Osmoregulatory Capacity Research Articles

Effects of domestic effluent discharges on mangrove crab physiology: Integrated energetic, osmoregulatory and redox balances of a key engineer species

Mangroves are increasingly used as biofiltering systems of (pre-treated) domestic effluents. However, these wastewater discharges may affect local macrofauna. This laboratory study investigates the effects of wastewater exposure on the mangrove spider crab Neosarmatium meinerti, a key engineering species which is known to be affected by waste waters in effluent-impacted areas. These effects were quantified by monitoring biological markers of physiological state, namely oxygen consumption, the branchial cavity ventilation rate, gill physiology and morphology, and osmoregulatory and redox balance. Adults acclimated to clean seawater (SW, 32 ppt) and freshwater (FW, ∼0 ppt) were compared to crabs exposed to wastewater for 5 h (WW, ∼0 ppt). Spider crabs exposed to WW increased their ventilation and whole-animal respiration rates by 2- and 3-fold respectively, while isolated gill respiration increased in the animals exposed to FW (from 0.5 to 2.3 and 1.1 nmol O2 min−1 mg DW−1 for anterior and posterior gills, respectively) but was not modified in WW-exposed individuals. WW exposure also impaired crab osmoregulatory capacity; an 80 mOsm kg−1 decrease was observed compared to FW, likely due to decreased branchial NKA activity. ROS production (DCF fluorescence in hemolymph), antioxidant defenses (superoxide dismutase and catalase activities) and oxidative damage (malondialdehyde concentration) responses varied according to animal gender. Overall, this study demonstrates that specific physiological parameters must be considered when focusing on crabs with bimodal breathing capacities. We conclude that spider crabs exposed to WW face osmoregulatory imbalances due to functional and morphological gill remodeling, which must rapidly exhaust energy reserves. These physiological disruptions could explain the ecological changes observed in the field.

Cold tolerance is linked to osmoregulatory function of the hindgut in Locusta migratoria.

There is growing evidence that maintenance of ion and water balance determines cold tolerance in many insects. The hindgut of terrestrial insects is critical for maintaining organismal homeostasis as it regulates solute and water balance of the haemolymph. Here, we used ex vivo everted gut sacs of Locustamigratoria to examine the effects of temperature (0-30°C), thermal acclimation, hypoxia, and ionic and osmotic forces on bulk water and ion (Na+, K+ and Cl-) movement across the rectal epithelium. These findings were related to simultaneous in vivo measurements of water and ion balance in locusts exposed to similar temperatures. As predicted, we observed a critical inhibition of net water and ion reabsorption at low temperature that was proportional to the in vivo loss of water and ion homeostasis. Further, cold-acclimated locusts, which are known to defend ion and water balance at low temperature, were characterised by improved reabsorptive capacity at low temperature. These findings strongly support the hypothesis that transport mechanisms in the hindgut at low temperature are essential for cold tolerance. The loss of osmoregulatory capacity at low temperature was primarily caused by reduced active transport, while rectal paracellular permeability to fluorescein isothiocyanate dextran was unchanged at 0 and 30°C. During cold exposure, water reabsorption was independent of major cation gradients across the epithelia, while a reduction in mucosal Cl- availability and an increase in mucosal osmolality markedly depressed water reabsorption. These findings are discussed in the context of existing knowledge and with suggestions for future physiological studies on cold acclimation and adaptation in insects.

