Masatomi Hosoi

Taurine transporter from the giant Pacific oyster Crassostrea gigas: function and expression in response to hyper- and hypo-osmotic stress

Taurine is the primary osmolyte in marine molluscs, whose cellular osmo-conforming process is vital for environmental adaptation because of a lack of osmotic homeostasis. Here, cDNA cloning and expression, and functional analyses of taurine transporter (TAUT) from the giant Pacific oyster are reported on. The deduced amino-acid sequence of oyster TAUT (oy TAUT) showed 47–51% identity to those of vertebrate TAUT, whereas identity among the vertebrates is 78–95%. Functional analysis of oyTAUT expressed in Xenopus oocytes revealed that oyTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration, compared with vertebrate TAUT. Taken together with similar functional properties of TAUT from mussel, indicated by our previous study, it is possible that these functional features reflect the internal environment of the molluscs (i.e. higher taurine and NaCl concentrations). Oyster taurine transporter mRNA expression was induced by not only hyper-osmotic stress, similar to other TAUT, but also hypo-osmotic stress. It is speculated that the expression in response to hypo-osmotic stress was induced by a substantial decrease in tissue taurine content following the decrease in the internal osmolality.

Expression and functional analysis of mussel taurine transporter, as a key molecule in cellular osmoconforming

SUMMARY Most aquatic invertebrates adapt to environmental osmotic changes primarily by the cellular osmoconforming process, in which osmolytes accumulated in their cells play an essential role. Taurine is one of the most widely utilized osmolytes and the most abundant in many molluscs. Here, we report the structure, function and expression of the taurine transporter in the Mediterranean blue mussel (muTAUT), as a key molecule in the cellular osmoconforming process. Deduced amino acid sequence identity among muTAUT and vertebrate taurine transporters is lower (47-51%) than that among vertebrate taurine transporters (>78%). muTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration than vertebrate taurine transporters. This seems to reflect the internal environment of the mussel; higher NaCl and taurine concentrations. In addition to the hyperosmotic induction that has been reported for cloned taurine transporters, the increase in muTAUT mRNA was unexpectedly observed under hypoosmolality, which was depressed by the addition of taurine to ambient seawater. In view of the decrease in taurine content in mussel tissue under conditions of hypoosmolality reported previously, our results lead to the conclusion that muTAUT does not respond directly to hypoosmolality, but to the consequent decrease in taurine content. By immunohistochemistry, intensive expression of muTAUT was observed in the gill and epithelium of the mantle, which were directly exposed to intensive osmotic changes of ambient seawater.

Osmo-responsive expression of oyster amino acid transporter gene and analysis of the regulatory region involved

To elucidate the involvement of amino acid transporter in osmotic adaptation of oysters, the expression of the amino acid transporter gene in response to environmental osmotic changes was investigated. As expected, the expression of the amino acid transporter gene was increased by hyper-osmotic stress, probably to elevate intracellular osmolality. Unexpectedly, the expression was also increased by hypo-osmotic stress, and the level of expression was higher than that induced by hyper-osmotic stress. To identify the region regulating the expression of the oyster amino acid transporter gene in response to changes in environmental osmolality, the 5′-flanking region of approximately 2.3 kb upstream from the translation start site was cloned. Expression vectors with luciferase as a reporter gene driven by 5′-flanking regions with different lengths were constructed and their promoter activities were compared. As a result, the osmo-responsive regulatory region responding to osmolality by both hyper- and hypo-osmolality was found within 132 bp from the transcription start site.

