Reproducibility and Consistency of Proteomic Experiments on Natural Populations of a Non-Model Aquatic Insect

Population proteomics has a great potential to address evolutionary and ecological questions, but its use in wild populations of non-model organisms is hampered by uncontrolled sources of variation. Here we compare the response to temperature extremes of two geographically distant populations of a diving beetle species (Agabus ramblae) using 2-D DIGE. After one week of acclimation in the laboratory under standard conditions, a third of the specimens of each population were placed at either 4 or 27°C for 12 h, with another third left as a control. We then compared the protein expression level of three replicated samples of 2–3 specimens for each treatment. Within each population, variation between replicated samples of the same treatment was always lower than variation between treatments, except for some control samples that retained a wider range of expression levels. The two populations had a similar response, without significant differences in the number of protein spots over- or under-expressed in the pairwise comparisons between treatments. We identified exemplary proteins among those differently expressed between treatments, which proved to be proteins known to be related to thermal response or stress. Overall, our results indicate that specimens collected in the wild are suitable for proteomic analyses, as the additional sources of variation were not enough to mask the consistency and reproducibility of the response to the temperature treatments.     

Plasmid Frequency Fluctuations in Bacterial Populations from Chemically Stressed Soil Communities

The frequency of plasmids in chemically stressed bacterial populations was investigated by individually adding various concentration of kanamycin, ampicillin, and mercuric chloride to soil samples. Viable bacterial populations were enumerated, soil respiration was monitored for up to 6 weeks as an indicator of physiological stress, and bacterial isolates from stressed and control soils were screened for the presence of plasmids. Low levels of the chemical stress factors did not for the most part significantly alter population viability, soil respiration, or plasmid frequency. Exposure to high stress levels of mercury and ampicillin, however, resulted in altered numbers of viable organisms, soil respiration, and plasmid frequency. Plasmid frequency increased in response to ampicillin exposure but was not significantly changed after exposure to kanamycin. In mercuric chloride-stressed soils, there was a decrease in plasmid frequency despite an increase in overall mercury resistance of the isolates, suggesting that mercury resistance in these populations is largely, if not completely, chromosome encoded. Chemical stress did not cause an increase in plasmid-mediated multiple resistance. A genetic response (change in plasmid frequency) was not found unless a physiological (phenotypic) response (change in viable cells and respiratory activity) was also observed. The results indicate that a change in plasmid frequency is dependent on both the amount and type of chemical stress.     

Viability and Estimation of Shelf-Life of Bacterial Populations

Mathematical concepts associated with the exponential and probit models are developed, and the similarity of the two methods is discussed. Because of its greater flexibility in design, the probit method was used to estimate the shelf-life for four bacterial populations, wet and dry spores of Bacillus anthracis and wet and dry cells of Pasteurella tularensis. On the basis of data gained by storing these organisms at high temperature, the probit method was used to predict the time at which 50% viability would occur for cells stored at 3 C. A plane passing through a three-space showing change in percentage viability of bacteria was formulated by a multiple regression method. With this functional technique, the percentage viability, expressed as a probit, was linearily related to a logarithm of storage time and storage temperature. The use of this method to study the effect of controlled variables on the viability of cells is demonstrated by comparing the effect of viability associated with three additives used prior to drying. The results of the test gave shelf-life estimates which were too low for all additives; however, the order of stability was ranked properly as confirmed by long-term tests.     

Gene Flow in Natural Populations of DROSOPHILA MELANOGASTER with Special Reference to Lethal Allelism Rates and Protein Variation

A simultaneous survey of 14 protein loci, together with frequencies and within- and between-population allelism rates of lethal chromosomes, was carried out in five (four Japanese and one Korean) natural populations and one cage population of Drosophila melanogaster. It was found that lethal allelism rates decrease rapidly as geographic distance between two populations increases, while variation at protein loci shows a remarkable similarity over all populations examined. These findings suggest that there are very high levels of gene flow in these natural populations and that selection at protein loci which can maintain substantial geographic variation, if present, is overshadowed by gene flow. There is no indication that invasion of D. melanogaster to the Far East occurred so recently that the frequencies of lethal chromosomes are still in nonequilibrium.     

Bayesian Procedures for the Estimation of Mutation Rates from Fluctuation Experiments

Bayesian procedures are developed for estimating mutation rates from fluctuation experiments. Three Bayesian point estimators are compared with four traditional ones using the results of 10,000 simulated experiments. The Bayesian estimators were found to be at least as efficient as the best of the previously known estimators. The best Bayesian estimator is one that uses (1/m(2)) as the prior probability density function and a quadratic loss function. The advantage of using these estimators is most pronounced when the number of fluctuation test tubes is small. Bayesian estimation allows the incorporation of prior knowledge about the estimated parameter, in which case the resulting estimators are the most efficient. It enables the straightforward construction of confidence intervals for the estimated parameter. The increase of efficiency with prior information and the narrowing of the confidence intervals with additional experimental results are investigated. The results of the simulations show that any potential inaccuracy of estimation arising from lumping together all cultures with more than n mutants (the jackpots) almost disappears at n = 70 (provided that the number of mutations in a culture is low). These methods are applied to a set of experimental data to illustrate their use.     