Effects of acclimation to high environmental temperatures on intermediary metabolism and osmoregulation in the sub-Antarctic notothenioid Eleginops maclovinus

Eleginops maclovinus is a sub-Antarctic fish endemic to the South American coast and is a monotypic species of the Eleginopidae family. A scarce amount of scientific information exists regarding the effects of temperature on E. maclovinus physiology. In this study, E. maclovinus specimens were acclimated for 2 weeks at 10 °C (control), 14, and 18 °C. Posterior assessments were performed for the intermediary metabolism of carbohydrates, lipids, and amino acids (in the plasma, liver, gills, and kidney) and on the osmoregulatory capacity [plasma osmolality and gill, kidney, and foregut Na+, K+-ATPase (NKA) activities]. In the plasma, only lactate and total amino acid levels were affected by the temperature. NKA activity presented a linear relationship with the increased temperatures. The liver exhibited no change in the carbohydrates metabolism, but the glycogen/glucose metabolite levels did differ. Amino acid metabolism showed a direct relationship between temperature and GDH and Asp-AT activities, and an inverse relationship between temperature and Ala-AT activity. Lipid metabolism was similar to Ala-AT. In the gills, carbohydrate metabolism (G6PDH and HK activities) presented a direct relationship with the increased temperatures. In the kidney, carbohydrate metabolism (only G6PDH activity) was greater at higher temperatures, and amino acid metabolism (GDH activity) showed a direct relationship with temperature. Lipid metabolism (G3PDH activity) was greatest at 14 °C, presenting significant differences compared to 10 °C. The obtained results suggest that E. maclovinus can acclimate to the projected thermal increase of climate change, with a reorganization of the intermediary metabolism components being tissue-dependent and osmoregulatory effects being affected by NKA activity and greater rates of nitrogenous waste extrusion.

Thinking in the sustainability of Nothofagus antarctica silvopastoral systems, how differ the responses of seedlings from different provenances to water shortage?

Nothofagus antarctica, is the most important species of the silvopastoral systems of southern Patagonia. However, there is limited information on the ecophysiological response mechanisms of seedlings for survival under different resource availability regimes, and whether such processes may differ between provenances. In this study, we evaluated the functional response to water shortage of seedlings of four N. antarctica provenances grown under controlled conditions. After an initial full irrigation period, seedlings were subjected to a water shortage cycle. Measurements of the diameter at the base of the stem, net photosynthesis (An), stomatal conductance to water vapour (gs), predawn water potential (Ψpd), osmotic potential at maximum and zero turgor (Π100; Π0), relative water content at zero turgor (RWC0) and maximum modulus of elasticity (Emax) were carried out during the experimental period. Differences between provenances concerning An and gs rates were recorded under full irrigation conditions, but were not detected in association with drought stress. All provenances had similar osmotic potential values (Π100 and Π0) at full irrigation, but water shortage led to significant osmotic potential differences between provenances. During the whole experimental period, no significant differences on RWC0 and Emax values were recorded among provenances. Under full irrigation conditions, all N. antarctica provenances had a similar physiological performance which suggests that environmental conditions may be the main driver for phenotypic differences in this species. From the osmotic adjustment results gathered, it is derived that a major osmoregulation capacity in response to water shortage is a common feature of all the N. antarctica provenances evaluated.

Phenotypic plasticity in gene expression and physiological response in red drum Sciaenops ocellatus exposed to a long-term freshwater environment

Red drum (Sciaenops ocellatus) is a euryhaline fish commonly found in the Gulf of Mexico and along the Atlantic coast of North America. Because of high commercial demand and its euryhaline characteristics, aquaculture of this species has diversified from marine to low-salinity aquaculture systems. In recent years, interest in the feasibility of producing red drum in inland freshwater systems has grown and this prompted us to investigate its osmoregulatory capacity after rearing for 8months in a freshwater aquaculture system. We compared the activities of several genes and enzymes involved in the osmoregulatory process in freshwater-acclimatized (FW) and seawater (SW) red drum. The gene expression profiles were variable: the expression of genes encoding Na+/K+-ATPase (NKA) and the cystic fibrosis transmembrane regulator (CFTR) was slightly higher in SW than FW fish, while phosphoenolpyruvate carboxykinase (PEPCK) and the glucocorticoid receptor messenger RNA (mRNA) levels were higher in FW red drum. The total plasma K concentration was 60.3% lower, and gill NKA activity was 63.5% lower in FW than in SW fish. PEPCK activity was twofold higher in FW than in SW red drum. Similarly, liver glycogen was 60% higher in FW fish. In summary, both gene expression and the enzyme activity data support the phenotypic plasticity of red drum and suggest that the limited capacity for ion homeostasis observed, in particular the low plasma K concentration, was due to the composition of freshwater and does not necessarily reflect a physiological inability to osmoregulate.

