Asian medaka fishes offer new models for studying mechanisms of seawater adaptation

Japanese medaka (Oryzias latipes) is a freshwater (FW) teleost that is popular throughout the world for laboratory use. In this paper, we discuss the utility of Japanese medaka and related species for studying mechanisms of seawater (SW) adaptation. In addition to general advantages as an experimental animal such as their daily spawning activity, transparency of embryos, short generation time and established transgenic techniques, Japanese medaka have some adaptability to SW unlike the strictly stenohaline zebrafish (Danio rerio). Since other species in the genus Oryzias exhibit different degrees of adaptability to SW, comparative studies between Japanese medaka, where molecular-biological and genetic information is abundant, and other Oryzias species are expected to present varying approaches to solving the problems of SW adaptation. We introduce some examples of interspecies comparison for SW adaptabilities both in adult fish and in embryos. Oryzias species are good models for evolutionary, ecological and zoogeographical studies and a relationship between SW adaptability and geographic distribution has been suggested. Medaka fishes may thus deliver new insights into our understanding of how fish have expanded their distribution to a wide variety of osmotic environments.     

Permeability properties and occludin expression in a primary cultured model gill epithelium from the stenohaline freshwater goldfish

Techniques for the primary culture of fish gill epithelia on permeable supports have provided ‘reconstructed’ gill models appropriate for the study of gill permeability characteristics in vitro. Models developed thus far have been derived from euryhaline fish species that can tolerate a wide range of environmental salinity. This study reports on procedures for the primary culture of a model gill epithelium derived from goldfish, a stenohaline freshwater (FW) fish that cannot tolerate high environmental salt concentrations. The reconstructed goldfish gill epithelium was cultured on permeable filter inserts and using electron microscopy and immunocytochemical techniques, was determined to be composed exclusively of gill pavement cells. When cultured under symmetrical conditions (i.e. with culture medium bathing both apical and basolateral surfaces), epithelial preparations generated appreciable transepithelial resistance (TER) (e.g. 1,150 ± 46 Ωcm²) within 36–42 h post-seeding in inserts. When apical medium was replaced with FW (asymmetrical conditions to mimic conditions that occur in vivo), epithelia exhibited increased TER and elevated paracellular permeability. Changes in permeability occurred in association with altered occludin-immunoreactive band position by western blot and no change in occludin mRNA abundance. We contend that the goldfish gill model will provide a useful in vitro tool for examining the molecular components of a stenohaline fish gill epithelium that participate in the regulation of gill permeability. The model will allow molecular observations to be made together with assessment of changing physiological properties that relate to permeability. Together, this will allow further insight into mechanisms that regulate gill permeability in fishes.     

Age/size effects on juvenile green sturgeon, Acipenser medirostris, oxygen consumption, growth, and osmoregulation in saline environments

The green sturgeon, Acipenser medirostris, is an anadromous species that migrates from freshwater (FW) to seawater (SW) relatively early in its life history, although the ages and sizes of juveniles at SW entry are not known. Developmental constraints of osmoregulatory organs may either prohibit (i.e., due to salinity tolerance limits) or minimize (i.e., due to substantial osmoregulatory or ionoregulatory energetic costs) SW entry in small fish. Interestingly, larger green sturgeon are often encountered in brackish water (BW) estuaries, perhaps due to an energetic advantage in occupying these near-isosmotic environments. To test hypotheses concerning fish-size effects on the energetic costs of occupying habitats of different salinities, we measured oxygen consumption rates in green sturgeon representing three age groups (100, 170, and 533 days post hatch; dph), which were acclimated for 5 weeks to one of three salinities (FW, <3‰; BW, 10‰; or SW, 33‰). Also, after 7 weeks, final wet masses were compared and blood and muscle tissue samples were taken to assess osmoregulatory abilities. There were no differences in body-mass-adjusted oxygen consumption rates between any salinities or ages, indicating that the energetic costs were not prohibitively high to occupy any of these salinities. The only mortalities occurred in the 100 dph SW group, where 23% of the fish died, from apparent starvation. Final wet masses were comparable between FW and BW for each age group and with the 533 dph SW group, but were lower in SW groups at 100 and 170 dph. Similarly, osmoregulatory abilities, in terms of plasma osmolality, Na⁺, K⁺, lactate, and protein concentrations, and muscle water content, were comparable in FW and BW groups at all ages, and with the SW group at 533 dph. These results indicated an age/body size effect in hyperosmotic adaptability, and that juvenile green sturgeon may be found in FW or BW at any age, but only have the ability to enter SW by 1.5 years (75 cm, 1.5 kg) of age.     

