Geographic variation in acid stress tolerance of the moor frog,Rana arvalis. I. Local adaptation

Abstract Spatially varying directional selection together with restricted gene flow among populations is expected to lead to local adaptation. One environmental factor that potentially causes strong directional selection, but is little explored in evolutionary terms, is naturally and anthropogenically induced acidity. We studied local adaptation to acidity in four Swedish populations (two originating from areas that have suffered from severe anthropogenic acidification during the 1900s and two from areas which have remained neutral due to higher buffering capacity) of the moor frog Rana arvalis in a laboratory experiment by investigating whether differences in acid tolerance correspond to population origin. Embryos were raised from fertilization to hatching at three different pH levels (pH 4.0, 4.25 and 7.5), corresponding to levels experienced by these populations in nature, and acid stress tolerance was measured in terms of embryonic survival, hatchling size, and age. Evidence for local adaptation in all of these traits was found, the acid origin embryos having higher survival and less impaired growth performance under acid conditions than the neutral origin embryos. Our estimated rates of divergence (0.007–0.102 haldanes) suggest a rapid adaptation process in response to anthropogenic environmental change, and that the different traits have evolved at relatively similar rates.     

Geographic variation in acid stress tolerance of the moor frog,Rana arvalis. II. Adaptive maternal effects

Abstract The knowledge about the relative contributions of additive genetic and maternal effects, as well as the proximate determinants of maternal effects variation, on population differentiation remains elusive. Likewise, although embryonic performance is often an important component of fitness, it has been relatively little explored in respect to population differentiation. By conducting reciprocal crosses between an acid and a neutral origin population of moor frogs ( Rana arvalis ), we investigated the relative importance of additive genetic versus maternal effects in local adaptation to acidity in embryonic traits. Furthermore, by performing removal experiments of gelatinous egg capsules (jelly), we evaluated the possibility that differences in the extraembryonic membranes might explain the interpopulation variation in embryonic acid tolerance found in this and earlier studies. Embryos were raised from fertilization to hatching at three different pH levels (pH 4.0, 4.25, and 7.5) in the laboratory, and acid stress tolerance was measured in terms of embryonic survival, growth and development (i.e., size and age at hatching). The results show that the higher acid tolerance of acid population embryos (in terms of survival) was maternally determined, indicating adaptive maternal effects. The jelly removal experiment revealed that adaptation to acidity in embryonic survival may arise through variation related to structure/composition of the egg capsules. There was no evidence for a genetic basis in acid tolerance in sublethal effects, but additive and nonadditive genetic effects were found in embryonic growth and development, independently of treatment. The results indicate a role for maternal effects in local adaptation to acidity in amphibians, and genetically based differences in early life-histories among the populations.     

Survival of a Salmonella typhimurium poultry isolate in the presence of propionic acid under aerobic and anaerobic conditions

Propionic acid is commonly found as a fermentation product in the gastrointestinal tracts of food animals and has also been used to limit the microbial contaminants in animal feeds. Because propionic acid is known to have antibacterial activity, the propionic acid encountered by foodborne pathogens during their life cycles may play an important role in inhibiting the survival of the pathogens. The survival patterns of Salmonella typhimurium poultry isolate were determined both in aerobic and anaerobic tryptic soy broth (TSB; pH 5.0 or 7.0) containing various concentrations of propionic acid (0-200 mM). The levels of recovered cells were consistently greater at pH 7.0 compared to those at pH 5.0. For the first 4 days, the levels were significantly decreased by incubation under anaerobic conditions as compared to aerobic condition at pH 7.0 (P<0.05). However, there were fluctuations of cell populations with different patterns depending on both concentrations and growth conditions. To characterize the nature of the capability which allowed the cell multiplication following decreases in cell population during incubation at pH 7.0, the cells isolated from the outgrowth cultures were tested for survival in aerobic or anaerobic TSB (pH 5.0 or pH 7.0) containing propionic acid (50 mM). The outgrowth isolates did not show significant differences in the level of recovered cells in the presence of propionic acid when compared to the wild type strain (P>0.05), suggesting that the cells in the outgrowth cultures did not harbour mutation(s) conferring increased resistance to propionic acid. In addition, the level of recovered cells of isogenic rpoS mutant strain of S. typhimurium was not significantly different from that of the wild type strain in the same assay conditions (P<0.05). The results of this study show that the bactericidal activity of propionic acid on S. typhimurium can be affected by environmental conditions such as acidic pH levels and anaerobiosis in food materials and gastrointestinal tracts. However, S. typhimurium is also able to multiply in the presence of sublethal concentrations of propionic acid at neutral pH during prolonged incubation under both aerobic and anaerobic conditions.     

