EFFECTS OF ACIDIC SOLUTIONS ON SEXUAL REPRODUCTION OF PTERIDIUM AQUILINUM

Experiments were performed to determine the effects of acidic solutions on spermatozoid motility and fertilization of gametophytes of Pteridium aquilinum. Buffered solutions (0.0025 m ) were used to simulate exposures to acidic precipitation for up to a 3.5 hr exposure. Experimental results suggest that the spermatozoid population can be subdivided into several groups with respect to pH sensitivity; about 25% spermatozoids are immobile one min after exposure to pH 6.1 buffer while about an equal percentage remain motile after 30 min exposure to buffer of pH 5.1. Between these two response extremes are two other subpopulations. One is quite sensitive to pH but shows some recovery if pH is between 5.6 and 6.1, while the second subpopulation does not seem to exhibit any motility recovery at all but is more resistant to acidity than the first subpopulation. To complement experiments that evaluate spermatozoid responses, experiments were performed to view the process of fertilization under controlled environmental conditions as well as under the canopy of a forest. Fertilization of gametophytes in uncovered petri dishes under a forest canopy was similar to results in aseptic culture after gametophytes were exposed to various pH levels and 86.6 μM sulfate. Although there were some differences between results obtained under aseptic culture conditions and cultures maintained under a forest canopy, it is evident that a lower buffer pH decreased fertilization. Fertilization at pH 4.5 and 3.6 was about one-half that occurring at pH 6.1. Fertilization in gametophytes exposed to pH 3.0 was about 10-20% of that occurring at pH 6.1. Addition of 86.6 μM sulfate decreased fertilization under all culture conditions. These experimental results suggest that fertilization in P. aquilinum may be used as a bioindicator of contaminants in rainwater. The results demonstrate that spermatozoid motility (and the process of fertilization) is more acid sensitive than gametophytic and sporophytic tissues.     

Sensitivity of sea urchin fertilization to pH varies across a natural pH mosaic

In the coastal ocean, temporal fluctuations in pH vary dramatically across biogeographic ranges. How such spatial differences in pH variability regimes might shape ocean acidification resistance in marine species remains unknown. We assessed the pH sensitivity of the sea urchin Strongylocentrotus purpuratus in the context of ocean pH variability. Using unique male–female pairs, originating from three sites with similar mean pH but different variability and frequency of low pH (pH_T ≤ 7.8) exposures, fertilization was tested across a range of pH (pH_T 7.61–8.03) and sperm concentrations. High fertilization success was maintained at low pH via a slight right shift in the fertilization function across sperm concentration. This pH effect differed by site. Urchins from the site with the narrowest pH variability regime exhibited the greatest pH sensitivity. At this site, mechanistic fertilization dynamics models support a decrease in sperm–egg interaction rate with decreasing pH. The site differences in pH sensitivity build upon recent evidence of local pH adaptation in S. purpuratus and highlight the need to incorporate environmental variability in the study of global change biology.     