Expression of HSP70 in response to heat-shock and its cDNA cloning from Mediterranean blue mussel

Expression of HSP70 in response to heat-shock was investigated at the protein and mRNA levels in Mediterranean blue mussel. Western and Northern blot analyses revealed that HSP70 was expressed following heat-shock in the mantle at both protein and mRNA levels, suggesting that gene expression of HSP70 is implicated in the cellular response to heat-shock stress in mussel. It was then attempted to clone HSP70 cDNA in order to determine the primary structure of mussel HSP70. As a result, two full-length cDNA encoding HSP70 were isolated from a cDNA library prepared from the heat-shocked mantle. The isolated cDNA consist of single open reading frames of 2067 bp and 1911 bp which encode proteins of 689 amino acids and 637 amino acids, respectively. Both HSP70 cDNA encode an ATPase do main, and a substrate-binding do main in addition to a Glu-Glu-Val-Asp (EEVD) peptide motif that is specific for cytosolic HSP70. These findings suggest that the cDNA clones obtained in the present study encode cytosolic HSP70.

Scallop DMT functions as a Ca2+ transporter

We identified a DMT (divalent metal transporter) homologous protein that functions as a Ca2+ transporter. Scallop DMT cDNA encodes a 539‐amino‐acid protein with 12 putative membrane‐spanning domains and has a consensus transport motif in the fourth extracellular loop. Since its mRNA is significantly expressed in the gill and intestine, it is assumed that scallop DMT transports Ca2+ from seawater by the gill and from food by the intestine. Scallop DMT lacks the iron‐responsive element commonly found in iron‐regulatory proteins, suggesting that it is free of the post‐transcriptional regulation from intracellular Fe2+ concentration. Scallop DMT distinctly functions as a Ca2+ transporter unlike other DMTs, however, it also transports Fe2+ and Cd2+ similar to them.

Expression of taurine transporter in response to hypo-osmotic stress in the mantle of Mediterranean blue mussel

Expression of taurine transporter in response to osmotic stress was investigated at the protein level in the mantle of the Mediterranean blue mussel by using the specific antibody raised against the carboxy-terminal region of the deduced amino acid sequence of mussel taurine transporter. Immunohistochemical observation revealed that taurine transporter was expressed in the mantle and the expression was up-regulated in response to hypo-osmotic stress, while down-regulated in response to hyper-osmotic stress. Western blot analysis revealed major protein bands corresponding to 62 kDa and 65 kDa. In response to hypo-osmotic stress, the 62 kDa band became more intense, while it became less intense when the ambient osmolality was elevated. These results suggested that the 62 kDa taurine transporter would be implicated in hypo-osmotic adaptation.

Evaluation of PCR methods for fixed bivalve larvae

Investigating the spatio-temporal patterns of planktonic larvae is fundamental to studies regarding stock assessment and larval dispersal of commercial and non-commercial, i.e. invasive or rare marine invertebrates. Because of the difficulty involved in morphological identification of marine invertebrate larvae, various molecular methods based on PCR have been used to enhance taxonomic resolution. In previous studies, different methods for the preservation or pretreatment of larvae were applied in each case. However, no comparative studies have been conducted to determine the optimal method for PCR testing for bivalve larvae, and no information is available regarding the selection of an appropriate method. This study compared the PCR success rate of 6 pretreatment methods for larvae of the Mediterranean blue mussel, which was preserved using different fixatives (70% ethanol, 100% ethanol, 70% acetone and 10% formalin). The results revealed that the success rate of PCR was different for each pretreatment; moreover, the use of ammonium sulphate and Tween 20 buffer with proteinase K digestion was found to be the most effective method. Some pretreatments showed lower success rates for long-fixed larvae than for short-fixed larvae for formalin-fixed larvae; however, the success rate of PCR amplification for ethanol-fixed larvae pretreated by this method did not decrease through i-year fixation. In addition, this pretreatment showed a high success rate for different fixation periods. These findings suggest that the selection of the pretreatment method is critically important for successfully amplifying larval DNA and that the pretreatment involving the use of ammonium sulphate prior to PCR amplification enables the use of fixatives for preserving bivalve larvae. This method will be utilized in various field studies and molecular genetic studies.