Effects of different salinities on the osmoregulatory capacity of Mediterranean sticklebacks living in freshwater

AbstractThree‐spined sticklebacks (Gasterosteus aculeatus L.) of the Camargue region (Rhone delta, northern Mediterranean Sea) occupy coastal mesohaline lagoons and freshwater canals. In this study, the acclimation capacity to different salinities of a population living in freshwater canals was investigated. Freshwater (FW; 5 ‰) individuals were compared to fish acclimated for at least 2 weeks to brackish water (BW; 15 ‰) and seawater (SW; 30 ‰). Blood osmotic pressure and gill Na+/K+‐ATPase (NKA) gene expression of the α1 subunit and α1a and α1b isoforms were determined at these salinities. NKA protein expression in the gill ionocytes and the remodelling of these cells were also investigated through NKA immunolabelling and electron microscopy. Blood osmolalities of fish acclimated to FW, BW and SW were significantly different. Branchial NKA α1a and α1b expressions also differed, with a higher expression of NKA α1b in fish acclimated to SW. Gill ionocytes in fish acclimated to FW were located along the lamellae and at their base and appeared elongated, whereas these cells were restricted to gill filaments in fish acclimated to SW with a round shape. Finally, electron microscopy revealed three different types of apical structures for these ionocytes: a honeycomb‐like structure and a dome shape in FW, and deeply encrypted in SW. This study shows that ionocyte cell type and morphology, and gill NKA expression are salinity‐dependent. This branchial remodelling is directly linked to the physiological homeostatic status reached by the fish. It also highlights that sticklebacks of the Camargue region can rapidly acclimate to different salinities and can easily migrate between FW canals and mesohaline coastal lagoons.

Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.

Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.

The euryhaline crabUca tangerishowed metabolic differences to sex and environmental salinity

This study constitutes a first attempt to investigate sex differences in osmoregulatory capacity and metabolic responses in relation to hyper- and hypo-osmoregulation in the intertidal euryhaline crabUca tangeri. Adult male and female specimens from Cadiz Bay, Spain (36°23′–37′N 6°8′–15′W), were acclimated to three different environmental salinities (12, 33 and 55 psu) during 7 days, and several parameters were assessed in haemolymph (osmolality, glucose, amino acids, triglycerides and lactate) as well as in metabolic key organs (hepatopancreas, anterior and posterior gills: glycogen, free glucose, amino acids and triglycerides). Specimens from both sex exhibited high and similar hyper- and hypo-osmoregulatory capacities. However, metabolite levels were differentially affected upon acclimation to low and high salinity in several metabolic organs and haemolymph of male and females: (i) glycogen in gills, (ii) free glucose in gills and hepatopancreas, (iii) amino acids in hepatopancreas, (iv) triglycerides in haemolymph, hepatopancreas and posterior gills, and (v) lactate in haemolymph. The results suggest the occurrence of differential metabolic adjustments upon hyper- and hypo-osmoregulation related to sex in the intertidal euryhaline crabU. tangeri.