Sperm motility and seminal fluid composition in the burbot, Lota lota

Sperm motility and composition of the seminal fluid in Lota lota were investigated. Fives after motility initiation, 88.2 ± 12.4% of the spermatozoa were motile, their mean average path swimming velocity was 61.6 ± 16.3 μm s^?1 and their principal swimming type the linear motion (77.4 ± 20.9%). In distilled water the rate of motile spermatozoa decreased to 0% in 40s. In 25–50 mosmol kg^?1 electrolyte (NaCl) or non-electrolyte (glucose, sucrose) solutions, motility was prolonged for 10s and these solutions can therefore increase the efficiency of artificial fertilization when used for sperm motility activation. When semen was diluted in electrolyte or non-electrolyte solutions with osmolalities higher than 50 mosmol kg^?1, sperm motility rates and swimming velocities decreased, and at osmolalities of 400 mosmol kg^?1 motility was completely suppressed. In the seminal fluid with an osmolality of 290.08 ± 45.22 mosmol kg^?1, sodium levels of 139.86 ± 23.79 mmol × 1^?1, potassium levels of 11.59 ± 2.45 mmol × 1^?1 and calcium levels of 0.20 ± 0.08 mmol × 1^?1, sperm motility was inhibited. Under in vitro conditions, artificial saline solutions resembling the seminal plasma composition and 400 mosmol kg^?1 NaCl or glucose solutions were useful as motility inhibiting solutions for predilution of semen. Sperm motility was not affected by pH 7.5–9.0, but at pH 6 the motility rate and the swimming velocity were reduced; seminal fluid pH was 8.47 ± 0.02. Therefore buffering of the artificial saline solutions can provide more stabile conditions for semen during storage and activation. Temperature optimum of semen was between 2 and 5°C. At higher temperatures semen became spontaneously motile. Therefore, controlled temperature conditions are an important factor for handling of semen. The qualitative, organical composition of seminal fluid was similar as in other fresh water teleosts.     

Metal Reduction at Low pH by a Desulfosporosinus species: Implications for the Biological Treatment of Acidic Mine Drainage

We isolated an acid-tolerant sulfate-reducing bacterium, GBSRB4.2, from coal mine-derived acidic mine drainage (AMD)-derived sediments. Sequence analysis of partial 16S rRNA gene of GBSRB4.2 revealed that it was affiliated with the genus Desulfosporosinus. GBSRB4.2 reduced sulfate, Fe(III) (hydr)oxide, Mn(IV) oxide, and U(VI) in acidic solutions (pH 4.2). Sulfate, Fe(III), and Mn(IV) but not U(VI) bioreduction led to an increase in the pH of acidic solutions and concurrent hydrolysis and precipitation of dissolved Al³⁺. Reduction of Fe(III), Mn(IV), and U(VI) in sulfate-free solutions revealed that these metals are enzymatically reduced by GBSRB4.2. GBSRB4.2 reduced U(VI) in groundwater from a radionuclide-contaminated aquifer more rapidly at pH 4.4 than at pH 7.1, possibly due to the formation of poorly bioreducible Ca-U(VI)-CO₃ complexes in the pH 7.1 groundwater.     

LEAF SURFACE AND HISTOLOGICAL PERTURBATIONS OF LEAVES OF PHASEOLUS VULGARIS AND HELIANTHUS ANNUUS AFTER EXPOSURE TO SIMULATED ACID RAIN

Initial injury to adaxial leaf surfaces of Phaseolus vulgaris and Helianthus annuus occurred near trichomes and stomata after exposure to simulated sulfate acid rain. Lesion frequency was not correlated with density of either stomata or trichomes but was correlated with degree of leaf expansion. The number of lesions per unit area increased with total leaf area. Results suggest that characteristics of the leaf indumentum such as development of trichomes and guard cells and/or cuticle thickness near these structures may be involved in lesion development. Adaxial epidermal cell collapse was the first event in lesion development. Palisade cells and eventually spongy mesophyll cells collapsed after continued, daily exposure to simulated rain of low pH. Lesion development on Phaseolus vulgaris followed a specific course of events after exposure to simulated rain of known composition, application rate, drop size frequency, drop velocities, and frequency of exposures. These results allow development of further experiments to observe accurately other parameters, such as nutrient inputs and nutrient leaching from foliage, after exposure to simulated sulfate acid rain.     