Freshwater bivalve Corbicula sandai uses free amino acids as osmolytes under hyperosmotic condition

Aquatic invertebrates that lack advanced homeostatic mechanisms to maintain their internal osmolality, which are present in teleost fish, adapt to environmental osmotic stress mainly at the cellular level. It has been reported that some free amino acids (FAAs) including alanine, glycine, proline, taurine and glutamic acid accumulate in intracellular spaces and are used as osmolytes by molluscs. Numerous studies on marine and brackishwater bivalves have reported changes in the FAA content when the animals are subjected to osmotic stresses. Studies have reported biochemical analysis of enzymes related to osmolyte metabolism and transmembrane transport of osmolytes. On the other hand, osmolyte systems in freshwater bivalves have rarely been reported, probably because the necessity of an osmolyte system is apparently less in freshwater bivalves due to their lower and more stable internal osmolality. However, a few studies have indicated that despite the lower necessity, freshwater bivalves possess the ability to increase some FAAs under hyperosmotic conditions. In the freshwater clam Lampsilis teres, the FAA content in the gills increased with acclimation to high salinity. In Corbicula manilensis, a congeneric species of C. sandai investigated in this present study, the FAA pool increased from 20 to 115 mmol/g dry weight after a 70-h exposure to water of salinity 5.0 (0.5% saline water). In the present study, we demonstrated the effects of hypersalinity on the internal osmolality and FAA content in the freshwater bivalve Corbicula sandai. This bivalve is endemic to the freshwater Lake Biwa, and thus, is a genuine freshwater species. On the other hand, the abovementioned species inhabit the middle or lower regions of rivers where the possibility of the occurrence of a hypersaline condition can not be excluded. Our results first suggest that the hyperosmotic–responsive osmolyte system using FAA is sustained even in a species that has a genuine freshwater habitat. Specimens of C. sandai, donated by Shiga Prefecture Fisheries Experimental Station, Hikone, Shiga were maintained in fresh water at 15°C with aeration for 7–10 days with no feeding before use. Shell lengths ranged 17–20 mm. The bivalves were then exposed to hypersaline conditions. Diluted artificial sea water (Marine Merit, Matsuda, Osaka, Japan) was used as hypersaline water. Bivalves acclimated to fresh water were transferred to the hypersaline water with either 1.0 (0.1%) or 3.0 (0.3%) salinity. Ten specimens were removed at 0, 2, 8, 24 and 72 h. No bivalve died in hypersaline water in 72 h. Tissues from the edge of the mantle lobe, gill and adductor muscle were excised. Tissue water content was obtained by the wet–dry-weight method. In three of ten specimens, the FAA content was determined as previously described. The 19 amino acids described in Table 1 were identified and their levels were determined.The osmolality of the hemolymph and tank water were determined by freezing point depression using a semimicroosmometer (Knauer, Berlin, Germany). Figure 1 shows the changes in the osmolality of the hemolymph and ambient water. The average hemolymph osmolality of C. sandai acclimated to fresh water was 58.4 mOsm/kg H2O. The osmolality of fresh water was 3 mOsm/kg H2O, that of hypersaline water with 1.0 salinity was approximately 35 mOsm/kg H2O, and that of 3.0 saline water ranged 95–98 mOsm/kg H2O. Therefore, to the hemolymph of C. sandai, 3.0 salinity water was hyperosmotic while 1.0 water was hypo-osmotic. In the 1.0 saline water, the hemolymph osmolality did not show any significant change after a 72-h exposure. In the 3.0 saline water, the hemolymph *Corresponding author: Tel: 81-75-753-6446. Fax: 81-75-753-6446. Email: toyohara@kais.kyoto-u.ac.jp Present address: Department of Ecosystem Studies, School of Environmental Science, The University of Shiga Prefecture, Hikone, Shiga 522-8533, Japan. Received 15 August 2007. Accepted 12 November 2007. FISHERIES SCIENCE 2008; 74: 1339–1341