Physiological performance and serum Na+, K+ Ca2 + and Mg2 + regulation of black tiger prawn (Penaeus monodon Fabricius 1798) reared in varying Na+/K+ ratios of inland saline water

A 28-day trial was conducted to investigate the serum ionic regulation of juvenile black tiger prawns (Penaeus monodon Fabricius 1798) of 1.2±0.1g mean initial weight, reared in varying Na+/K+ ratios of inland saline water (ISW). The serum Na+, K+, Ca2+ and Mg2+ regulation, osmoregulatory capacity (OC), isosmotic points, survival, growth and condition indices of P. monodon were measured when P. monodon were reared in four different ISW types having Na+ and K+ ratios of 119.4 (represented by raw ISW control having higher Na+/K+ ratio), 6.5, 12,0 and 16.2. The first water type of ISW (control) was at 5ppt (ISW5) and the other 3 were at the salinities of 5ppt (ISWK5), 10ppt (ISWK10) and 15ppt (ISWK15) which were fortified with K+ equivalent to [K+] in the ocean water of 25ppt. P. monodon juveniles reared in ISW5 and ISWK15 regulated their serum Na+ and K+ stronger than juveniles reared in ISWK5 and ISWK10 and brought the serum Na+/K+ ratios closer to the normal Na+/K+ ratio of OW. Serum Ca2+ and Mg2+ of the juveniles reared in ISWK10, were regulated strongly over the trial period. However, the juveniles reared in ISW5 and ISWK15 had increased serum Ca2+ at day 28. After 14days of rearing, the serum osmolality of juveniles decreased significantly from 640.2 and 649.6mOsm to 550.1 and 562.2mOsm in ISWK5 and ISWK10 respectively. In contrast, the serum osmolality and OC of juveniles reared in ISW5 (control) remained constant over the 14day period but then increased significantly toward the end of the trial. During the initial 14days of rearing, 20.8 and 20.5% of the juveniles survived in ISWK5 and ISWK10 respectively. However, 100% of juveniles died at the end of the trial. In contrast, the juveniles reared in ISW5 and ISWK15 were able to survive until the end of the trial with a survival rate of 37.5 and 62%, respectively. At the end of the trial, the SGR and condition indices of juveniles reared at ISW5 and ISWK15 showed no significant difference (P<0.05). At day 14, the amount of food ingested by the juveniles reared in ISWK5 and ISWK10 were significantly lower than the juveniles reared in ISW5 and ISWK15. The exoskeleton moisture content (EM %) and dry exoskeleton index (Ebd) of juveniles reared in ISW5 were significantly higher and lower respectively, than the EM % and Ebd of juveniles reared in ISWK15. This trial suggested that P. monodon juveniles faced unfavourable medium Na+ and K+ ratio in ISW through strong regulation of [Na+] and [K+] which enabled the prawns to establish normal ratios of Na+ and K+ in their haemolymph ranging from 21.42 to 25.97mg/L which were similar to the normal haemolymph ratio of Na+ and K+ of prawns reared in OW ranging from 22.3 to 29.9.

Homeostatic responses of crustaceans to salinity changes

The wide distribution of crustaceans from freshwater through brackish into marine waters and from salt marshes to terrestrial zones shows the exceptional capacities of this animal group to cope with salinity changes. Osmoregulation is the main physiological mechanism that maintains the hydromineral homeostasis of these animals. The difference of osmoregulation capacities between an osmoconformer and an osmoregulator species is mainly explained by anisosmotic extracellular regulation (AER) and/or isosmotic intracellular regulation (IIR). This review will also discuss the particular acquisition of specific osmoregulation capacities in crustaceans from the first embryonic stages to the adult. Several findings revealed that gills and antennal glands are the primordial hydromineral regulation tissues, and they present the same structure and function in all crustaceans, except in shrimps. These tissues contain ionocytes that harbor membrane pumps implicated in ion regulation. Na+/K+-ATPase, the major of these membrane proteins, acts upon rapid ionic changes, usually in cooperation with several other membrane pumps. Also some proteins, belonging to the detoxification and antioxidant systems, seem implicated in the regulation mechanisms after salinity change. This review will resume and discuss the current knowledge on salinity regulation in this large and important animal group.

Evaluating dispersal potential of an invasive fish by the use of aerobic scope and osmoregulation capacity.