Transcriptomic differences between euryhaline and stenohaline malaria vector sibling species in response to salinity stress

Evolution of osmoregulatory systems is a key factor in the transition of species between fresh‐ and saltwater habitats. Anopheles coluzzii and Anopheles merus are stenohaline and euryhaline malaria vector mosquitoes belonging to a larger group of sibling species, the Anopheles gambiae complex, which radiated in Africa within the last 2 million years. Comparative ecological genomics of these vector species can provide insight into the mechanisms that permitted the rapid radiation of this species complex into habitats of contrasting salinity. Here, we use RNA‐Seq to investigate gene expression differences between An. coluzzii and An. merus after briefly exposing both young and old larval instars of each species to either saltwater (SW) or freshwater (FW). Our study aims to identify candidate genes and pathways responsible for the greater SW tolerance of An. merus. Our results are congruent with the ability of gene induction to mediate salinity tolerance, with both species showing increasing amounts of differential gene expression between SW and FW as salt concentrations increase. Besides ion transporters such as AgAE2 that may serve as effectors for osmoregulation, we also find mitogen‐activated protein kinases that may serve in a phosphorylation signalling pathway responding to salinity, and report potential cross‐talk between the mosquito immune response and osmoregulation. This study provides a key step towards applying the growing molecular knowledge of these malaria vectors to improve understanding of their ecological tolerances and habitat occupancy.     

Salinity tolerance of two tropical fishes, Oreochromis aureus and O. niloticus. I. Biochemical and morphological changes in the gill epithelium

Cichlids of the genus Oreochromis are fish of economic importance in African countries. They tolerate brackish water, however, with great variations between species. In this work, two species, both from the Ivory Coast but of different origins, O. niloticus (field and laboratory strains) and O. aureus (field strain) were compared during osmotic challenges (10, 20 and 30%o salinity) in order to provide physiological support for their specific behaviour when confronted with natural hypertonic environments. Tolerance to salinity was assessed by correlated observations on gill structure, plasma sodium levels and gill Na⁺/K⁺ ATPase activity. In fresh water (FW), all fish presented a gill epithelium structure characteristic of FW stenohaline fish: no chloride cells (CC) on the lamellae and few CC on the filaments. An increase in external salinity induced the proliferation of CC on filaments, a feature typical of seawater teleosts. This change in gill structure was accompanied by an increase of gill Na⁺/K⁺ ATPase activity. In the most tolerant strains, plasma Na⁺ did not change, indicating successful ion regulation in the hypertonic media. With regard to potential interest of field strains in fish culture, O. aureus acclimated more easily to brackish water than O. niloticus . Interestingly, O. niloticus , kept for several generations in the laboratory, performed best in our challenge studies. Plasma Na⁺ levels and gill CC proliferation upon transfer to an isotonic medium may be the parameters of choice when testing these fish for their response to a salinity change.     

Day-night behavior in river entry of kutum and its relation to melatonin

Reproduction in most fish species is a scheduled phenomenon and spawning occurs at a precise time ensuring maximum survival of offspring. During the spawning season, reproductive activities of fish species and their daily entry to rivers are influenced by environment. Number of Rutilus frisii kutum entered a river discharging to Caspian Sea was analyzed to investigate the association between timing of river entry and day–night changes in environmental variables. Four thousand five hundred and sixty seven ascending adult kutum were collected in Khoshkrood River between March and May 2012. Physicochemical parameters were recorded and Melatonin levels were measured in a hundred migratory kutum over day and night during the spawning period. A significant effect of daytime on river entry was observed, as more fish entered the river at night. Melatonin levels decreased to the minimum level during peak days of migration, suggesting variations in melatonin levels can help to harmonize the daily rhythm in kutum migration.     

Effects of environmental salinity and 17alpha-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus

Effects of environmental salinity and 17α-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3–5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.     

The metabolic effects of prolonged starvation and refeeding in sturgeon and rainbow trout

The present study examines the particular metabolic strategies of the sturgeon Acipenser naccarii in facing a period of prolonged starvation (72 days) and subsequent refeeding (60 days) compared to the trout Oncorhynchus mykiss response under similar conditions. Plasma metabolites, endogenous reserves, and the activity of intermediate enzymes in liver and white muscle were evaluated. This study shows the mobilization of tissue reserves during a starvation period in both species with an associated enzymatic response. The sturgeon displayed an early increase in hepatic glycolysis during starvation. The trout preferentially used lactate for gluconeogenesis in liver and white muscle. The sturgeon had higher lipid-degradation capacity and greater synthesis of hepatic ketone bodies than the trout, although this latter species also showed strong synthesis of ketone bodies during starvation. During refeeding, the metabolic activity present before starvation was recovered in both fish, with a reestablishment of tissue reserves, plasmatic parameters (glucemia and cholesterol), and enzymatic activities in the liver and muscle. A compensatory effect in enzymes regarding lipids, ketone bodies, and oxidative metabolism was displayed in the liver of both species. There are metabolic differences between sturgeon and trout that support the contention that the sturgeon has common characteristics with elasmobranchs and teleosts.     

