Response in Growth, Scute Development, and Whole-Body Ion Composition of Acipenser fulvescens Reared in Water of Differing Chemistries.

In fishes, environmental ion availability can have substantial effects on growth and development. This study examined the development of Lake Sturgeon in response to the varying environmental ion availability that they experience as part of a conservation stocking program. We reared sturgeon in natural water from the Coosa River, which had higher concentrations of Mg2+, Na+, and Zn2+ than standard hatchery conditions, while [Ca2+] at the Warm Springs National Fish Hatchery was 2× higher than in the Coosa River. Eggs were hatched in each water type and the larvae were sampled at time points before and after yolk absorption during the first 8 weeks of development. Total length and weight in WSNFH larvae were significantly higher than larvae in Coosa River water starting at 8 dph, indicating that growth was dependent on the different environmental ion levels. Concentrations of the ions of interest were also determined for whole-body acid digests of the exposed Lake Sturgeon. We found that Lake Sturgeon reared in Coosa River water had significantly higher magnesium and zinc than Lake Sturgeon reared in WSNFH water (p < 0.05), while calcium was significantly higher in WSNFH than Coosa River water. This difference shows that different environmental ion concentrations also impact the overall development of larval Lake Sturgeon.

Environmental calcium and variation in yolk sac size influence swimming performance in larval lake sturgeon (Acipenser fulvescens).

In many animal species, performance in the early life stages strongly affects recruitment to the adult population; however, factors that influence early life history stages are often the least understood. This is particularly relevant for lake sturgeon, Acipenser fulvescens, living in areas where environmental calcium concentrations are declining, partly due to anthropogenic activity. As calcium is important for muscle contraction and fatigue resistance, declining calcium levels could constrain swimming performance. Similarly, swimming performance could be influenced by variation in yolk sac volume, because the yolk sac is likely to affect drag forces during swimming. Testing swimming performance of larval A. fulvescens reared in four different calcium treatments spanning the range of 4-132 mg l-1 [Ca2+], this study found no treatment effects on the sprint swimming speed. A novel test of volitional swimming performance, however, revealed reduced swimming performance in the low calcium environment. Specifically, volitionally swimming larvae covered a shorter distance before swimming cessation in the low calcium environment compared with the other treatments. Moreover, sprint swimming speed in larvae with a large yolk sac was significantly slower than in larvae with a small yolk sac, regardless of body length variation. Thus, elevated maternal allocation (i.e. more yolk) was associated with reduced swimming performance. Data suggest that larvae in low calcium environments or with a large yolk sac exhibit reduced swimming performance and could be more susceptible to predation or premature downstream drift. Our study reveals how environmental factors and phenotypic variation influence locomotor performance in a larval fish.

Measuring intestinal fluid transport in vitro: Gravimetric method versus non-absorbable marker.

The gut sac is a long-standing, widely used in vitro preparation for studying solute and water transport, and calculation of these fluxes requires an accurate assessment of volume. This is commonly determined gravimetrically by measuring the change in mass over time. While convenient this likely under-estimates actual net water flux (Jv) due to tissue edema. We evaluated whether the popular in vivo volume marker [(14)C]-PEG 4000, offers a more representative measure of Jvin vitro. We directly compared these two methods in five teleost species (toadfish, flounder, rainbow trout, killifish and tilapia). Net fluid absorption by the toadfish intestine based on PEG was significantly higher, by almost 4-fold, compared to gravimetric measurements, compatible with the latter under-estimating Jv. Despite this, PEG proved inconsistent for all of the other species frequently resulting in calculation of net secretion, in contrast to absorption seen gravimetrically. Such poor parallelism could not be explained by the absorption of [(14)C]-PEG (typically <1%). We identified a number of factors impacting the effectiveness of PEG. One was adsorption to the surface of sample tubes. While it was possible to circumvent this using unlabelled PEG 4000, this had a deleterious effect on PEG-based Jv. We also found sequestration of PEG within the intestinal mucus. In conclusion, the short-comings associated with the accurate representation of Jv by gut sac preparations are not overcome by [(14)C]-PEG. The gravimetric method therefore remains the most reliable measure of Jv and we urge caution in the use of PEG as a volume marker.