Non-indigenous species (NIS) can impact marine biodiversity and ecosystem structure and function. Once introduced into a new region, secondary dispersal is limited by the physiology of the organism in relation to the ambient environment and by complex interactions between a suite of ecological factors such as presence of predators, competitors, and parasites. Early prediction of dispersal potential and future ‘area of impact’ is challenging, but also a great asset in taking appropriate management actions. Aerobic scope (AS) in fish has been linked to various fitness-related parameters, and may be valuable in determining dispersal potential of aquatic invasive species in novel environments. Round goby, Neogobius melanostomus, one of the most wide-ranging invasive fish species in Europe and North America, currently thrives in brackish and fresh water, but its ability to survive in high salinity waters is unknown to date. We show that AS in round goby is reduced by 30% and blood plasma osmolality increased (indicating reduced capacity for osmoregulation) at salinities approaching oceanic conditions, following slow ramping (5 PSU per week) and subsequent long-term acclimation to salinities ranging between 0 and 30 PSU (8 days at final treatment salinities before blood plasma osmolality measurements, 12–20 additional days before respirometry). Survival was also reduced at the highest salinities yet a significant proportion (61%) of the fish survived at 30 PSU. Reduced physiological performance at the highest salinities may affect growth and competitive ability under oceanic conditions, but to what extent reduced AS and osmoregulatory capacity will slow the current 30 km year-1 rate of advance of the species through the steep salinity gradient from the brackish Baltic Sea and into the oceanic North Sea remains speculative. An unintended natural experiment is in progress to test whether the rate of advance slows down. At the current rate of advance the species will reach the oceanic North Sea by 2018/2019, therefore time for taking preventative action is short.

Similarity of osmoregulatory capacity in coastal and inland alligator gar

The alligator gar Atractosteus spatula is a primitive fish species, occupying a wide range of temperature and salinity habitats. Long-distance movements are limited, leading to genetic differentiation between inland and coastal populations. Unknown is whether physiological capacity differs between geographically separated populations, particularly for traits important to osmoregulation in saline environments. Alligator gar from inland and coastal populations were reared in a similar environment and exposed to temperature (10, 30°C) and salinity (0, 20ppt) extremes to determine whether iono- and osmoregulatory ability differed between populations. There were few differences in osmoregulatory ability between populations, with similar gill, blood and gastrointestinal tract osmoregulatory parameters. Blood plasma osmolality, ion concentrations, intestinal pH and bicarbonate base concentrations, intestinal fluid osmolality, ion concentrations and gill Na+, K+-ATPase (NKA) activity were similar between populations. Notably, gar from both populations did not osmoregulate well at low temperature and high salinity, with elevated plasma osmolality and ion concentrations, low gill NKA, and little evidence of gastrointestinal tract contribution to ionic and base regulation based on a lack of intestinal fluid and low base content. Therefore, the hypothesis that coastal gar would have improved osmotic regulatory ability in saline environments as compared to inland alligator gar was not supported, suggesting physiological capacity may be retained in primitive species possibly due to its importance to their persistence through time.

Divergence in physiological factors affecting swimming performance between anadromous and resident populations of brook charr Salvelinus fontinalis.

In this study, an anadromous strain (L) and a freshwater-resident (R) strain of brook charr Salvelinus fontinalis as well as their reciprocal hybrids, were reared in a common environment and submitted to swimming tests combined with salinity challenges. The critical swimming speeds (Ucrit ) of the different crosses were measured in both fresh (FW) and salt water (SW) and the variations in several physiological traits (osmotic, energetic and metabolic capacities) that are predicted to influence swimming performance were documented. Anadromous and resident fish reached the same Ucrit in both FW and SW, with Ucrit being 14% lower in SW compared with FW. The strains, however, seemed to use different underlying strategies: the anadromous strain relied on its streamlined body shape and higher osmoregulatory capacity, while the resident strain had greater citrate synthase (FW) and lactate dehydrogenase (FW, SW) capacity and either greater initial stores or more efficient use of liver (FW, SW) and muscle (FW) glycogen during exercise. Compared with R♀ L♂ hybrids, L♀ R♂ hybrids had a 20% lower swimming speed, which was associated with a 24% smaller cardio-somatic index and higher physiological costs. Thus swimming performance depends on cross direction (i.e. which parental line was used as dam or sire). The study thus suggests that divergent physiological factors between anadromous and resident S. fontinalis may result in similar swimming capacities that are adapted to their respective lifestyles.