Compensatory growth in sub‐yearling Siberian sturgeon,Acipenser baerii Brandt, 1869: Effects of starvation and refeeding on growth,feed utilization and body composition

The capacity of sub‐yearling Siberian sturgeon (Acipenser baerii Brandt, 1869) (19.7 ± 0.8 g) to show compensatory growth was assessed for a 40‐day period for the effects of short‐term starvation and refeeding on growth, feeding performance and body composition. After acclimation, 25 experimental fish were randomly distributed among twelve 500‐L cylindrical fiberglass tanks with a flow‐through system. The fish were subjected to four different feeding regimes: control, which was fed four times daily to apparent satiation; T₁: four periods of 2 days starvation alternating with 8 days re‐feeding; T₂: two periods of 4 days starvation alternating with 16 days refeeding; T₃: an 8 days starvation period followed by 32 days refeeding. At the end of the experiment, the deprived fish attained body weights comparable to those attained by the control fish. There were no differences in growth and feeding performances between the deprived and the control fish. Total protein and lipid contents of the control fish were significantly higher than that of T₁ and T₂ fish at the end of the experiment (P < 0.05). A significant difference in the energy content was observed between T₂ and the control. Siberian sturgeon exhibited complete compensation, indicating a high ability of the deprived fish to grow sufficiently to fully compensate for weight loss during starvation. The results suggested that the feeding schedule involving starvation–refeeding cycles could be a promising feed management option for the culture of this species.     

<Emphasis Type="Italic">Takifugu obscurus</Emphasis> is a euryhaline fugu species very close to <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and suitable for studying osmoregulation

Background  

The genome sequence of the pufferfish Takifugu rubripes is an enormously useful tool in the molecular physiology of fish. Euryhaline fish that can survive both in freshwater (FW) and seawater (SW) are also very useful for studying fish physiology, especially osmoregulation. Recently we learned that there is a pufferfish, Takifugu obscurus, common name "mefugu" that migrates into FW to spawn. If T. obscurus is indeed a euryhaline fish and shares a high sequence homology with T. rubripes, it will become a superior animal model for studying the mechanism of osmoregulation. We have therefore determined its euryhalinity and phylogenetic relationship to the members of the Takifugu family.     

Prolactin regulation in the coho salmon,Oncorhynchus kisutch

Summary Parr and smolt sea water acclimated coho salmon,Oncorhynchus kisutch were subjected to gradual and direct transfers to fresh water. Plasma osmotic pressure, Na⁺, K⁺, Ca⁺⁺ and Mg⁺⁺ were similar in freshwater (FW) fish and seawater (SW) transferred controls for the 24 h following transfer. In spite of the similarity in osmotic pressure and ion levels, plasma cortisol concentrations were significantly increased immediately following salinity change while both pituitary and plasma prolactin decreased indicating enhanced secretion by the pituitary and clearance from the blood. In vitro experiments showed greater incorporation of tritiated leucine into prolactin (PRL) cells immediately after transfer to FW while prolactin injections into intact fish lowered activity in rostral pars distalis (RPD) cells as measured by the same technique, providing evidence of hormonal feedback. These experiments show that the increased synthesis and release of PRL that occurs in coho following movement into FW is not obviously correlated with plasma osmotic pressure, Na⁺ or Ca⁺⁺ concentrations as has been observed in other species of teleosts.Abbreviations FW freshwater - SW seawater - PRL prolactin - RPD rostral pars distalis     

Fatty‐Acid Profile of Total and Polar Lipids in Cultured Rainbow Trout (Oncorhynchus mykiss) Raised in Freshwater and Seawater (Croatia) Determined by Transmethylation Method

Fatty acids from total lipids and polar lipids in cultured rainbow trout (Oncorhynchus mykiss) raised in seawater (SW) and freshwater (FW) were identified and quantified from the muscle samples in January, April, and July. The highest total lipid and polar lipid amounts were found in April. July contents of total lipids were low, but percent of the polyunsaturated fatty acids (PUFAs) was high in SW and FW environment (particularly n‐3 PUFAs). Variety of 17 fatty acids was identified by GC‐FID after transmethylation. The predominant fatty acids in rainbow trout from SW and FW were: docosahexaenoic acid among n‐3 PUFAs, palmitic acid among saturated fatty acids (SFAs), and oleic acid among monounsaturated fatty acids (MUFAs). Appreciably higher n‐3/n‐6 ratio was found in total lipids in April (6.40, FW fish) and in polar lipids in July (18.76; SW fish). High n‐3/n‐6 ratio in total lipids and polar lipids of rainbow trout from SW and FW, besides beneficial n‐3/n‐6 ratio in the commercial fish food, could be characteristic for the local environmental conditions (Croatia).     