Evidence of circadian rhythm, oxygen regulation capacity, metabolic repeatability and positive correlations between forced and spontaneous maximal metabolic rates in lake sturgeon Acipenser fulvescens.

Animal metabolic rate is variable and may be affected by endogenous and exogenous factors, but such relationships remain poorly understood in many primitive fishes, including members of the family Acipenseridae (sturgeons). Using juvenile lake sturgeon (Acipenser fulvescens), the objective of this study was to test four hypotheses: 1) A. fulvescens exhibits a circadian rhythm influencing metabolic rate and behaviour; 2) A. fulvescens has the capacity to regulate metabolic rate when exposed to environmental hypoxia; 3) measurements of forced maximum metabolic rate (MMR(F)) are repeatable in individual fish; and 4) MMR(F) correlates positively with spontaneous maximum metabolic rate (MMR(S)). Metabolic rates were measured using intermittent flow respirometry, and a standard chase protocol was employed to elicit MMR(F). Trials lasting 24 h were used to measure standard metabolic rate (SMR) and MMR(S). Repeatability and correlations between MMR(F) and MMR(S) were analyzed using residual body mass corrected values. Results revealed that A. fulvescens exhibit a circadian rhythm in metabolic rate, with metabolism peaking at dawn. SMR was unaffected by hypoxia (30% air saturation (O(2sat))), demonstrating oxygen regulation. In contrast, MMR(F) was affected by hypoxia and decreased across the range from 100% O(2sat) to 70% O(2sat). MMR(F) was repeatable in individual fish, and MMR(F) correlated positively with MMR(S), but the relationships between MMR(F) and MMR(S) were only revealed in fish exposed to hypoxia or 24 h constant light (i.e. environmental stressor). Our study provides evidence that the physiology of A. fulvescens is influenced by a circadian rhythm and suggests that A. fulvescens is an oxygen regulator, like most teleost fish. Finally, metabolic repeatability and positive correlations between MMR(F) and MMR(S) support the conjecture that MMR(F) represents a measure of organism performance that could be a target of natural selection.

Regulation of calcium transport in the early life stages of an ancient fish, Acipenser fulvescens.

The freshwater, cartilaginous lake sturgeon (Acipenser fulvescens) encounters its greatest calcium (Ca(2+)) demand in the early life stages. In this study we examined Ca(2+) regulation of lake sturgeon larvae reared at three levels of environmental [Ca(2+)]-0.1, 0.2, and 1.5 mmol L(-1)-from hatch until after the transition to exogenous feeding. Examination of skin, gill, and yolk sac with scanning electron microscopy (SEM) indicated that the density and surface area of mitochondria-rich cells (MRCs) varies over developmental time but that availability of environmental Ca(2+) affected only MRC density. SEM results also demonstrated that Ca(2+) transport is adjusted in localization over the course of development, with the transition to primarily branchial uptake occurring earlier in the highest environmental [Ca(2+)]. Net whole-animal Ca(2+) flux was primarily dependent on influx rate. The increase in whole-body Ca(2+) uptake following the transition to exogenous feeding was greatest in larval lake sturgeon acclimated to low environmental [Ca(2+)], and it suggests that intestinal absorption may supplement enhanced branchial uptake in Ca(2+)-limited fish.

Mechanisms of calcium absorption by anterior and posterior segments of the intestinal tract of juvenile lake sturgeon.

Rapid growth in juvenile fish increases calcium demand, and the intestine may play a role in calcium homeostasis at this life stage, in addition to branchial and renal transport. This study examined calcium flux in the gastrointestinal tract (GIT) of freshwater juvenile lake sturgeon acclimated to 0.14, 0.34, and 2.26mmol L(-1) environmental calcium. Net Ca(2+) flux did not differ due to environmental [Ca(2+)] in either the anterior or posterior intestine. Blocking the apical epithelial calcium channel (ECaC) with ruthenium red (RR, 8.5µmol L(-1)) significantly decreased Ca(2+) influx in the anterior intestine, but exposure to the plasma membrane Ca(2+)-ATP-ase (PMCA) inhibitor trifluoperazine (TFP, 10mmol L(-1)) had no effect at any environmental [Ca(2+)], nor did inhibition of the Na(+)-Ca(2+) exchanger (NCX) with KB-R7943 (10µmol L(-1)). Neither RR nor TFP affected Ca(2+) uptake by the posterior intestine in any of the treatment groups, but KB-R7943 reduced net calcium flux in the posterior intestine at all environmental [Ca(2+)]. Thus, basolateral Ca(2+) influx in the posterior GIT of lake sturgeon relies more heavily on NCX than PMCA. Furthermore, the differing pharmacological effects in the anterior and posterior intestine suggest that the dominant transporters responsible for calcium uptake vary over the length of the GIT in lake sturgeon.

