Warmed Winter Water Temperatures Alter Reproduction in Two Fish Species.

We examined the spawning success of Fathead Minnows (Pimephales promelas) and Johnny Darters (Etheostoma nigrum) exposed to elevated winter water temperatures typical of streams characterized by anthropogenic thermal inputs. When Fathead Minnows were exposed to temperature treatments of 12, 16, or 20 °C during the winter, spawning occurred at 16 and 20 °C but not 12 °C. Eggs were deposited over 9 weeks before winter spawning ceased. Fathead Minnows from the three winter temperature treatments were then exposed to a simulated spring transition. Spawning occurred at all three temperature treatments during the spring, but fish from the 16° and 20 °C treatment had delayed egg production indicating a latent effect of warm winter temperatures on spring spawning. mRNA analysis of the egg yolk protein vitellogenin showed elevated expression in female Fathead Minnows at 16 and 20 °C during winter spawning that decreased after winter spawning ceased, whereas Fathead Minnows at 12 °C maintained comparatively low expression during winter. Johnny Darters were exposed to 4 °C to represent winter temperatures in the absence of thermal inputs, and 12, 16, and 20 °C to represent varying degrees of winter thermal pollution. Johnny Darters spawned during winter at 12, 16, and 20 °C but not at 4 °C. Johnny Darters at 4 °C subsequently spawned following a simulated spring period while those at 12, 16, and 20 °C did not. Our results indicate elevated winter water temperatures common in effluent-dominated streams can promote out-of-season spawning and that vitellogenin expression is a useful indicator of spawning readiness for fish exposed to elevated winter temperatures.

Metabolism and antioxidant defense in the larval chironomid Tanytarsus minutipalpus: adjustments to diel variations in the extreme conditions of Lake Magadi.

Insect larvae are reported to be a major component of the simple but highly productive trophic web found in Lake Magadi (Kenya, Africa), which is considered to be one of the most extreme aquatic environments on Earth. Previous studies show that fish must display biochemical and physiological adjustments to thrive under the extreme conditions of the lake. However, information for invertebrates is lacking. In the present study, the occurrence of the larval chironomid Tanytarsus minutipalpus is reported in Lake Magadi for the first time. Additionally, changes in larval metabolism and antioxidant defense correlated with diel variations in the extremely hostile environmental conditions of the lake are described. Wide variations in water temperature (20.2-29.3°C) and dissolved oxygen content (3.2-18.6 mg O2 l-1) were observed at different times of day, without significant change in water pH (10.0±0.03). Temperature and dissolved oxygen were higher at 13:00 h (29.3±0.4°C and 18.6±1.0 mg O2 l-1) and 19:00 h (29.3±0.8°C and 16.2±1.6 mg O2 l-1) and lower at 01:00 h (21.1±0.1°C and 10.7±0.03 mg O2 l-1) and 07:00 h (20.2±0.4°C and 3.2±0.7 mg O2 l-1). Significant and parallel increases in parameters related to metabolism (cholinesterase, glucose, cholesterol, urea, creatinine and hemoglobin) and the antioxidant system (SOD, GPx, GR, GSH and GSSG) were observed in larvae collected at 13:00 h. In contrast, no significant changes were observed in pro-oxidants (ROS and NO), TOSC and oxidative damage parameters (LPO and DNA damage). Therefore, the observed increases in temperature and dissolved O2 content in Lake Magadi were associated with changes in the antioxidant system of T. minutipalpus larvae. Adjustments performed by the chironomid larvae were efficient in maintaining body homeostasis, as well as protecting biomolecules against oxidative damage, so that oxidative stress did not occur. GSH-GSSG and GPx-GR systems appeared to play an essential role in the adjustments displayed by the chironomid larvae during the diel changes in the extreme conditions of Lake Magadi.

Mammalian metabolic rates in the hottest fish on earth.