Elevated pCO2 increases sperm limitation and risk of polyspermy in the red sea urchin Strongylocentrotus franciscanus

Anthropogenic carbon dioxide (CO₂) emissions and the resultant acidification of surface ocean waters are predicted to have far‐reaching consequences for biological processes in the marine environment. For example, because changes in pH and pCO₂ can alter sperm performance, ocean acidification may be accompanied by reductions in the success of fertilization in marine broadcast spawners. Several studies have attempted to determine the effects of elevated pCO₂ on marine invertebrate fertilization success, albeit with differing results. These conflicts may stem from the use of inappropriate sperm–egg contact times and, in several cases, the lack of measurements over a range of sperm concentrations extending from sperm‐limited conditions to polyspermy scenarios. In our study, we used biologically realistic sperm–egg contact times and a full range of sperm concentrations to assess the effect of elevated pCO₂ on fertilization in the broadcast spawning sea urchin, Strongylocentrotus franciscanus. Fertilization experiments were carried out in seawater bubbled with CO₂ to 400 (control), 800, and 1800 ppm. Using a fertilization kinetics model, we estimate that elevated pCO₂ levels both increased sperm limitation and reduced the efficiency of fast blocks to polyspermy. Thus, elevated pCO₂ decreased the range of sperm concentrations over which high fertilization success was likely. Given the inherent difficulties in achieving high fertilization success in broadcast spawners, raised pCO₂ levels are likely to exacerbate low fertilization success in low‐density populations or in areas with high water turbulence.     

In vitro capacitation and fertilizing ability of ejaculated rabbit sperm treated with lysophosphatidylcholine

Four experiments were replicated 1) to establish dose-response relationships between lysophosphatidylcholine (LPC), sperm motility, and the acrosome reaction (AR), 2) to evaluate the influence of rabbit serum (RS) on these endpoints, 3) to compare buck differences in induction of the AR, and 4) to examine fertilizing ability in vitro of sperm tested under the first three objectives. Semen was collected from Dutch-belted rabbits, washed once by centrifugation, resuspended, and preincubated for 2 or 4 hr in a chemically defined medium (DM), DM plus 20% RS, or BSA-free DM plus 20% RS at 37°C. At the end of preincubation LPC was added to the preincubated sperm at concentrations of from 0 to 100 μg/ml. Sperm were examined .5–4 hr later for AR and sperm motility. For in vitro fertilization, sperm and ova were coincubated in DM up to 24 hr after insemination and in a more complex medium for another 24 hr. Addition of LPC to 4-hr-preincubated sperm was more effective for inducing the AR than addition to 2-hr-preincubated sperm. A significant increase (P < .05) in the AR occurred in 15 and 30 min following exposure to 100 and 80 μg of LPC per ml, respectively, but the higher concentration of LPC decreased sperm motility. Addition of 20% RS to DM with or without BSA surprisingly inhibited the AR but maintained sperm motility, as expected. Bucks differed (P < .05) in the initial percentage and the induced percentage of AR sperm. For the AR the optimal concentration of LPC per ml was 80 μg, but for in vitro fertilization 60 μg tended to be superior.     

THE FERTILIZING CAPACITY OF SEA URCHIN SPERM RAPIDLY DECREASES AFTER INDUCTION OF THE ACROSOME REACTION*

When immotile, flagella-less sperm were added to acid-dejellied eggs of Strongylocentrotus purpuratus 11% of the eggs fertilized. Addition of soluble egg jelly increased the percentage fertilization to 90.5. Over 50% of the sperm exposed to egg jelly had undergone the acrosome reaction compared to only 3–5% in the absence of jelly. Egg jelly was added to flagella-less sperm to induce the acrosome reaction and dejellied eggs added at various times thereafter. The fertilizing capacity of the sperm decreased with first order kinetics with 50% loss by 23 sec after induction of the acrosome reaction. Intact, motile sperm bind to formaldehyde-fixed eggs with maximum binding occurring 40 sec after sperm addition. After 40 sec the sperm begin to detach from the fixed eggs and by 240 sec none remain attached. Sperm detachment from fixed eggs and loss of fertilizing capacity after the acrosome reaction show a close temporal correlation.     