Fundulus heteroclitus acutely transferred from seawater to high salinity require few adjustments to intestinal transport associated with osmoregulation.

The common killifish, Fundulus heteroclitus, has historically been a favorite organism for the study of euryhalinity in teleost fish. Despite the species' large range of salinity tolerance, studies of osmoregulation in high salinity are rare, with most previous studies focused on fish transferred between freshwater and seawater. Similarly, while branchial transport properties have been studied extensively, there are relatively few studies investigating the role of the intestine in osmoregulation in killifish. This study sought to characterize the fluid and ion transport occurring in the intestinal tract of killifish adapted to seawater, and furthermore to investigate the adjustments that occur to these mechanisms following acute transfer to high salinity (70ppt). In vivo samples of blood plasma and intestinal fluids of seawater-acclimated killifish indicated absorption of Na(+), Cl(-), and water, the relative impermeability of the intestine to Mg(2+) and SO(4)(2-), and active secretion of HCO(3)(-) into the intestinal lumen. The details of these processes were investigated further using in vitro techniques of isolated intestinal sac preparations and an Ussing chamber pH-stat titration system. However, these methods were discovered to be of limited utility under physiologically relevant conditions due to tissue deterioration. Results that could be validly interpreted suggested that there are few changes to intestinal transport following transfer to high salinity, and that adjustments to epithelial permeability occur in the first 24h post-transfer.

Intestinal transport following transfer to increased salinity in an anadromous fish (Oncorhynchus mykiss).

The ability to transition from freshwater to seawater environments is an intrinsic requirement of the life history of some fish species, including the anadromous rainbow trout (Oncorhynchus mykiss). The differences between hyper- and hypoosmoregulation are developed quickly (in hours to days), and at all scales, from gene expression to organ function. In this study, intestinal ion and water transport was examined in O. mykiss following acute transfer from freshwater (FW) to 70% seawater (SW). Plasma [Mg²+] increased at 24h post-transfer but recovered by 72 h. In the intestinal fluids, total CO2 was found to increase with SW exposure/acclimation, while [Na+] decreased after 24h of SW exposure. Overall, in vitro experiments demonstrated the importance of base secretion to epithelial water uptake, and suggested that the primary physiological adjustments occurred 24-72 h after acute SW transfer. The mRNA expression of ion transporters important for intestinal osmoregulation and maintenance of acid-base balance was also investigated. A Na+/H+ exchanger (NHE2) and anion exchanger (SLC26a6) were hypothesized to be involved in the transport of acid-base equivalents, Na+, and Cl⁻, but were not uniformly expressed across tissue samples, and expression, where present, did not change following salinity transfer. NHE1, however, was expressed in all examined tissues (gill, kidney, anterior intestine, and pyloric cecae), but exhibited no changes in expression following acute salinity transfer.

Concentration of MgSO4 in the intestinal lumen of Opsanus beta limits osmoregulation in response to acute hypersalinity stress.