The Magadi tilapia, Alcolapia grahami, a small cichlid fish of Lake Magadi, Kenya lives in one of the most challenging aquatic environments on earth, characterized by very high alkalinity, unusual water chemistry, and extreme O2, ROS, and temperature regimes. In contrast to most fishes which live at temperatures substantially lower than the 36-40 °C of mammals and birds, an isolated population (South West Hot Springs, SWHS) of Magadi tilapia thrives in fast-flowing hotsprings with daytime highs of 43 °C and night-time lows of 32 °C. Another population (Fish Springs Lagoon, FSL) lives in a lagoon with fairly stable daily temperatures (33-36 °C). The upper critical temperatures (Ctmax) of both populations are very high; moreover the SWHS tilapia exhibit the highest Ctmax (45.6 °C) ever recorded for a fish. Routine rates of O2 consumption (MO2) measured on site, together with MO2 and swimming performance at 25, 32, and 39 °C in the laboratory, showed that the SWHS tilapia exhibited the greatest metabolic performance ever recorded in a fish. These rates were in the basal range of a small mammal of comparable size, and were all far higher than in the FSL fish. The SWHS tilapia represents a bellwether organism for global warming.

Predictors for dioxin accumulation in residents living in Da Nang and Bien Hoa, Vietnam, many years after Agent Orange use.

Agent Orange (AO) was the main defoliant used by the US in Vietnam from 1961 to 1971; AO was contaminated with dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, or TCDD). Three major dioxin "hot spots" remain from previous AO storage and use at former US bases at Bien Hoa, Da Nang, and Phu Cat, posing potential health risks for Vietnamese living on or near these hot spots. We evaluated potential risk factors contributing to serum TCDD levels in Vietnamese residents at and near contaminated sites in Da Nang and Bien Hoa, Vietnam. We used multiple linear regression to analyze possible associations of blood dioxin concentrations with demographic, socioeconomic, lifestyle, and dietary risk factors for residents living on or near these hot spots. For the Da Nang study, fish farming on the site, living on property flooded from monsoon rains, and age were among the factors showing significant positive associations with serum TCDD concentrations. For the Bien Hoa study, fish farmers working at this site and their immediate family members had significantly higher serum TCDD concentrations. Our results suggest that water-related activities, especially fish-farming, at the hot spots increased the risk of exposure to dioxin.

Rh proteins and NH4(+)-activated Na+-ATPase in the Magadi tilapia (Alcolapia grahami), a 100% ureotelic teleost fish.

The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l(-1)). The Magadi tilapia is the only 100% ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins (Rhag, Rhbg and Rhcg2) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg, Rhcg2 and Na(+),K(+)-ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH4(+) (versus K(+)), suggesting it can function as an NH4(+)-transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH4(+)-activated Na(+)-ATPase function.

Transepithelial potential in the Magadi tilapia, a fish living in extreme alkalinity.

We investigated the transepithelial potential (TEP) and its responses to changes in the external medium in Alcolapia grahami, a small cichlid fish living in Lake Magadi, Kenya. Magadi water is extremely alkaline (pH = 9.92) and otherwise unusual: titratable alkalinity (290 mequiv L(-1), i.e. HCO(3) (-) and CO(3) (2-)) rather than Cl(-) (112 mmol L(-1)) represents the major anion matching Na(+) = 356 mmol L(-1), with very low concentrations of Ca(2+) and Mg(2+) (<1 mmol L(-1)). Immediately after fish capture, TEP was +4 mV (inside positive), but stabilized at +7 mV at 10-30 h post-capture when experiments were performed in Magadi water. Transfer to 250% Magadi water increased the TEP to +9.5 mV, and transfer to fresh water and deionized water decreased the TEP to -13 and -28 mV, respectively, effects which were not due to changes in pH or osmolality. The very negative TEP in deionized water was attenuated in a linear fashion by log elevations in [Ca(2+)]. Extreme cold (1 vs. 28°C) reduced the positive TEP in Magadi water by 60%, suggesting blockade of an electrogenic component, but did not alter the negative TEP in dilute solution. When fish were transferred to 350 mmol L(-1) solutions of NaHCO(3), NaCl, NaNO(3), or choline Cl, only the 350 mmol L(-1) NaHCO(3) solution sustained the TEP unchanged at +7 mV; in all others, the TEP fell. Furthermore, after transfer to 50, 10, and 2% dilutions of 350 mmol L(-1) NaHCO(3), the TEPs remained identical to those in comparable dilutions of Magadi water, whereas this did not occur with comparable dilutions of 350 mmol L(-1) NaCl-i.e. the fish behaves electrically as if living in an NaHCO(3) solution equimolar to Magadi water. We conclude that the TEP is largely a Na(+) diffusion potential attenuated by some permeability to anions. In Magadi water, the net electrochemical forces driving Na(+) inwards (+9.9 mV) and Cl(-) outwards (+3.4 mV) are small relative to the strong gradient driving HCO(3) (-) inwards (-82.7 mV). Estimated permeability ratios are P (Cl)/P (Na) = 0.51-0.68 and [Formula: see text] = 0.10-0.33. The low permeability to HCO(3) (-) is unusual, and reflects a unique adaptation to life in extreme alkalinity. Cl(-) is distributed close to Nernst equilibrium in Magadi water, so there is no need for lower P (Cl). The higher P (Na) likely facilitates Na(+) efflux through the paracellular pathway. The positive electrogenic component is probably due to active HCO(3) (-) excretion.