Mouse Uterine 24p3 Protein as a Suppressor of Sperm Acrosome Reaction

Under in vitro conditions, incubation with 0.3% bovine serum albumin (BSA) and 1.8 mM CaCl₂ induces mouse sperm capacitation and increases the consequential acrosome-reaction. The effect of mouse uterine 24p3 protein on such stimulated sperm has been investigated to understand the biological function of the 24p3 protein. Variations in the intracellular pH (pHi), calcium concentration, cAMP levels and tyrosine phosphorylation in cytosol were determined and on in vitro mouse fertilization was evaluated. The presence of 24p3 protein reduced the response of sperm to BSA and calcium by suppressing the elevation of intracellular pH, calcium uptake, cAMP accumulation and protein tyrosine phosphorylation of BSA/calcium-stimulated sperm and showed inhibitory effect on mouse in vitro fertilization. The results indicated the inhibition of the BSA-stimulated sperm acrosome reaction by 24p3 protein then suppressed sperm fertilization. We suggested that the 24p3 protein acts as an in vitro inhibitor of the acrosome reaction in BSA stimulated sperm and this might be an anti-fertilization factor in vitro.     

FLAGELLAR MOTILITY IS NOT INVOLVED IN THE INCORPORATION OF THE SPERM INTO THE EGG AT FERTILIZATION*

Cinemicrography of sea urchin fertilization reveals that the fertilizing sperm is one of the first sperm to attach to the egg. Just before the cortical reaction the fertilizing sperm ceases motility and then is incorporated into the egg without flagellar beating. The rate of incorporation is 5–11 μm/sec and is constant. Lytechinus pictus sperm rendered immotile by azide treatment can bind to and fertilize eggs but binding, and therefore fertilization, is blocked by azide treatment of Strongylocentrotus purpuratus gametes.     

Effects of acidic fog on productivity of celery and lettuce and impact on incidence and severity of diseases

The effects of acidic fog on productivity of celery and lettuce were examined using a portable fogging apparatus to expose field plots to simulated fog episodes. Acidic solutions were formulated to simulate fog reported for southern California. Celery (Apium graveolens var. dulec cv. Bishop) and lettuce (Lactuca sativa cv. Mesa 659) were exposed in separate experiments once or twice each week during crop growth and maturation to 1 - to 2-h of fog at specific acidity levels between pH 1.6 and 3.8. Necrosis occurred as small, irregularly shaped white (celery) or brown (lettuce) lesions on the most exposed portions of the plants. Fog necrosis sufficient to reduce marketability of celery was evident after single exposures at pH 2.4. Although injury occurred on wrapper leaves of lettuce at acidity levels as high as pH 3.4, marketability was reduced from foliar necrosis only after exposure to acidity levels of pH 2.6 or lower. Repeated exposure of lettuce to acidity below pH 2.0 was necessary to reduce yield. The field experiments demonstrated that acidic fog influenced natural incidence of disease. At low pH fog exposure, celery had increased occurrence of basal stalk rot and lettuce exhibited increased bacterial soft rot. Indirect effects of acidic fog on host-pathogen relationships and subsequent crop marketability may be more important than the direct effects of acidic fog exposure on plant tissue.     

Does seasonal reproduction occur at the optimal time for fertilization in the polychaetes Arenicola marina L. and Nereis virens Sars?

Summary

Many marine invertebrates exhibit highly seasonal and synchronised reproduction, with offspring production often being confined to just two or three days each year. Several models have been proposed to explain the fitness benefits of this reproductive pattern, many of which assume enhanced offspring survival due to temperature constraints placed on fertilization and development at other times of the year. In this investigation the temperature limits and optimum for fertilization were determined for two polychaete species, Arenicola marina and Nereis virens. These two polychaete species are exposed to the same environmental conditions throughout the year, yet breed at very different times. Other seasonal impacts on fertilization, i.e., reduced salinity due to rainfall and the effect of sub-zero temperatures on sperm of A. marina, were also investigated. In both A. marina and N. virens fertilization success was significantly influenced by temperature, with the maximum success recorded at 15–18°C. The ambient seawater temperature at the time of natural spawning for both worms is around 10°C, which means that both species are spawning right at the lower limit for maximum fertilization. Salinity and exposure of A. marina sperm to sub-zero temperatures were also found to influence fertilization success, but only at levels that would not be experienced by these polychaetes under natural conditions at the time of spawning. These results suggest there must be additional selective pressures acting on the fitness of these two polychaetes causing A. marina to breed later than, and N. virens to breed earlier than, the optimum time for fertilization. A. marina apparently waits as late as possible to maximise adult fecundity and survival. N. virens breeds as soon as it can achieve high fertilization to maximise larval and juvenile competitiveness.     