Cytogenetic Approach to Estimation of Developmental Stability of the Natural Populations of Coregonid Fish

A comparative cytogenetic analysis of Coregonid fish was carried out in the river and lake ecosystems of the northern Tyumen' District. The occurrence of chromosomal aberrations in embryonic cells of fish steadily increased during the period from 1989 until 2001. Possible causes of this dynamic are discussed.     

Estimation and Testing of Tumor Incidence Rates in Experiments Lacking Cause-of-Death Data

Approximate nonparametric maximum likelihood estimation of the tumor incidence rate and comparison of tumor incidence rates between treatment groups are examined in the context of animal carcinogenicity experiments that have interval sacrifice data but lack cause-of-death information. The estimation procedure introduced by MALANI and VAN RYZIN (1988), which can result in a negative estimate of the tumor incidence rate, is modified by employing a numerical method to maximize the likelihood function iteratively, under the constraint that the tumor incidence rate is nonnegative. With the new procedure, estimates can be obtained even if sacrifices occur anywhere within an interval. The resulting estimates have reduced standard error and give more power to the test of two heterogeneous groups. Furthermore, a linear contrast of more than two groups can be tested using our procedure. The proposed estimation and testing methods are illustrated with an experimental data set.     

The Response of Three Bacterial Populations to Pollution in a Stream

Abstract Practical methods for biomonitoring of natural systems are still under development. Bacteria are potentially useful indicators of water quality because of their species diversity and ability to rapidly respond to changing environmental conditions. In this study, bacterial populations from unpolluted and polluted stream sites in two watersheds were compared to determine their suitability for use as environmental indicators. Upper Three Runs Creek and Four Mile Creek headwaters have had little anthropogenic disturbance, as opposed to lower Four Mile Creek which received thermal, radioactive, and chemical perturbations. Chemical and physical measurements provided evidence that seepage from holding ponds polluted Four Mile Creek. Polluted sites did not have altered total bacterial numbers but had decreased numbers of colony-forming units. Abundances of three bacterial species, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, were determined by colony hybridization with species-specific rDNA probes. Contribution of A. calcoaceticus to the assemblage was higher at polluted sites, which indicated either tolerance of polluted conditions or the ability to utilize compounds existing at these sites to reach larger populations. No differences in B. cepacia populations were detected, and differences in P. putida populations could not be attributed solely to disturbance. The pollution of Four Mile Creek induced differences in bacterial populations that could be monitored using the described approach. Received: 24 September 1996; Accepted: 20 December 1996     

Estimation of Bacterial Nitrate Reduction Rates at In Situ Concentrations in Freshwater Sediments

A method was developed to follow bacterial nitrate reduction in freshwater sediments by using common high-performance liquid chromatographic equipment. The low detection limit (14 pmol) of the method enabled us to study concentration profiles and reaction kinetics under natural conditions. Significant nitrate concentrations (1 to 27 μM) were observed in the sediment of Lake Vechten during the nonstratified period; the concentration profiles showed a successive depletion of oxygen, nitrate, and sulfate with depth. The profiles were restricted to the upper 3 cm of the sediment which is rich in organics and loosely structured. Nitrate reduction in the sediment-water interface followed first-order reaction kinetics at in situ concentrations. Remarkably high potential nitrate-reducing activity was observed in the part of the sediment in which nitrate did not diffuse. This activity was also observed throughout the whole year. Estimates of K_m varied between 17 and 100 μM and V_max varied between 7.2 and 36 μmol cm⁻³ day⁻¹ for samples taken at different depths. The diffusion coefficient of nitrate ([10 ± 0.4] × 10⁻⁶ cm² s⁻¹) across the sediment-water interface was estimated by a constant-source technique and applied to a mathematical model to estimate the net nitrate reduction during the nonstratified period. In this period, observed nitrate reduction rates by the model, 0.2 to 0.4 mmol m⁻² day⁻¹, were lower than those found for oxygen (27 mmol m⁻² day⁻¹) and sulfate (0.4 mmol m⁻² day⁻¹). During the summer stratification, nitrate was absent in the sediment and reduction could not be estimated by the model.     

Antibiotics Interfere with the Evolution of Plasmid Stability

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Estimation of a Difference in Finite Populations with Missing Observations

Sampling strategies for the difference are constructed when missing observations are present. Two different situations are analyzed. One of them is related with a non random device settled by the statistician for reducing costs. The other is a non response problem. An unbiased minimum variance estimator is obtained in the first case and an approximation to it is deduced. The unbiased estimation in the second is associated with subsampling tactics.     