Discordance between genetic structure and morphological, ecological, and physiological adaptation in Lake Magadi tilapia.

The Magadi tilapia (Alcolapia grahami, formerly Oreochromis alcalicus grahami) is a remarkable example of teleost life in an extreme environment. Typical conditions include water pH=10, titration alkalinity>300 mM, osmolality=525 mOsm, temperatures ranging from 23 degrees to 42 degrees C, and O(2) levels fluctuating diurnally between extreme hyperoxia and anoxia. A number of relatively small tilapia populations are present in various thermal spring lagoons around the margin of the lake separated by kilometers of solid trona crust (floating Na(2)CO(3)) underlain by anoxic water. Despite the apparent isolation of different populations, annual floods may provide opportunities for exchange of fish across the surface of the trona and subsequent gene flow. To assess the question of isolation among Lake Magadi populations, we analyzed the variable control region of the mitochondrial DNA (mtDNA) from six lagoons. A total of seven mtDNA haplotypes, including three common haplotypes, were observed in all six populations. Several of the Lake Magadi populations showed haplotype frequencies indicative of differentiation, while others showed very little. However, differentiation among lagoon populations was discordant with their geographical distribution along the shoreline. All populations exhibited the unusual trait of 100% ureotelism but specialized morphological and physiological characteristics were observed among several of the lagoon systems. In addition, distinct differences were observed in the osmolality among the lagoons with levels as high as 1,400-1,700 mOsm kg(-1), with corresponding differences in the natural levels of whole-body urea. These levels of osmotic pressure proved fatal to fish from less alkaline systems but remarkably were also fatal to the fish that inhabited lagoons with this water chemistry. Upon more detailed inspection, specific adaptations to differential conditions in the lagoon habitat were identified that allowed survival of these cichlids. Additional evidence against potential for gene flow among lagoons despite the sharing of common mtDNA haplotypes was that the osmolality of floodwaters following a heavy rain showed lethal levels exceeding 1,700 mOsm kg(-1). In isolation, different mtDNA haplotypes would be predicted to go to fixation in different populations due to rapid generation times and the small effective population sizes in a number of lagoons. We propose a model of balancing selection to maintain common mtDNA sequences through a common selection pressure among lagoons that is based on microhabitats utilized by the tilapia.

Physiological adaptations of the gut in the Lake Magadi tilapia, Alcolapia grahami, an alkaline- and saline-adapted teleost fish.