Induction of Tetraploid Gynogenesis in the European Sea Bass (Dicentrarchus labrax L.)

A preliminary study on tetraploid gynogenetic induction in the European sea bass was performed by pressure-blocking the second polar body release and the first cleavage in eggs fertilized with ultraviolet-irradiated sperm. Fertilization of eggs with genetically inactivated sperm produced only haploid development that terminated around hatching. Pressure treatments (8.500 psi for 2 min) applied at 6 and 65 min after fertilization (a.f.) produced variable levels (7–95%) of tetraploid larvae at hatching. A small proportion of mosaics (3.8n/4.2n) was also recorded.     

Cryopreservation of sperm in farmed Australian greenlip abalone Haliotis laevigata

This study investigated factors important to the development of the liquid nitrogen (LN) vapor sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata, including (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature (height above LN surface); (3) thawing temperature; (4) sperm to egg ratio; and (5) sugar supplementation, using sperm motility, fertilization rate or integrity/potential of sperm components and organelles as quality assessment indicators. Results suggested that among the single CPAs evaluated 6% dimethyl sulfoxide (Me2SO) would be the most suitable for sperm cryopreservation in this species. The highest post-thaw sperm motility was achieved with the sperm that had been exposed to LN vapor for 10 min at 5.2 cm above the LN surface, thawed and recovered in 60 and 18 °C seawater bathes, respectively after at least 2 h storage in LN. The highest fertilization rates were achieved at a sperm to egg ratio of 10,000:1 or 15,000:1. Addition of 1% glucose or 2% sucrose produced significantly higher post-thaw sperm motility than 6% Me2SO alone. Among the three cryoprotectant solutions further trialled, 6% Me2SO + 1% glucose produced the highest fertilization rate of 83.6 ± 3.7%. Evaluation of sperm has shown that the addition of glucose could significantly improve the sperm plasma membrane integrity and mitochondrial membrane potential. These results demonstrated a positive role of glucose in the improvement of sperm cryopreservation in farmed greenlip abalone.     

THE RELEASE MECHANISM AND LOCOMOTOR BEHAVIOR OF EQUISETUM SPERM

When antheridia of gametophytes of Equisetum hyemale L. are placed in water, only spermatid cells are released. These spermatid cells have from 6–12 fibrils radiating from 2–4 loci on the cell wall. One sperm is released from each spermatid cell. With high speed cinemicrography, it can be shown that sperm flagella beat three dimensionally with a continuous, traveling helical wave. A flagellar beat cycle is completed every 0.03 sec. In water the sperm swim rapidly at a rate of 300 μ per sec and traverse a helical path of long wavelength and shallow amplitude. Calcium is essential for normal locomotor behavior. When calcium is chelated by ethylenediaminetetraacetate (EDTA), the sperm spin in place. All other monovalent and divalent ionic solutions tested cause the sperm to swim abnormally. Monovalent ionic solutions prevent the drastic change in sperm mobility patterns resulting from chelation by EDTA.     