Cloning and molecular characterization of the betaine aldehyde dehydrogenase involved in the biosynthesis of glycine betaine in white shrimp (Litopenaeus vannamei)

The enzyme betaine aldehyde dehydrogenase (BADH) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (GB), a very efficient osmolyte accumulated during osmotic stress. In this study, we determined the nucleotide sequence of the cDNA for the BADH from the white shrimp Litopenaeus vannamei (LvBADH). The cDNA was 1882 bp long, with a complete open reading frame of 1524 bp, encoding 507 amino acids with a predicted molecular mass of 54.15 kDa and a pI of 5.4. The predicted LvBADH amino acid sequence shares a high degree of identity with marine invertebrate BADHs. Catalytic residues (C-298, E-264 and N-167) and the decapeptide VTLELGGKSP involved in nucleotide binding and highly conserved in BADHs were identified in the amino acid sequence. Phylogenetic analyses classified LvBADH in a clade that includes ALDH9 sequences from marine invertebrates. Molecular modeling of LvBADH revealed that the protein has amino acid residues and sequence motifs essential for the function of the ALDH9 family of enzymes. LvBADH modeling showed three potential monovalent cation binding sites, one site is located in an intra-subunit cavity; other in an inter-subunit cavity and a third in a central-cavity of the protein. The results show that LvBADH shares a high degree of identity with BADH sequences from marine invertebrates and enzymes that belong to the ALDH9 family. Our findings suggest that the LvBADH has molecular mechanisms of regulation similar to those of other BADHs belonging to the ALDH9 family, and that BADH might be playing a role in the osmoregulation capacity of L. vannamei.

Osmoregulatory capacity, health status and growth as functions of moult stages from various weight classes in marron (Cherax cainii) and yabbies (Cherax destructor)

The study investigated the relationship between five moult stages and the osmoregulatory capacities (OC), moisture content of hepatopancreas (HM%), hepatosomatic indices (HIwet, HIdry) and growth of 6 weight classes of 235 marron (Cherax cainii) and 235 yabbies (Cherax destructor) under laboratory conditions. In each moult stage, OC increased linearly with the increase in wet body weight. Intermoult stage C showed the highest OC significantly lower than premoult stages D0, D1, D2 and postmoult stage AB in every weight class in both crayfish. HM% decreased from AB to D2, while both HIwet and HIdry reduced from C to D2. Percentage dry matter of whole body carcass was highest in stage C and lowest during the AB stage with a significant difference between the two species. Larger than 15-g marron and yabbies differ with each other in OC, HIwet, and HIdry, moult intervals and growth rates.

Hyperglycemic and osmotic effects of dopamine and recombinant hormone CHH-B1 in the Pacific white shrimp Litopenaeus vannamei

The effects of dopamine (DA) on crustacean hyperglycemic hormone (CHH) release and osmoregulation were investigated in the white shrimp Litopenaeus vannamei. Application of 2 μg of the recombinant CHH-B1His hormone or 2 × 10−6 mol L−1 of DA to intact shrimp caused an increase in the hemolymph glucose levels 1 h post-injection, suggesting that DA might stimulate hyperglycemia through CHH release from the sinus glands. This assumption was supported by similar experiments using bilaterally eyestalk-ablated shrimp. Additionally, rCHH-B1His restored the osmoregulatory capacity (OC) of shrimp under hyperosmotic conditions to basal values 2 h post-injection, whereas DA led to an OC decrease in shrimp at all sampling times. These neuroendocrine factors may be involved in the control of metabolism and osmoregulation in L. vannamei and could be important for its adaptation to different environmental conditions.