Suggestions as to Quantitative Measurements of Plasmid Loss

Suggestions are made for the estimation of plasmid loss rates in bacterial populations that multiply by binary fission. The plasmid loss rate is defined as the probability of a division of a plasmid-carrying individual giving birth to one plasmid-free and one plasmid-carrying daughter cell. The unit of time is consequently the interdivision time for plasmid-carrying individuals. The cautions that have to be taken when the population is cultivated in serial transfer (the usual stability experiment) are discussed.     

Experimental Evolution of Gene Duplicates in a Bacterial Plasmid Model

The fate of gene duplicates subjected to diversifying selection was tested experimentally in a bacterial system. The wild-type TEM-1 β-lactamase gene confers resistance to ampicillin but not to cefotaxime. Point mutations confer cefotaxime resistance, but they compromise ampicillin resistance. Thus, selection for both drug resistances in a bacterium with two copies of β-lactamase should favor the divergence of one copy to improve cefotaxime resistance while maintaining the other copy to preserve ampicillin resistance. This selection was performed on a bacterium with identical sequences of β-lactamase on two separate, compatible plasmids. As expected, one plasmid evolved increased cefotaxime resistance when appropriately strong cefotaxime selection was applied. However, the cefotaxime-resistant plasmid maintained sufficient ampicillin resistance to tolerate the concentration of ampicillin used, and the other plasmid was lost. Hosts carrying both the cefotaxime-resistant and wild-type plasmids were then subjected to various higher concentrations of both drugs to find conditions that would ensure the maintenance of both plasmids. In a striking contradiction to our model, no such conditions were found. The fitness cost of carrying both plasmids increased dramatically as antibiotic levels were raised, and either the wild-type plasmid was lost or the cells did not grow. This study highlights the importance of the cost of duplicate genes and the quantitative nature of the tradeoff in the evolution of gene duplication through functional divergence. Reviewing Editor: Dr. Margaret Riley     

Environmental Factors Correlated with Size of Bacterial Populations in a Polluted Stream

Samples of water were taken from a polluted zone of the Gallinas River and analyzed as to numbers of total bacteria, coliforms, and fecal streptococci. Environmental factors measured were temperature, pH and concentrations of detergent, nitrate plus nitrite nitrogen, sulfate, chloride, bicarbonate, and phosphate. Thirty-two observations were made from 12 March through 22 July 1971. Stepwise multiple linear regression analyses of the data were carried out by computer to determine which of the environmental factors were significantly correlated with numbers of bacteria present. A multiple linear regression equation was constructed for each bacteriological parameter as a function of significant variables only. Log total bacteria was correlated positively with bicarbonate, phosphate, and detergent concentrations. Log coliforms was correlated positively with phosphate and sulfate concentrations and negatively with chloride concentration. Log fecal streptococci was correlated positively with bicarbonate and chloride concentrations.     

Simulating Genetic Circuits in Bacterial Populations with Growth Heterogeneity

We computationally study genetic circuits in bacterial populations with heterogeneities in the growth rate. To that end, we present a stochastic simulation method for gene circuits in populations of cells and propose an efficient implementation that we call the “Next Family Method”. Within this approach, we implement different population setups, specifically Chemostat-type growth and growth in an ideal Mother Machine and show that the population structure and its statistics are different for the different setups whenever there is growth heterogeneity. Such dependence on the population setup is demonstrated, in the case of bistable systems with different growth rates in the stable states, to have distinctive signatures on quantities including the distributions of protein concentration and growth rates, and hysteresis curves. Applying this method to a bistable antibiotic resistance circuit, we find that as a result of the different statistics in different population setups, the estimated minimal inhibitory concentration of the antibiotic becomes dependent on the population setup in which it is measured.     

The Loss of the Kinetoplast in Trypanosomes, with Special Reference to Trypanosoma evansi

An account is given of the spontaneous occurrence and prolonged maintenance of strains of Trypanosoma evansi devoid of the kinetoplast. The structure and nature of this organelle—which is regarded as a plastid—are discussed and previous work on its loss is reviewed. Among the six akinetoplastic strains studied, two were kept under continuous observation for about 17 years, in the course of which many thousands of trypanosomes were examined, without a single normal flagellate being detected. In these cases, therefore, the aberrant condition had become permanently fixed as a heritable character. Another strain of T. evansi first exhibited considerable fluctuation in the number of akinetoplastic forms (1–70%) but subsequently reverted to the normal state. The perpetuation of the akinetoplastic condition appears to be due to failure of the kinetoplast to divide, with the result that trypanosomes thus incapacitated divide irregularly, giving rise to akinetoplastic forms, which continue to breed true. This may lead to the production, on the one hand, of fluctuating strains, in which the akinetoplastic and normal trypanosomes compete for survival; and, on the other hand, of totally akinetoplastic strains. The loss of the kinetoplast is comparable to the loss of plastids in phytoflagellates. In a discussion of the genetic aspects of this phenomenon it is regarded as a mutation determined by plastogenes, for the mutant trypanosomes appear suddenly, breed true, and give rise to a new race. Moreover, the kinetoplast once lost does not arise de novo. It is suggested that T. equinum had originated from an akinetoplastic strain of T. evansi produced by such mutation.