We describe the gut physiology of the Lake Magadi tilapia (Alcolapia grahami), specifically those aspects associated with feeding and drinking while living in water of unusually high carbonate alkalinity (titratable base=245 mequiv l(-1)) and pH (9.85). Drinking of this highly alkaline lake water occurs at rates comparable to or higher than those seen in marine teleosts. Eating and drinking take place throughout the day, although drinking predominates during hours of darkness. The intestine directly intersects the esophagus at the anterior end of the stomach forming a 'T', and the pyloric sphincter, which comprises both smooth and striated muscle, is open when the stomach is empty and closed when the stomach is full. This unique configuration (a functional trifurcation) allows imbibed alkaline water to bypass the empty stomach, thereby avoiding a reactive mixing with acidic gastric fluids, and minimizes interference with a full stomach. No titratable base was present in the stomach, where the mean pH was 3.55, but the intestine was progressively more alkaline (foregut 6.96, midgut 7.74, hindgut 8.12, rectum 8.42); base levels in the intestinal fluid were comparable to those in lake water. The gut was highly efficient at absorbing water (76.6%), which accompanied the absorption of Na(+) (78.5%), titratable base (80.8%), and Cl(-) (71.8%). The majority of Na(+), base and water absorption occurred in the foregut by an apparent Na(+) plus base co-transport system. Overall, more than 70% of the intestinal flux occurred via Na(+) plus base co-transport, and less than 30% by Na(+) plus Cl(-) co-transport, a very different situation from the processes in the intestine of a typical marine teleost.

Structural changes in gills of Lost River suckers exposed to elevated pH and ammonia concentrations.

The Lost River sucker (Deltistes luxatus) is a federally listed, endangered fish that occurs primarily in Upper Klamath Lake-a hypereutrophic lake in southern Oregon, USA. A decline of the sucker population in the lake over the past few decades has been partly attributed to adverse water quality conditions, including elevated pH and ammonia concentrations that occur during summer cyanobacterial blooms. We quantitatively analyzed structural changes in gills of larval Lost River suckers after they were exposed to elevated pH and ammonia concentrations for 30 d. Exposure to pH as high as 10 caused no observed structural changes. However, lamellar thickness and O(2) diffusion distance increased significantly (P<0.05) at ammonia concentrations that did not significantly decrease survival, growth, whole-body ion concentrations, or swimming performance. Additionally, we qualitatively observed increases in the frequency of hyperplasic and hypertrophic mucous cells, tissue damage, epithelial lifting, and infiltration of white blood cells into paracellular lymphatic spaces at the highest sublethal ammonia concentration. These observed gill changes typically indicate compromised respiratory and ionoregulatory capacity, although such effects were not manifested in the assays we performed. Regardless, these structural gill changes appear to be a more sensitive indicator of exposure to elevated ammonia concentrations than are more traditional sublethal indices. Therefore, gill histopathology might be a relevant early-warning monitoring tool of the health of Lost River suckers in Upper Klamath Lake, and other species in similar eutrophic systems.

Obligatory urea production and the cost of living in the Magadi tilapia revealed by acclimation to reduced salinity and alkalinity.

Alcolapia grahami is a unique ureotelic tilapia that lives in the highly alkaline, saline Lake Magadi, Kenya (pH, approximately 10.0; alkalinity, approximately 380 mmol L(-1); Na(+), approximately 350 mmol L(-1); Cl(-), approximately 110 mmol L(-1); osmolality, approximately 580 mosm kg(-1)). The fish survived well upon gradual exposure to dilute lake water (down to 1%, essentially freshwater). Urea excretion continued, and there was no ammonia excretion despite favorable conditions, indicating that ureotelism is obligatory. Levels of most ornithine-urea cycle enzymes in the liver were unchanged relative to controls kept for the same period in 100% lake water. The fish exhibited good abilities for hypo- and hyperregulation, maintaining plasma Na(+), Cl(-), and osmolality at levels typical of marine and freshwater teleosts in 100% and 1% lake water, respectively. Plasma total CO(2) did not change with environmental dilution. Routine oxygen consumption (Mo(2)) was extremely high in 100% lake water but decreased by 40%-68% after acclimation to dilute lake water. At every fixed swimming speed, Mo(2) was significantly reduced (by 50% at high speeds), and critical swimming speed was elevated in fish in 10% lake water relative to 100% lake water. Osmotic and Cl(-) concentration gradients from water to plasma were actually increased, and osmotic and Na(+) gradients were reversed, in 10% and 1% dilutions relative to 100% lake water, whereas acid-base gradients were greatly reduced. We suggest that approximately 50% of the animal's high metabolic demand originates from the cost of acid-base regulation in the highly alkaline Lake Magadi. When this load is reduced by environmental dilution, the energy saved can be diverted to enhanced swimming performance.