Sanae Hasegawa

Excellent salinity tolerance of Mozambique tilapia (Oreochromis mossambicus): elevated chloride cell activity in the branchial and opercular epithelia of the fish adapted to concentrated seawater.

Abstract Changes in morphology and cellular activity of the chloride cells in branchial and opercular epithelia were examined in tilapia, Oreochromis mossambicus, adapted to fresh water (FW), seawater (SW) and concentrated SW (180% SW). The tilapia are adaptable to a wide range of salinity, maintaining the plasma osmolality within physiological levels. Gill Na+, K+-ATPase activity was remarkably increased in response to elevated environmental salinity. Using immunocytochemical staining with an antiserum specific for α-subunit of Na+, K+-ATPase, chloride cells were detected on the afferent half of the filament epithelia. The size of immunoreactive chloride cells was twice larger in SW and four times larger in 180% SW than in FW. Confocal laser scanning microscopic observations revealed the frequent occurrence of chloride cell complexes under hypersaline conditions. By electron microscopy, a deeply invaginated apical crypt and well-developed tubular network were observed in chloride cells of SW- and 180% SW-adapted fish, indicating enhanced cellular activity. Chloride cells present in the opercular membrane were also developed in response to increased salinity. These findings suggest that highly activated chloride cells in branchial and opercular epithelia may be responsible for salt secretion in hyperosmotic environments. The excellent salinity tolerance of tilapia appears to be attributed to their ability to develop chloride cells in response to increased environmental salinity.

Intestinal water absorption through aquaporin 1 expressed in the apical membrane of mucosal epithelial cells in seawater-adapted Japanese eel

SUMMARY To elucidate the mechanisms associated with water absorption in the intestine, we compared drinking and intestinal water absorption in freshwater- and seawater-adapted Japanese eels, and investigated a possible involvement of aquaporin (AQP) in the absorption of water in the intestine. Seawater eels ingested more water than freshwater eels, the drinking rate being 0.02 ml kg-1 h-1 in fresh water and 0.82 ml kg-1 h-1 in sea water. In intestinal sacs prepared from freshwater and seawater eels, water absorption increased in time- and hydrostatic pressure-dependent manners. The water absorption rates were greater in seawater sacs than in freshwater sacs, and also greater in the posterior intestine than in the anterior. In view of the enhanced water permeability in the intestine of seawater eel, we cloned two cDNAs encoding AQP from the seawater eel intestine, and identified two eel homologues (S-AQP and L-AQP) of mammalian AQP1. S-AQP and L-AQP possessed the same amino acid sequence, except that one amino acid was lacking in S-AQP and two amino acids were substituted. Eel AQP1 was expressed predominantly in the intestine, and the expression levels were higher in seawater eel than in freshwater eel. Immunocytochemical studies revealed intense AQP1 immunoreaction in the apical surface of columnar epithelial cells in seawater eel, in which the immunoreaction was stronger in the posterior intestine than in the anterior. In contrast, the immunoreaction was faint in the freshwater eel intestine. Preferential localization of AQP1 in the apical membrane of epithelial cells in the posterior intestine of seawater eel indicates that this region of the intestine is responsible for water absorption, and that AQP1 may act as a water entry site in the epithelial cells.

Isolation and properties of chum salmon prolactin.

A highly purified prolactin (PRL) was isolated from the chum salmon pituitary by extraction with acid acetone, gel filtration on Sephadex G-25 and ion-exchange chromatography on CM-Sephadex C-25 with a yield of 1 mg/g of wet tissue. It was 10-15 times more potent than ovine PRL in sodium-retaining activity for juvenile rainbow trout adapted to 50% seawater. The salmon PRL emerged as a single and symmetrical peak on Sephadex G-100 with Ve/Vo = 2.0. Polyacrylamide gel electrophoresis revealed only one band at pH 4.3, whereas no band was seen at pH 7.5. The isoelectric point was estimated to be 10.3 by gel electric focusing. The circular dichroism spectrum of the salmon PRL was similar to that of tilapia PRL, showing an alpha-helix content of 50%. The salmon PRL had a molecular weight of 23,400 daltons by gel filtration and 22,300 daltons by sodium dodecyl sulfate gel electrophoresis, with a single NH2-terminal residue, isoleucine, and a single COOH-terminal residue, half-cystine. In the sequence comparison with those of mammalian PRLs and growth hormones, the clusters of invariant residues were found in both terminal regions, although the disulfide at NH2-terminal of mammalian PRLs was missing. Specific salmon PRL antisera were prepared in rabbits giving a precipitin reaction against the salmon PRL and a pituitary extract of tilapia in agar diffusion but no cross reaction with purified mammalian PRLs. The antibody was localized specifically in PRL cells of the chum salmon pituitary.

Development of multicellular complexes of chloride cells in the yolk-sac membrane of tilapia (Oreochromis mossambicus) embryos and larvae in seawater.