Carry-over effects of embryonic acid conditions on development and growth of Rana temporaria tadpoles

1. Conditions experienced during the early stages of development may have carry‐over effects on performance during later life. The egg laying period and embryonic development of temperate and boreal zone amphibians often coincides with peak acidity resulting from spring snow‐melt, but the effects of acid conditions during embryonic stage on subsequent performance are unknown. 2. We investigated the potential carry‐over effects of acidity during the embryonic stage on performance up to metamorphosis in the common frog (Rana temporaria) tadpoles. There were four combinations of acid (4.5) and neutral (7.5) pH treatments applied to the egg and larval stages in a factorial laboratory experiment. In addition, we studied the difference in embryonic and larval tolerance of acidity between two populations originating from circumneutral (pH 6.6) and acidic conditions (pH 4.8). 3. The effects of acid conditions during the embryonic stage were sublethal, as indicated by delayed development and reduced size. Under acid conditions, tadpoles that had been raised in neutral water as embryos at first grew more slowly than tadpoles raised under acid conditions as embryos. At metamorphosis, no effects of embryonic acidity were detectable indicating that tadpoles were able to compensate fully for the initial reduction in growth. 4. Acid conditions during the larval period had a strongly negative effect on survival, size and age at metamorphosis. The amount of food consumed was lower under acid conditions, suggesting that reduced food consumption was at least partly responsible for the negative effects. 5. Although the two populations differed in the length of larval period, there was no indication of a differential response to the treatments in any of the metamorphic traits studied. 6. These results suggest that, although moderate acid conditions during embryonic development affect growth and development negatively, this influence does not persist after conditions have returned to normal. However, even moderately acid conditions during the larval period may have a strong negative influence on survival and performance of the tadpoles.     

The fish populations of some streams in Wales and northern England in relation to acidity and associated factors

Electrofishing and water quality surveys were carried out on 60 upland moorland streams in central and north Wales and the Peak District of England (south Pennines). A number of biological characteristics of the fish populations were recorded, including species representation, population density and biomass and, for brown trout, Salmo trutta L., only, growth, condition factor and diet. The stream waters were also analysed over a 2-year period for pH, calcium, aluminium and the heavy metals copper, zinc and lead.
Mean pH levels in the streams ranged from 5.0 to 7.6, with calcium concentrations in the range 0.80–27 mg 1⁻¹. Trace metal concentrations (Al, Cu, Zn, Pb) tended to be elevated in the more acid streams. The latter had fewer fish species, and a higher proportion was fishless, 73% at pH < 5.5 compared with 6% at pH >6.5. Trout and eels, Anguilla anguilla L., predominated at lower pH values, but at reduced population levels.
Dietary composition and stomach fullness of trout were similar throughout the stream pH range. More than 40% of the diet was derived from surface and terrestrial sources, supplementing autochthonous items. Growth (as back-calculated length to end of first year of life) and condition factor did not vary significantly with respect to stream acidity. It is thus concluded that the impoverishment of trout populations in the more acid streams was not a second-order effect acting through the lower trophic levels.
Water quality acting directly on fish would appear to account for the poor status of salmonid fisheries in acid streams. Absence or paucity of salmonids was related to high levels of labile monomeric aluminium (>40 μgl⁻¹) or Cu-Zn-Pb toxicity (>0.4 t-LC50 to rainbow trout, Salmo gairdneri Richardson). Limiting values in this study correspond well with published laboratory and field data.     

Growth and nutrient concentrations of alfalfa and common bean as influenced by soil acidity

Growth and nutrient utilization of alfalfa (Medicago sativa L. cv. Arc) and common bean (Phaseolus vulgaris L. cv. Carioca) were studied in an acid soil adjusted to eight levels of soil acidity by lime addition. Application of lime significantly (P<0.05) increased shoot and root growth for both species. However, common bean was far less sensitive to soil acidity than alfalfa. Maximum alfalfa growth was obtained at a soil pH of 5.8 and maximum bean growth was achieved at pH 5.0. Root and shoot growth of both legumes was positively correlated (P<0.01) with soil pH, exchangeable Ca and exchangeable Mg and negatively correlated (P<0.01) with soil exchangeable Al. Common bean had a lower internal P requirement for maximum growth and was more efficient than alfalfa in taking up Ca and Mg. These characteristics would contribute to the favorable growth of common bean in acid-infertile soils.     

Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea (Camellia sinensis) plants

BACKGROUND AND AIMS: Tea (Camellia sinensis) is considered to be acid tolerant and prefers ammonium nutrition, but the interaction between root zone acidity and N form is not properly understood. The present study was performed to characterize their interaction with respect to growth and mineral nutrition. METHODS: Tea plants were hydroponically cultured with NH4+, NO3- and NH(4+) + NO3-, at pH 4.0, 5.0 and 6.0, which were maintained by pH stat systems. KEY RESULTS: Plants supplied with NO3- showed yellowish leaves resembling nitrogen deficiency and grew much slower than those receiving NH4+ or NH(4+) + NO3- irrespective of root-zone pH. Absorption of NH4+ was 2- to 3.4-fold faster than NO3- when supplied separately, and 6- to 16-fold faster when supplied simultaneously. Nitrate-grown plants had significantly reduced glutamine synthetase activity, and lower concentrations of total N, free amino acids and glucose in the roots, but higher concentrations of cations and carboxylates (mainly oxalate) than those grown with NH4+ or NH(4+) + NO3-. Biomass production was largest at pH 5.0 regardless of N form, and was drastically reduced by a combination of high root-zone pH and NO3-. Low root-zone pH reduced root growth only in NO(3-)-fed plants. Absorption of N followed a similar pattern as root-zone pH changed, showing highest uptake rates at pH 5.0. The concentrations of total N, free amino acids, sugars and the activity of GS were generally not influenced by pH, whereas the concentrations of cations and carboxylates were generally increased with increasing root-zone pH. CONCLUSIONS: Tea plants are well-adapted to NH(4+)-rich environments by exhibiting a high capacity for NH4+ assimilation in their roots, reflected in strongly increased key enzyme activities and improved carbohydrate status. The poor plant growth with NO3- was largely associated with inefficient absorption of this N source. Decreased growth caused by inappropriate external pH corresponded well with the declining absorption of nitrogen.     

Growth and survival of Escherichia coli O157:H7 under acidic conditions.

The effect of pH reduction with acetic (pH 5.2), citric (pH 4.0), lactic (pH 4.7), malic (pH 4.0), mandelic (pH 5.0), or tartaric (pH 4.1) acid on growth and survival of Escherichia coli O157:H7 in tryptic soy broth with 0.6% yeast extract held at 25, 10, or 4 degrees C for 56 days was determined. Triplicate flasks were prepared for each acid treatment at each temperature. At 25 degrees C, populations increased 2 to 4 log10 CFU/ml in all treatments except that with mandelic acid, whereas no growth occurred at 10 or 4 degrees C in any treatments except the control. However, at all sampling times, higher (P < 0.05) populations were recovered from treatments held at 4 degrees C than from those held at 10 degrees C. At 10 degrees C, E. coli O157:H7 was inactivated at higher rates in citric, malic, and mandelic acid treatments than in the other treatments. At the pH values tested, the presence of the organic acids enhanced survival of the pathogen at 4 degrees C compared with the unacidified control. E. coli O157:H7 has the ability to survive in acidic conditions (pH, > or = 4.0) for up to 56 days, but survival is affected by type of acidulant and temperature.     

Why plants grow poorly on very acid soils: are ecologists missing the obvious?