Marine teleosts constantly lose water to their surrounding environment, a problem exacerbated in fish exposed to salinity higher than normal seawater. Some fish undergo hypersaline exposures in their natural environments, such as short- and long-term increases in salinity occurring in small tidal pools and other isolated basins, lakes, or entire estuaries. Regardless of the degree of hypersalinity in the ambient water, intestinal absorption of monovalent ions drives water uptake to compensate for water loss, concentrating impermeable MgSO(4) in the lumen. This study considers the potential of luminal [MgSO(4)] to limit intestinal water absorption, and therefore osmoregulation, in hypersalinity. The overall tolerance and physiological response of toadfish (Opsanus beta) to hypersalinity exposure were examined. In vivo, fish in hypersaline waters containing artificially low [MgSO(4)] displayed significantly lower osmolality in both plasma and intestinal fluids, and increased survival at 85 parts per thousand, indicating improved osmoregulatory ability than in fish exposed to hypersalinity with ionic ratios similar to naturally occurring ratios. Intestinal sac preparations revealed that in addition to the osmotic pressure difference across the epithelium, the luminal ionic composition influenced the absorption of Na(+), Cl(-), and water. Hypersalinity exposure increased urine flow rates in fish fitted with ureteral catheters regardless of ionic composition of the ambient seawater, but it had no effect on urine osmolality or pH. Overall, concentrated MgSO(4) within the intestinal lumen, rather than renal or branchial factors, is the primary limitation for osmoregulation by toadfish in hypersaline environments.

Acid-base regulation in the plainfin midshipman (Porichthys notatus): an aglomerular marine teleost.

The plainfin midshipman (Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid-base regulation during respiratory acidosis either by facilitating or constraining HCO(3)(-) accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid-base status during exposure to increasing levels of hypercapnia (nominally 1-5% CO(2)). Fish exhibited a well-developed ability to increase plasma HCO(3)(-) levels with values of 39.8 ± 2.8 mmol l(-1) being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO(2) = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO(3)(-) during hypercapnia was associated with marked increases in branchial net acid excretion (J (NET)H(+)) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in J (NET)H(+) was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na(+)/K(+)-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO(3)(-) concentrations of 92.2 ± 4.8 mmol l(-1). At the highest level of hypercapnia, rectal fluid HCO(3)(-) levels were increased significantly to 141.8 ± 7.4 mmol l(-1) but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l(-1)). Thus, the impressive accumulation of plasma HCO(3)(-) to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO(3)(-) excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO(3)(-) concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO(3)(-). The apparent inability of P. notatus to effectively excrete HCO(3)(-) following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts.

Effects of salinity on intestinal bicarbonate secretion and compensatory regulation of acid-base balance in Opsanus beta.

Marine teleosts have extracellular fluids less concentrated than their environment, resulting in continual water loss, which is compensated for by drinking, with intestinal water absorption driven by NaCl uptake. Absorption of Cl(-) occurs in part by apical Cl(-)/HCO(3)(-) exchange, with HCO(3)(-) provided by transepithelial transport and/or by carbonic anhydrase-mediated hydration of endogenous epithelial CO(2). Hydration of CO(2) also liberates H(+), which is transported across the basolateral membrane. In this study, gulf toadfish (Opsanus beta) were acclimated to 9, 35 and 50 ppt. Intestinal HCO(3)(-) secretion, water and salt absorption, and the ensuing effects on acid-base balance were examined. Rectal fluid excretion greatly increased with increasing salinity from 0.17+/-0.05 ml kg(-1) h(-1) in 9 ppt to 0.70+/-0.19 ml kg(-1) h(-1) in 35 ppt and 1.46+/-0.22 ml kg(-1) h(-1) in 50 ppt. Rectal fluid composition and excretion rates allowed for estimation of drinking rates, which increased with salinity from 1.38+/-0.30 to 2.60+/-0.92 and 3.82+/-0.58 ml kg(-1) h(-1) in 9, 35 and 50 ppt, respectively. By contrast, the fraction of imbibed water absorbed decreased from 85.9+/-3.8% in 9 ppt to 68.8+/-3.2% in 35 ppt and 61.4+/-1.0% in 50 ppt. Despite large changes in rectal base excretion from 9.3+/-2.7 to 68.2+/-20.4 and 193.2+/-64.9 mumol kg(-1) h(-1) in 9, 35 and 50 ppt, respectively, acute or prolonged exposure to altered salinities was associated with only modest acid-base balance disturbances. Extra-intestinal, presumably branchial, net acid excretion increased with salinity (62.0+/-21.0, 229.7+/-38.5 and 403.1+/-32.9 mumol kg(-1) h(-1) at 9, 35 and 50 ppt, respectively), demonstrating a compensatory response to altered intestinal base secretion associated with osmoregulatory demand.