Abstract.Morphological changes in the chloride cells (CCs) in the yolk-sac membrane of euryhaline tilapia (Oreochromis mossambicus) embryos and larvae were examined in relation to environmental salinity. Half of a brood of embryos spawned in fresh water (FW) were transferred directly to seawater (SW) 1 day before hatching; the other half was maintained in FW. The embryos and larvae in both FW and SW contained a rich population of CCs in the yolk-sac membrane; the CCs were visualized by whole-mount immunocytochemistry with an antiserum specific for Na+,K+-ATPase. The sectional areas of CCs increased markedly following SW transfer, whereas they remained small in the embryos and larvae maintained in FW. Scanning electron microscopy showed that the apical opening of CCs was enlarged in the fish transferred to SW. Transmission electron microscopy revealed enhanced cellular activity in SW, as evidenced by well-developed mitochondria and tubular systems. The CCs in SW frequently formed a multicellular complex, consisting of a main CC and one or two accessory cells. Accessory cells interdigitated with the main cells and extended their cytoplasmic processes to the apex of the main cell. The three-dimensional arrangement of the cells participating in the complex was identified by confocal laser scanning microscopy. Such complexes were rarely observed in FW fish. The activated CCs in the yolk-sac membrane in the SW fish probably function as ion-extruding sites during embryonic and larval stages until gill CCs become functional.

Isolation of two forms of growth hormone secreted from eel pituitaries in vitro

Two forms of growth hormone (GH) were purified by chromatofocusing of medium from cultured Japanese eel (Anguilla japonica) pituitaries. The pituitaries were organ-cultured in Eagles minimum essential medium with Earles salts. Following polyacrylamide gel electrophoresis of the medium at pH 9.5, two prominent bands were seen with Rf 0.36 and 0.29; they were designated as eGHI and eGHII, respectively. Seven-hundred fifty milliliters of medium, in which 260 pituitaries were cultured for 6-10 weeks, was concentrated by DIAFLO membrane (YM-5) and subjected to gel filtration on a Sephadex G-75 column and to chromatofocusing on a PBE-94 column. eGHI and II were finally purified by gel filtration on a Sephadex G-75 column, yielding 2.0 mg of eGHI and 1.3 mg of eGHII. Both eGHI and eGHII were equipotent to ovine GH in promoting growth of juvenile rainbow trout. The putative GH-producing cells in the proximal pars distalis of the eel pituitary were stained specifically with antisera raised against eGHI or eGHII; no cross-reactivity was seen in the follicular prolactin cells in the rostral pars distalis. As determined by gel isoelectric focusing, eGHI and eGHII have isoelectric points of 6.3 and 6.7, respectively. Identical molecular masses of 23,000 Da were determined by sodium dodecyl sulfate gel electrophoresis. Their amino acid compositions strongly resembled each other; comparison of the partial N-terminal amino acids indicates that sequence 1 to 36 of GHII is exactly the same as 4 to 39 of GHI.

Osmoregulatory ability of chum salmon,Oncorhynchus keta, reared in fresh water for prolonged periods

The osmoregulatory ability of chum salmon (Oncorhynchus keta), reared in fresh water for a prolonged period, was examined by transferring them directly to seawater and then back to fresh water. When fry and juveniles weighing 0.3–125g, reared in fresh water for 1.5–13 months, were transferred directly to seawater, they adjusted their plasma Na+ concentration to the seawater-adapted level within 12–24h. When they were transferred back to fresh water after having been adapted to seawater for 2 weeks, the plasma Na+ level gradually decreased during the first 12–24h, and then increased to reattain the initial freshwater level after 5–7 days. No mortality was observed during the experiment except among the smallest fry weighing about 0.3g after transfer to seawater (2.1%). The maintenance of good osmoregulatory ability of the chum salmon for a prolonged period in fresh water seems to be unique among Pacific salmon, with the possible exception of the pink salmon.Changes in plasma levels of hormones during the transfer experiments were recorded in juveniles reared in fresh water for 13 months. Prolactin levels increased maximally 3 days after transfer from seawater to fresh water, as would be expected from its well-established role in freshwater adaptation in several euryhaline teleosts. In addition, an increase in plasma growth hormone was observed during the first 12h after seawater transfer, along with a tendency towards a decrease during freshwater transfer, suggesting an important role for this hormone in seawater adaptation. There were no consistent changes in plasma levels of thyroxine and cortisol during freshwater to seawater or seawater to freshwater transfer.

