Shrimps inhabiting coastal waters can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp () at 1.7%, 3.4% and 4.0% salinities. After acclimating for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA sequencing analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after 24 h of acclimation. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. In particular, the increase of the expression level of the gene encoding glutamate-ammonia ligase, which functions in glutamine metabolism, appeared to be associated with the increased glutamine concentration at high salinity. Furthermore, the increased alanine concentration at high salinity was likely associated with the decrease in the expression levels of the the gene encoding alanine-glyoxylate transaminase. Thus, there is a possibility that changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are regulated by changes in the expression levels of genes related to amino acid metabolism.
3Shrimps inhabiting around the coastal area can survive in a wide range of salinity. 49 However, the molecular mechanisms involved in their adaptation to different 50 environmental salinity have remained largely unknown. In the present study, we reared 51 kuruma shrimp Marsupenaeus japonicus at 1.7 %, 3.4 % and 4.0 % salinity. After rearing 52 for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal 53 muscle, and performed RNA-seq analysis on this muscle. The concentrations of free 54 amino acids were clearly altered depending on salinity after rearing for 24 h. Glutamine 55 and alanine concentrations were markedly increased following the increase of salinity. In 56 association with such changes, many genes related to amino acid metabolism changed 57 their expression levels. Notably, the increased glutamine content at high salinity appeared 58 to be relevant to the increase of the expression level of the gene encoding 59 glutamate-ammonia ligase which functions in the glutamine metabolism. Furthermore, 60 the alanine content increased at high salinity was likely to be associated with the decrease 61 in the expression levels of the alanine-glyoxylate transaminase gene. Thus, the changes in 62 the concentration of free amino acids for osmoregulation in kuruma shrimp are 63 considered to be regulated by the changes in the expression levels of genes related to 64 amino acid metabolism. 65 66 67 68 69 70 71 109 2015; Zhao et al., 2012). It has been reported that swimming crab Portunus 110 trituberculatus reared for ten days at different salinity changed the expression levels of 111 osmoregulation-related genes such as those encoding ion transporters and amino acid 112 metabolism-related proteins in their gills (Lv et al., 2013). 113 As mentioned above, many genes including ion and amino acid transporters seem to 114 participate in adaptation of crustaceans to the salinity change. However, the molecular 115 mechanisms involved have still remained unclear, since the regulatory mechanisms 116 underlying the changes of free amino acid concentrations are not well understood. 117 In the present study, we targeted kuruma shrimp as experimental animals, which are 118 widely cultured and commercially available, important species. We reared the shrimp 119 samples at different salinity and determined free amino acid concentrations in their 120 6 abdominal muscle. In addition, we performed RNA-seq analysis on the same samples by 121 using a next generation sequencer. 122 123 Materials and methods 124 Animals 125About 40 adult specimens of kuruma shrimp were obtained from Miyazaki Prefecture, 126 Japan, and cultured in 3.4 % salinity tank (60 l) of Kitasato University at 25 ºC for 3 days. 127 Then, they were divided into 3 groups using 60 l tanks each at 1.7 %, 3.4 % and 4.0 % 128
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