STEROID SULFATASE IN BRAIN: COMPARISON OF SULFOHYDROLASE ACTIVITIES FOR VARIOUS STEROID SULFATES IN NORMAL AND PATHOLOGICAL BRAINS, INCLUDING THE VARIOUS FORMS OF METACHROMATIC LEUKODYSTROPHY

Abstract– The enzymatic hydrolysis by brain homogenate of the sulfate esters of estrone, pregnenolone, dehydroepiandrosterone, testosterone, cholesterol and p-nitrophenol was studied. With homogenate of young rat brain, the pH optima of estrone sulfatase ⁴ 4 The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout.
and arysulfatase C (p-nitrophenyl sulfate as substrate) were 8.2 and all other steroid sulfatases had pH optima at 6.6. Apparent K_ms for these steroid sulfates were widely different. The highest K_m value was 32.2 μm for estrone sulfate and the lowest was 0.66 μm for testosterone sulfate; the K_m for p-nitrophenyl sulfate was 30 fold higher than for estrone sulfate. Specific activity was also highest with estrone sulfatase and lowest with testosterone sulfatase; specific activity with aryl sulfatase C was over 3 fold higher than with estrone sulfatase. Estrone sulfatase activity was inhibited noncompetitively by sulfate esters of dehydroepiandrosterone, pregnenolone, and cholesterol; on the other hand, other steroid sulfatases were inhibited by these latter three sulfates competitively. Developmental changes of these sulfohydrolase activities in rat brain were almost identical with the exception of testosterone sulfatase activity; the latter sulfatase had a peak activity at 30 days old, while all other sulfatase had a peak at 20 days old. Thermal stability of all these activities was identical. Testosterone sulfatase activity in neurological mouse mutants, jimpy, msd, and quaking mice, was less than one half of littermate controls, while other steroid sulfatase levels in these mutants' brain were normal. All sulfatase activities were diminished in the brain of a metachromatic leukodystrophy patient with multiple sulfatase deficiency. The brains of classical metachromatic leukodystrophy patients contained normal levels of all steroid sulfatases and arylsulfatase C, with the single exception of testosterone sulfatase which level was less than 50% of control.     

OBSERVATIONS ON THE MORPHOLOGY AND PHYSIOLOGY OF MARSILEA SPERM

The multiciliated sperm of the water fern Marsilea vestita was examined with a view to establishing its suitability as an experimental subject. Time-course experiments revealed spermatid development to be temperature dependent. Sterile techniques were devised for observation of sperm on both a population and an individual basis. Sperm discharge, active and senescing sperm were examined by phase-contrast microscopy. A regular pattern of senescence was ascertained. This included vacuolation of the cytoplasmic vesicle, loss of motility, and ultimate loss of the helical structure of the sperm coil. Sperm life spans were recorded using motility and O₂ uptake as criteria. Sperm populations are active 3–3½ hr at ambient temperature (22–25 C). Individual sperm are active less than 1 hr. Sperm suspensions show a decline in O₂ uptake which parallels the loss of motility. Various constituents affecting the life span were investigated. A twofold prolongation of the sperm life span occurred in the presence of 0.1 m sucrose. An ultrastructural examination of the mature sperm was made to aid in assessing its metabolic potential. The sperm shows little ultrastructural differentiation. The cytoplasmic vesicle is predominantly composed of starch-containing plastids. The main structural components of the sperm coil are a continuous mitochondrial band, an elongate nucleus, and a series of microtubules which separate the basal bodies from the nucleus and mitochondrion. A comparison of ultrastructural features common to Pteridium and Marsilea was made and factors affecting senescence discussed.     

Effect of different calcium concentration on sperm motility and fertilisation capacity of rainbow trout (Oncorhynchus mykiss)

Calcium is a prerequisite factor for triggering the sperm activation in salmonids. The aim of this study was to test different concentrations of Ca²⁺ on sperm motility activation and fertility of rainbow trout (Oncorhynchus mykiss). Semen samples activated with calcium free activator (0.0 mM) and increasing concentrations of CaCl₂ (6.3, 7.9, 9.5 and 11 Mm) were evaluated by computer-assisted sperm analysis (CASA). To assess fertility, eggs from three females were fertilized with semen and activated with each of the five solutions and incubated until four-cell stage when the rate fertility and the symmetry of the first blastomeres were evaluated. The results show that eliminating calcium from the activator solution significantly reduced the motility rate and fertility in comparison with the other treatments, while increasing the CaCl₂ from 6.3 to 11 mM generated high sperm motility (78.5 ± 5.8–95.4 ± 4.0%) and fertility (85.0 ± 9.3–96.0 ± 6.51%) rates, and low symmetry rates in the first blastomeres (<20%). No significant differences were found between solutions for the latter two variables, suggesting that the calcium concentrations were in balance with the other components of the medium and within the requirements for the fertilization of rainbow trout.     