The mayfly nymph Austrophlebioides pusillus Harker defies common osmoregulatory assumptions.

Osmoregulation is a key physiological function, critical for homeostasis. The basic physiological mechanisms of osmoregulation are thought to be well established. However, through a series of experiments exposing the freshwater mayfly nymph Austrophlebioides pusillus (Ephemeroptera) to increasing salinities, we present research that challenges the extent of current understanding of the relationship between osmoregulation and mortality. A. pusillus had modelled 96 h LC10, LC50 and LC99 of 2.4, 4.8 and 10 g l−1 added synthetic marine salt (SMS), respectively. They were strong osmoregulators. At aquarium water osmolality of 256 ± 3.12 mmol kg−1 (±s.e.; equivalent to 10 g l−1 added SMS), the haemolymph osmolality of A. pusillus was a much higher 401 ± 4.18 mmol kg−1 (±s.e.). The osmoregulatory capacity of A. pusillus did not break down, even at the salinity corresponding to their LC99, thus their mortality at this concentration is due to factors other than increased internal osmotic pressure. No freshwater invertebrate has been previously reported as suffering mortality from rises in salinity that are well below the iso-osmotic point. Recently, studies have reported reduced abundance/richness of Ephemeroptera with slightly elevated salinity. Given that salinization is an increasing global threat to freshwaters, there is an urgent need for studies into the osmophysiology of the Ephemeroptera to determine if their loss at locations with slightly elevated salinity is a direct result of external salinity or other, possibly physiological, causes.

Transcriptome Analysis of the Hepatopancreas in the Pacific White Shrimp (Litopenaeus vannamei) under Acute Ammonia Stress.

In the practical farming of Litopenaeus vannamei, the intensive culture system and environmental pollution usually results in a high concentration of ammonia, which usually brings large detrimental effects to shrimp, such as increasing the susceptibility to pathogens, reducing growth, decreasing osmoregulatory capacity, increasing the molting frequency, and even causing high mortality. However, little information is available on the molecular mechanisms of the detrimental effects of ammonia stress in shrimp. In this study, we performed comparative transcriptome analysis between ammonia-challenged and control groups from the same family of L. vannamei to identify the key genes and pathways response to ammonia stress. The comparative transcriptome analysis identified 136 significantly differentially expressed genes that have high homologies with the known proteins in aquatic species, among which 94 genes are reported potentially related to immune function, and the rest of the genes are involved in apoptosis, growth, molting, and osmoregulation. Fourteen GO terms and 6 KEGG pathways were identified to be significantly changed by ammonia stress. In these GO terms, 13 genes have been studied in aquatic species, and 11 of them were reported potentially involved in immune defense and two genes were related to molting. In the significantly changed KEGG pathways, all the 7 significantly changed genes have been reported in shrimp, and four of them were potentially involved in immune defense and the other three were related to molting, defending toxicity, and osmoregulation, respectively. In addition, majority of the significantly changed genes involved in nitrogen metabolisms that play an important role in reducing ammonia toxicity failed to perform the protection function. The present results have supplied molecular level support for the previous founding of the detrimental effects of ammonia stress in shrimp, which is a prerequisite for better understanding the molecular mechanism of the immunosuppression from ammonia stress.