Factors associated with soil acidity are considered to be limiting for plants in many parts of the world. This work was undertaken to investigate the role of the toxicity of hydrogen (H(+)) which seems to have been underconsidered by ecologists as an explanation of the reduced plant growth observed in very acid soils. Racial differences are reported in plant growth response to increasing acidity in the grass Holcus lanatus L. (Yorkshire-fog) and the tree Betula pendula Roth (Silver Birch). Soils and seeds were collected from four Scottish sites which covered a range of soils from acid (organic and mineral) to more base-rich. The sites and their pH (1:2.5 fresh soil:0.01 M CaCl(2)) were: Flanders Moss (FM), pH 3.2+/-0.03; Kippenrait Glen (KP), pH 4.8+/- 0.05; Kinloch Rannoch (KR), pH 6.1+/-0.16; and Sheriffmuir (SMM), pH 4.3+/-0.11. The growth rates of two races of H. lanatus, FM and KP, and three races of B. pendula (SMM, KP and KR) were measured in nutrient solution cultures at pH 2.0 (H. lanatus only), 3.0, 4.0, 5.0, and 5.6. Results showed races from acid organic soils (FM) were H(+)-tolerant while those from acid mineral soils (SMM) were Al(3+)-tolerant but not necessarily H(+)-tolerant. These results confirmed that populations were separately adapted to H(+) or Al(3+) toxicity and this was dependent upon the soil characteristics at their site of collection. The fact of plant adaptation to H(+) toxicity supports the view that this is an important factor in very acid soils.     

Tolerance to acid in pH 5.0-grown organisms of potentially pathogenic Gram-negative bacteria

S.A. NOJOUMI, D.G. SMITH AND R.J. ROWBURY. 1995. A wide range of potentially pathogenic species of Gram-negative bacteria were far more resistant to extreme acidity (pH 2.0–3.5) when cultured at pH 5.0 (habituated to acid) than after pH 7.0 culture. The differences were particularly great for Citrobacter spp., Enterobacter spp., Klebsiella spp. and for Vibrio parahaemolyticus ; substantial habituation was also observed for Proteus mirabilis and Aeromonas formicans but the effect was less marked for Serratia marcescens and Acinetobacter calcoaceticus . Growth at pH 5.0 was substantially poorer than at pH 7.0 for most of the above species and also for Salmonella typhimurium and Salm. enteritidis but phosphate markedly enhanced growth at pH 5.0 for many of these species without affecting growth at pH 7.0.     

Natural variation of <Emphasis Type="Italic">Arabidopsis thaliana </Emphasis>reveals that aluminum resistance and proton resistance are controlled by different genetic factors

Root growth of Arabidopsis thaliana is inhibited by proton rhizotoxicity in low ionic strength media when the pH of the medium is lower than 5.0. QTL analysis at pH 4.7 revealed that two major QTLs on chromosome 2 and 5 and an additional six epistatic interacting loci pairs control proton resistance in the Ler/Col recombinant inbred population. These genetic factors are independently associated with proton resistance in comparison to the known Al resistant QTL and epistases detected in the same RI population at 4 μM Al at pH 5.0. This indicates that different genetic factors regulate mechanisms of resistance to each stress in this plant species. No correlation was observed between proton resistance and Al resistance among 260 accessions indicating that there is no simple relationship between the genetic factors controlling each trait. Several accessions with different combinations of proton (pH 4.7) and Al (4 μM Al at pH 5.0) resistances were identified by phenotypic cluster analysis. Although this grouping was performed using root growth data, the degree of resistance was correlated with their sensitivity to short-term damage in the root tip, indicating that the same resistance mechanism controls proton resistance at different time scales. Resistant accessions grew better than sensitive ones in acid soil culture. This suggests that proton resistance in hydroponic conditions could be an important index in breeding programs to improve productivity in acid soil, at least in acid sensitive plant species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis of cyclopropane fatty acid and its effect on freeze-drying survival of Lactobacillus bulgaricus L2 at different growth conditions

In order to correlate cyclopropane fatty acid of the membrane of Lactobacillus bulgaricus L2 with freeze-drying survival at different growth conditions, fatty acid methyl esters (FAME) from extracts grown at difference fermentation pH (5.0, 5.5, 6.0, 6.5) and temperature (30, 35, 37, 39°C) were obtained and analyzed. Results showed that cultures grown at 30°C and pH 5.0, 35°C and pH 5.0, 39°C and pH 6.0 exhibited more resistance to the freeze-drying process than cultures grown in other conditions, cells cultured at 30°C and pH 5.0 had a highest survival rate. On the other hand, cells grown at 37°C displayed poor resistance to adverse conditions possible because of the lower cycC19:0 content. It was concluded that the improved cryotolerance observed during freeze-drying would be associated with an increase in cycC19:0 content and cycC19:0/SFA ratio and vice versa.     

