avoid pollution of the internal environment. It can perpetuate to aestivate under such conditions for more than a year. The aestivating lungfish can be aroused from aestivation by the addition of water. Upon arousal, the fish struggles out of the cocoon and swims, albeit sluggishly, to the water surface to gulp air. After arousal, it excretes the accumulated waste products, and feeds for repair and growth. Completion of aestivation occurs only if arousal is successful; if not, the animal have had apparently succumbed to certain factors during the maintenance phase. Feeding begins approximately 7?0 days after arousal, and the fish grow and develop as normal thereafter. It is apparent that adaptive (physiological, biochemical and molecular) changes in various organs of the aestivating African lungfish would vary during the three phases of aestivation. However, the majority of studies in the past focused only on the maintenance phase, and there is a dearth of information on the induction and arousal phases of aestivation [12]. Loong et al. [13] pioneered in using suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) to identify aestivation-specific gene clusters in the liver of P. annectens after 6 days (induction phase) of aestivation in a mucus cocoon in air (normoxia). They reported up- or down-regulation of CCX282-B supplement several gene clusters which were involved in urea synthesis, prevention of clot formation, activation of the lectin pathway for complement activation, conservation of minerals (e.g. iron and copper) and increased production of hemoglobin beta. Since there were up- and down-regulation of mRNA expressions of genes related to ribosomal proteins and translational elongation factors, there could be simultaneous increases in protein degradationPLOS ONE | DOI:10.1371/journal.pone.0121224 March 30,2 /Differential Gene Expression in the Liver of the African Lungfishand protein synthesis during 6 days of aestivation, confirming the importance of reconstruction of protein structures in preparation for the maintenance phase of aestivation [13]. The liver is involved in diverse metabolic activities which include detoxification, oxidative defense, urea synthesis, carbohydrate and amino acid metabolism, and iron and copper metabolism. Even during the maintenance phase of aestivation, the liver has to continue functioning to ICG-001 side effects detoxify ammonia to urea; only then, would the aestivating fish be able to mobilize protein and amino acid as an energy source for survival during the aestivation process. Therefore, in this study, we continued to examine the effects of 6 months of aestivation and 1 day arousal from 6 months of aestivation on the up- and down-regulation of genes in the liver of P. annectens using SSH PCR. SSH involves two types of cDNAs: testers (with treatment) and drivers (control). In order to examine differential gene expression in the liver during the maintenance phase (6 months) of aestivation (tester), liver of fish kept in fresh water was used as the driver. Results obtained would indicate changes in gene expression in aestivating fish with reference to non-aestivating fish. However, in order to examine differential gene expression in the liver during the arousal phase (1 day arousal from 6 months of aestivation) of aestivation (tester), liver of fish that had undergone 6 months of aestivation in air were used as driv.The fish has to preserve the biological structures and sustain a slow rate of waste production to avoid pollution of the internal environment. It can perpetuate to aestivate under such conditions for more than a year. The aestivating lungfish can be aroused from aestivation by the addition of water. Upon arousal, the fish struggles out of the cocoon and swims, albeit sluggishly, to the water surface to gulp air. After arousal, it excretes the accumulated waste products, and feeds for repair and growth. Completion of aestivation occurs only if arousal is successful; if not, the animal have had apparently succumbed to certain factors during the maintenance phase. Feeding begins approximately 7?0 days after arousal, and the fish grow and develop as normal thereafter. It is apparent that adaptive (physiological, biochemical and molecular) changes in various organs of the aestivating African lungfish would vary during the three phases of aestivation. However, the majority of studies in the past focused only on the maintenance phase, and there is a dearth of information on the induction and arousal phases of aestivation [12]. Loong et al. [13] pioneered in using suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) to identify aestivation-specific gene clusters in the liver of P. annectens after 6 days (induction phase) of aestivation in a mucus cocoon in air (normoxia). They reported up- or down-regulation of several gene clusters which were involved in urea synthesis, prevention of clot formation, activation of the lectin pathway for complement activation, conservation of minerals (e.g. iron and copper) and increased production of hemoglobin beta. Since there were up- and down-regulation of mRNA expressions of genes related to ribosomal proteins and translational elongation factors, there could be simultaneous increases in protein degradationPLOS ONE | DOI:10.1371/journal.pone.0121224 March 30,2 /Differential Gene Expression in the Liver of the African Lungfishand protein synthesis during 6 days of aestivation, confirming the importance of reconstruction of protein structures in preparation for the maintenance phase of aestivation [13]. The liver is involved in diverse metabolic activities which include detoxification, oxidative defense, urea synthesis, carbohydrate and amino acid metabolism, and iron and copper metabolism. Even during the maintenance phase of aestivation, the liver has to continue functioning to detoxify ammonia to urea; only then, would the aestivating fish be able to mobilize protein and amino acid as an energy source for survival during the aestivation process. Therefore, in this study, we continued to examine the effects of 6 months of aestivation and 1 day arousal from 6 months of aestivation on the up- and down-regulation of genes in the liver of P. annectens using SSH PCR. SSH involves two types of cDNAs: testers (with treatment) and drivers (control). In order to examine differential gene expression in the liver during the maintenance phase (6 months) of aestivation (tester), liver of fish kept in fresh water was used as the driver. Results obtained would indicate changes in gene expression in aestivating fish with reference to non-aestivating fish. However, in order to examine differential gene expression in the liver during the arousal phase (1 day arousal from 6 months of aestivation) of aestivation (tester), liver of fish that had undergone 6 months of aestivation in air were used as driv.