Effects of fasting and re-alimentation on gill and intestinal morphology and indicators of osmoregulatory capacity in genetically selected sea bass (Dicentrarchus labrax) populations with contrasting tolerance to fasting

Fasting and refeeding occur naturally in predators but this is largely ignored when dealing with farmed fish. Therefore, the effects of 3-week fasting and re-alimentation (2.5% of the individual body mass) were investigated using two genetically selected populations (F2 generation) of 250g juvenile sea bass (Dicentrarchus labrax L.). Blood osmolarity, gill and intestinal morphology and expression of the sodium pump (Na+, K+-ATPase, NKA) were studied on two phenotypes showing different degrees of body mass loss during food deprivation: one group losing body mass rapidly during fasting (F+) and the other one limiting body mass loss during the same period (F−).Blood osmotic pressure significantly decreases due to re-alimentation in both groups, but this is compensated in the F+ group. In this group, gill ionocytes are smaller and less numerous, but a significantly higher NKA gene expression is noted in the gills in comparison to the F− individuals 48 and 72h after re-alimentation, and also in the posterior intestine 72h after re-alimentation. This most probably occurs to compensate for a higher salt intake during nutrient absorption in comparison to the F− group. Furthermore, refed F− fish absorb more lipids along the proximal anterior intestine, and take longer to digest than the F+ group, and show enterocyte vacuolization in the posterior intestine.Therefore, the two selected populations have different postprandial digestive strategies: the F− fish optimize feed efficiency first at the cost of optimal hydromineral adjustment, while the F+ group invests in osmoregulatory performance at the expense of digestive physiology. Statement of relevanceOur paper is highly relevant to the general field of commercial aquaculture. There is an increasing number of research articles dealing with fasting and refeeding in commercial fish and how to improve fish nutrition based on these physiological data and genetic selection.

A transcriptomic scan for potential candidate genes involved in osmoregulation in an obligate freshwater palaemonid prawn (Macrobrachium australiense).

BackgroundUnderstanding the genomic basis of osmoregulation (candidate genes and/or molecular mechanisms controlling the phenotype) addresses one of the fundamental questions in evolutionary ecology. Species distributions and adaptive radiations are thought to be controlled by environmental salinity levels, and efficient osmoregulatory (ionic balance) ability is the main mechanism to overcome the problems related to environmental salinity gradients.MethodsTo better understand how osmoregulatory performance in freshwater (FW) crustaceans allow individuals to acclimate and adapt to raised salinity conditions, here we (i), reviewed the literature on genes that have been identified to be associated with osmoregulation in FW crustaceans, and (ii), performed a transcriptomic analysis using cDNA libraries developed from mRNA isolated from three important osmoregulatory tissues (gill, antennal gland, hepatopancreas) and total mRNA from post larvae taken from the freshwater prawn, Macrobrachium australiense using Illumina deep sequencing technology. This species was targeted because it can complete its life cycle totally in freshwater but, like many Macrobrachium sp., can also tolerate brackish water conditions and hence should have genes associated with tolerance of both FW and saline conditions.ResultsWe obtained between 55.4 and 65.2 million Illumina read pairs from four cDNA libraries. Overall, paired end sequences assembled into a total of 125,196 non-redundant contigs (≥200 bp) with an N50 length of 2,282 bp and an average contig length of 968 bp. Transcriptomic analysis of M. australiense identified 32 different gene families that were potentially involved with osmoregulatory capacity. A total of 32,597 transcripts were specified with gene ontology (GO) terms identified on the basis of GO categories. Abundance estimation of expressed genes based on TPM (transcript per million) ≥20 showed 1625 transcripts commonly expressed in all four libraries. Among the top 10 genes expressed in four tissue libraries associated with osmoregulation, arginine kinase and Na+/K+- ATPase showed the highest transcript copy number with 7098 and 660, respectively in gill which is considered to be the most important organ involved in osmoregulation.DiscussionThe current study provides the first broad transcriptome from M. australiense using next generation sequencing and identifies potential candidate genes involved in salinity tolerance and osmoregulation that can provide a foundation for investigating osmoregulatory capacity in a wide variety of freshwater crustaceans.