Influence of Acidity on Growth and Biochemistry of Pennisetum clandestinum

Hydroponics were used to study the impact of acidity on growth, nutritive properties and metabolic changes in kikuyu grass (Pennisetum clandestinum Hochst). Four treatments (pH 6.0, 5.0, 4.0, and 3.0) were compared for effects on biomass, leaf and root length, crude protein, amino acid content and key enzymes of sugar metabolism. Reduction in biomass, root and leaf length, amino acid contents, glucose-6-phosphate dehydrogenase (G6PDH) and pyruvate kinase (PK) content was observed only at pH 3.0, in association with increased leaf proline content. Kikuyu grass is able to grow normally under mild acidity (down to at least pH 4.0).     

Dependence of erythromycin biosynthesis on the active acidity of the medium]

Dependence of erythromycin biosynthesis on the medium active acidity was studied by the following methods: by changing pH of the initial medium, by changing the concentration of the medium components determining the active acidity of the culture, by using buffer mixtures by automatic control of pH. It was found that pH of the initial medium within 5.7-8.1 had no effect on the culture growth. Biosynthesis of erythromycin markedly decreased at pH 6.3 or lower. The values of pH within 6.6-7.5 (optimal values 6.7-6.9) were favourable for the antibiotic biosynthesis. At pH 6.2-6.3 the antibiotic accumulation was equal to 5-10 per cent of the control.     

Conformational change of apolipoprotein A-I and HDL formation from model membranes under intracellular acidic conditions

The molecular mechanism by which nascent HDL forms via the interaction of apolipoprotein A-I (apoA-I) and transmembrane ABCA1 is poorly understood. Here, because ABCA1 has been reported to localize to acidic intracellular compartments, including the Golgi and endosome, we studied the interaction of apoA-I with model membranes under acidic conditions. Pure phosphatidylcholine liposomes were persistent against apoA-I at pH levels above 5.0, but were progressively transformed into reconstituted HDLs (rHDLs) by apoA-I at lower pH. Circular dichroism spectral measurements and 8-anilino-1-naphthalenesulfonic acid fluorescence measurements of lipid-free apoA-I ascribed this accelerated rHDL formation to the conformational change of the protein into a rather hydrophobic alpha-helical structure under acidic conditions. The addition of phosphatidylserine (PS) increased acidity at the bilayer surface and enabled the formation of discoidal rHDLs even at the pH of the endosome and slightly lower pH of the Golgi. These results suggest the following new scenario of nascent HDL formation: ABCA1, which colocalizes with apoA-I in acidic intracellular compartments, including the Golgi and endosome, increases acidity at the membrane surface on the luminal side by PS translocase activity and causes apoA-I to form nascent HDL.     

Molecular characterization of the acid-inducible asr gene of Escherichia coli and its role in acid stress response

Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli under acid shock conditions.     

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach

AIMS: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium. Methods and RESULTS: The effects of acidity on the survival of B. cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7. Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken. At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken. A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively. Between pH 5.0 and 5.3, B. cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h). CONCLUSIONS: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells. This resistance strongly depends on the type of food present in the GM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the probability that viable B. cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity.     

Susceptibility of starved, juvenile smallmouth bass, Micropterus dolomieui (Lacépède) to low pH

Juvenile smallmouth bass, Micropterus dolomieui , were starved over a period of 6 months in the laboratory and their survival in waters of low pH tested. The ability of the bass to withstand the toxic effects of hydrogen ion decreased with time. The LC50 (96-h) changed from pH 3.7 to pH 4.4 over the experimental period. During the final days, the decrease in the threshold was greatly accelerated. In the wild, some overwintering bass are subjected to pulses of water as low as pH 4.7 as a result of vernal snowmelt. Bass at the end of winter are often emaciated and are especially susceptible to acid stress.     

The influence of pH on the growth rate and viability of neutrophilic and acidophilic streptomycetes.

The influence of pH on the specific growth rates of two acidophilic and two neutrophilic soil streptomycetes was studied. The acidophiles had maximum growth rates over a broad range from pH 4.5 to 5.5, while the neutrophiles had clearly defined optima at pH 7.0. Mycelium of neutrophiles was less tolerant of acidity than that of acidophiles; both showed decreased viability at pH levels below those which allowed growth. Spores of neutrophiles and acidophiles were equally tolerant of acidity and this may allow the former to survive in acid soils. Both spores and mycelium of acidophiles remained viable at pH levels above those allowing growth.