Effect of hypo-osmotic environmental changes on the expression of gonadotropin-releasing hormone,its receptor,and gonadotropin hormone subunit mRNA in adult chum salmon (Oncorhynchus keta)

Migrating fish such as salmonids are affected by external environmental factors and salinity changes are particularly important, influencing spawning migration. The aim of this study was to test whether changes in salinity would affect the expression of the hypothalamic-pituitary-gonadal (HPG) axis hormones (gonadotropin-releasing hormones (GnRHs) [salmon GnRH and chicken GnRH-II], GnRH receptors [GnRHR1 and GnRHR5], and mRNA of the gonadotropin hormone [GTH] subunits [GTHα, follicle stimulating hormone β, and luteinizing hormone β]) in chum salmon (Oncorhynchus keta). Fish were progressively transferred from seawater (SW) through 50% SW to freshwater (FW), and the relationship between the osmoregulatory hormone prolactin (PRL) and sexual maturation was determined. The expression and activity of HPG hormones and their receptors, and levels of estradiol-17β and PRL increased after fish were transferred to FW, demonstrating that changes in salinity stimulate the HPG axis and PRL production in migrating chum salmon. These findings reveal details about the role of the endocrine system in maintaining homeostasis and stimulating sexual maturation and reproduction in response to salinity changes in this species.     

Diverse adaptability in oryzias species to high environmental salinity

The genus Oryzias containing freshwater (FW) and seawater (SW) species is a potential model for studying mechanisms of osmotic adaptation. In this study, we compared SW adaptability of four Oryzias species, O. javanicus, O. dancena, O. latipes and O. marmoratus inhabiting different osmotic environments. SW adaptability was evaluated at several stages of the lifecycle: (i) survival rates of adult fish after transfer from FW to 50%SW or SW, (ii) spawning ability in FW and SW, (iii) fertilization rates in FW and SW, and (iv) hatching rates in FW, 50%SW and SW. Results obtained agreed with the natural habitat of each species: O. javanicus, which inhabits SW or brackish water (BW), is fully adaptable to both SW and FW at all the stages examined. The BW species O. dancena also revealed high SW adaptability except for the hatching rate. O. marmoratus, confined in FW, exhibited low SW adaptability at all stages examined while O. latipes, another FW species, was adaptable to SW at most stages examined. Based on these results, the role of SW adaptability to the distribution area of each species is discussed.     

Effects of dietary trimethylamine on free amino acid and nonprotein nitrogen levels in muscle of the guppy, Poecilia reticulata, in relation to seawater adaptation

Guppies Poecilia reticulata acclimated to 100% seawater (SW) had lower taurine and alanine levels in muscle than fish kept in freshwater (FW). The glycine level, in contrast, was higher in SW fish than in FW fish. Levels of other free amino acids (FAA) were comparatively low and little different between fish adapted in FW and in SW. In both FW and SW fish almost all of muscle FAA showed little difference in levels between fish kept on diets containing three different levels of trimethylamine (TMA) (0, 223, and 334 mumol TMA/kg dry weight of diet). Total FAA and nonprotein nitrogen levels in muscle were unaffected by the difference in either the diet species or the ambient salinities. Muscle trimethylamine oxide levels were higher in SW fish than in FW fish. In both salinities, muscle trimethylamine oxide levels in fish on the diets containing 223 and 334 mumol TMA/kg were slightly greater than the level in fish on the TMA-free diet.     

Distribution of chloride cells in teleost larvae

Distribution and density of the chloride cells in the newly hatched larvae of teleosts vary depending on species and environmental salinity at hatching. In the euryhaline freshwater ayu (Plecoglossus altivelis), chloride cells are concentrated in the skin posterior to the pectoral fins and gradually decrease in number toward the head and tail. In the stenohaline sea water flounder (Kareius bicoloratus), most chloride cells are localized at the inner membrane of gill chambers and in the skin near the openings of gill chambers, but only a few cells appear in the skin of the yolk sac. In the stenohaline freshwater carp (Cyprinus carpio), only a few small chloride cells are scattered in the body skin. The density and abundance of chloride cells appears to be correlated with the different requirements for osmoregulation in teleost larvae.     