Short- and long-term exposure to methamidophos impairs spermatogenesis in mice

Methamidophos (MET) is a pesticide that has toxic properties, including effects on fertility. This study aimed to assess the joint action of treatment time and exposure to methamidophos on the male reproductive system. MET was orally administered to adult male Swiss mice at a dose of 0.004 mg.kg⁻¹ for 15 and 50 consecutive days. The following parameters were evaluated: weight of reproductive organs, spermatogenesis, sperm and Sertoli cell count, daily sperm production and sperm transit time. Short-term exposure to methamidophos induced a decrease in epididymal weight. The frequency of stages V–VI of spermatogenesis increased and the frequency of stage IX decreased. In the epididymis, sperm transit time (caput/corpus) was reduced and the relative sperm number (cauda) increased. Long-term exposure induced an increase in the frequencies of stages I–IV and V-VI and decreased the stages VII-VIII and IX. The number of Sertoli cells with evident nucleoli was reduced in both exposures. These results confirm the reproductive toxicity of MET.     

Adaptations of sodium balance to low pH in a sunfish (Enneacanthus obesus) from naturally acidic waters

Summary Net sodium flux (J _net), sodium influx (J _in), and sodium efflux (J _out) were measured in two sunfish,Enneacanthus obesus (acid-tolerant) andLepomis gibbosus (less acid-tolerant), during 24 h exposure to soft water of pH's 4.0 and 3.5.E. obesus exhibited a mild transitory disturbance at both pH's caused by inhibitedJ _in and slightly stimulatedJ _out. Body and plasma ion concentrations ofE. obesus were measured weekly during exposures for 5 weeks to acidified artificial soft water (ASW). Body sodium concentration declined 30% during 2 weeks exposure to pH 3.5 but no further during the next three weeks. Exposure to pH 4.0 had no effect on body sodium concentration during the entire 5 weeks. Plasma sodium concentration declined 15% over a 3 week period at pH 3.5; there was no further change in the next two weeks. Plasma potassium concentrations, which were measured after 4 and 5 weeks at pH's 5.8 and 3.5 in ASW, were not significantly different. In a separate two week long experiment, plasma sodium concentration ofE. obesus in ASW was correlated with pH between pH's 3.5 and 7.5. This effect was mainly due to increases above pretreatment levels at pH 4.5 and above. Increased ambient sodium and calcium concentrations had no effect on body sodium concentration ofE. obesus at pH 5.8, but mitigated the effects of exposure to pH 3.5. Increased calcium concentrations up to 25 M at pH 3.5 increased body sodium concentration, but higher concentrations had no additional effect. Body potassium concentration and body water concentration ofE. obesus were linearly related to body sodium concentration under a wide variety of external conditions. This suggests the presence of a mechanism by whichE. obesus regulates plasma sodium levels and body fluid compartments in response to sodium loss. In contrast toE. obesus, L. gibbosus showed larger sodium losses at low pH resulting from greater acceleration ofJ _out; those exposed at pH 3.5 died in less than 12 h.L. gibbosus also had reduced body and plasma sodium concentrations at pH 4.5 and below; those at pH 4.0 were the lowest. Body potassium concentration ofL. gibbosus was reduced in those fish exposed to pH 4.0 and below, but body water was increased. Thus there are striking differences in the ability to regulate ion and water balance at low pH between an acid-tolerant specialist (E. obesus) and a less acid-tolerant generalist (L. gibbosus).Abbreviations ASW artificial soft water - WBM wet body mass - DBM dry body mass