Changes in plasma hormone levels during loss of hypoosmoregulatory capacity in mature chum salmon (Oncorhynchus keta) kept in seawater

Returning chum salmon (Oncorhynchus keta) in northern Honshu Island, Japan, complete gonadal maturation while in the bay. Mature fish caught in the bay failed to survive in seawater for more than a week, whereas they adapted to fresh water efficiently. Mortality in seawater seems to be due primarily to an increased plasma osmolality. Maladaptation to seawater was more pronounced in the fish caught deep in the bay than those caught outside the bay, and also greater in females than in males. In close correlation with the increased plasma osmolality and electrolyte concentrations, plasma levels of cortisol and growth hormone increased in the fish kept in seawater. Cortisol and growth hormone may be secreted in response to the increased plasma osmolality and would not be the direct cause of the maladaptation to seawater. Plasma prolactin remained low in the seawater fish, indicating that the increased secretion of prolactin, a freshwater-adapting hormone, is not the cause of maladaptation to seawater either. On the other hand, when the fish caught in the river were kept in fresh water, an increase in plasma prolactin concentrations was seen, particularly in females, whereas no significant change was seen in plasma cortisol and growth hormone. Concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one in the female and of testosterone in both the male and female were extremely high in the bay fish and decreased slightly but significantly after 7 days in fresh water. The gonadal steroids may have inhibitory effects on osmoregulation in the mature salmon in seawater.

Hypoosmoregulatory ability of eyed-stage embryos of chum salmon

To evaluate the osmoregulatory ability of eyedstage embryos of laboratory-reared chum salmon, Oncorhynchus keta, we examined changes in osmolality of the perivitelline fluid and blood following transfer to 50 and 100% seawater (SW), together with morphological changes in chloride cells present in the yolk sac membrane. Transfer to SW did not cause any significant change in the whole egg weight. However, the embryos shrank when the eggs were transferred to SW, whereas the perivitelline space increased at the expense of the embryo. Osmolality of the perivitelline fluid increased rapidly to reach environmental levels 3 h after transfer, indicating that the egg shell is permeable to ions and water. Blood osmolality increased after transfer to SW, reached a peak level at 3 h, and then decreased gradually. The chloride cells in the yolk sac membrane became activated following transfer, as shown by increased cell size and frequent appearance of apical openings. These results indicate that the eyed-stage embryos of chum salmon possess hypoosmoregulatory ability and that chloride cells in the yolk sac membrane may be involved in salt extrusion, in place of gill chloride cells, during the late embryonic stage.

Acute salinity challenges in Mozambique and Nile tilapia: Differential responses of plasma prolactin, growth hormone and branchial expression of ion transporters

The responses of Mozambique and Nile tilapia acclimated to fresh water (FW) and brackish water (BW; 17 per thousand) were compared following acute salinity challenges. In both species, plasma osmolality increased to above 450 mOsm by 2h after transfer from FW to seawater (SW); these increases in osmolality were accompanied by unexpected increases in plasma prolactin (PRL). Likewise, PRL receptor gene expression in the gill also increased in both species. In Nile tilapia, hyperosmotic transfers (FW to BW and SW) resulted in increased plasma growth hormone (GH) and in branchial GH receptor gene expression, responses that were absent in Mozambique tilapia. Branchial gene expression of osmotic stress transcription factor 1 (OSTF1) increased in both species following transfer from FW to SW, whereas transfer from BW to SW induced OSTF1 expression only in the Nile tilapia. Branchial expression of Na(+)/Cl(-) cotransporter was higher in FW in both species than in BW. Branchial gene expression of Na(+)/K(+)/2Cl(-) cotransporter (NKCC) increased after transfer from BW to SW in Mozambique tilapia, whereas expression was reduced in the Nile tilapia following the same transfer. The difference in the SW adaptability of these species may be related to a limited capacity of Nile tilapia to up-regulate NKCC gene expression, which is likely to be an essential component in the recruitment of SW-type chloride cells. The differential responses of GH and OSTF1 may also be associated with the disparate SW adaptability of these two tilapiine species.

Developmental changes in drinking rate and ion and water permeability during early life stages of euryhaline tilapia, Oreochromis mossambicus, reared in fresh water and seawater

Euryhaline tilapia (Oreochromis mossambicus) can breed either in fresh water (FW) or in seawater, and the developing embryos and larvae withstand direct transfer from FW to SW or vice versa, before the development of osmoregulatory organs. In the study, developmental changes in drinking rate and ion and water permeability were examined after transfer of the embryos from FW to SW. Drinking was measured by accumulation of fluorescent beads in the intestine and also by 14C-dextran accumulation he whole body. The drinking rate increased steadily from 2 to 10 days after hatching, and the larvae in SW consistently imbibed more water than those in FW. The diffusional water permeability remained low during embryonic stages but increased markedly after hatching in both FW and SW; the water permeability was consistently less in SW-adapted embryos and larvae than those in FW. In contrast, the turnover rates of chloride ion in SW were 50 to 100 times greater than those in FW, and increased markedly after hatching. The drinking rate as well as ion and water permeability of the tilapia embryos and larvae in FW and SW were comparable with those reported for stenohaline species. These results clearly indicate that different water and ion regulatory mechanisms are operating in the tilapia embryos and larvae in FW and those in SW to convey their strong euryhalinity.