Effect of winter feeding and starvation on the growth performance of young‐of‐year (YOY) great sturgeon,Huso huso

The objective of this study was to evaluate the feeding rate of the great sturgeon (Huso huso) young of the year (YOY) and to investigate the effects of different feeding rates in maintaining the weight of fish during short periods of winter starvation. Six feeding rates of 0.2, 0.4, 0.6, 0.8, 1.0% body weight (BW) day^?1 and feeding to satiation were considered for the first experiment. Each feeding rate was randomly assigned to three replicate tanks, with continuous feeding throughout a 5‐week winter period of water temperatures below 10°C. Fifteen fish were held in each of 18 tanks with an average initial body weight of 219.6 ± 6.9 g. After 5 weeks of feeding, the best performance was observed in fish fed 1% BW day^?1, but negative growth was observed in fish fed 0.2% BW day^?1. In the second experiment, fish were deprived of feed for 3 weeks at winter temperatures. Weights and condition factors of all fish decreased during starvation, while the differences in mean weight before and after the starvation period were not significant in fish fed a level of 0.2% BW day^?1 and those fish fed to satiation. No mortality was recorded in either experiment. Results of this study indicate that a feeding rate of 1% BW day^?1 would be sufficient for commercial fish farming of YOY of this species to maintain them over winter. Also, to maintain fish weights and prevent weight loss in overwintering ponds, a feeding rate of around 0.3% BW day^?1 seems appropriate for hatcheries.     

Plasma thyroxine and cortisol profiles and gill and kidney Na+/K+-ATPase and SDH activities during acclimation of the catfish Heteropneustes fossilis (bloch) to higher salinity, with special reference to the effects of exogenous cortisol on hypo-osmoregulatory ability of the catfish

When the stenohaline catfish Heteropneustes fossilis was transferred from fresh water (FW) to 30% seawater (SW), the Na(+)/K(+)-ATPase activity significantly increased in the kidney, while in gills it remained more or less constant. A reverse pattern was observed for succinic dehydrogenase (SDH) activity inasmuch as it significantly increased in gills and remained unchanged in the kidney. Plasma osmolality significantly increased within 3 days of transfer to 30% SW and remained significantly higher throughout the duration of experiment. These results suggest that catfish gills may not be able to reverse their function from salt uptake in FW to salt excretion at higher salinity, and that the elimination of monovalent as well as divalent ions is performed by the kidney but not the gills. The significant decline in plasma cortisol (F) levels following transfer to higher salinity may not be due to reduced production but rather to an enhanced utilization and clearance rate, a conclusion supported by the fact that exogenous administration of cortisol acetate (FA) resulted in significant increases in branchial and renal Na(+)/K(+)-ATPase in FW and 30% SW. FA also improved the plasma osmotic regulatory ability of the catfish, possibly due to a change in branchial function from salt-absorption to salt excretion, as was evident from a significant increase in branchial Na(+)/K(+)-ATPase activity in the fish in 30% SW pretreated with FA for 5 days. Consistently higher levels of plasma thyroxine (T4) following transfer to higher salinity suggest the involvement of this hormone at higher salinity.     

Water and NaCl absorption by the intestine of the tilapiaSarotherodon mossambicus adapted to fresh water or seawater and the possible role of prolactin and cortisol

Summary Rates of intestinal water, sodium and chloride absorption in tilapia, adapted to fresh water (FW) and seawater (SW), were measured in vitro, using noneverted sacs made from the anterior, middle and posterior intestinal regions. The anterior intestine from SW fish showed considerably less water, sodium and chloride absorption compared with that seen in FW fish. The middle intestine showed either minimal absorption or some secretion in both FW and SW. In the posterior intestine, water absorption was only limitedly affected by SW-adaptation, but sodium and chloride absorption rates were significantly lower in SW fish. Reductions in water absorption were already evident in the anterior intestine 24 h after transfer to 1/3 SW but reached lower levels 3 to 5 days following transfer to 100% SW. Thus, the anterior intestine of tilapia responds to increased environmental salinity by decreasing uptake of ions, whereas the posterior intestine maintains similar water absorption in both FW and SW, although ion absorption is lower in SW.Prolactin administration to SW fish augmented sodium and water absorption in the anterior intestine but had no effect on chloride absorption. In contrast, cortisol administration to FW fish decreased absorption of sodium, chloride and water to levels usually seen in SW fish. The observed effects of these hormones in tilapia intestinal absorption may be confined to the specialized anterior intestinal region in this species; hormonal effects on the rest of the intestine were not examined.