A likelihood-based approach for assessment of extra-pair paternity and conspecific brood parasitism in natural populations.

Genotypes are frequently used to assess alternative reproductive strategies such as extra-pair paternity and conspecific brood parasitism in wild populations. However, such analyses are vulnerable to genotyping error or molecular artefacts that can bias results. For example, when using multilocus microsatellite data, a mismatch at a single locus, suggesting the offspring was not directly related to its putative parents, can occur quite commonly even when the offspring is truly related. Some recent studies have advocated an ad-hoc rule that offspring must differ at more than one locus in order to conclude that they are not directly related. While this reduces the frequency with which true offspring are identified as not directly related young, it also introduces bias in the opposite direction, wherein not directly related young are categorized as true offspring. More importantly, it ignores the additional information on allele frequencies which would reduce overall bias. In this study, we present a novel technique for assessing extra-pair paternity and conspecific brood parasitism using a likelihood-based approach in a new version of program cervus. We test the suitability of the technique by applying it to a simulated data set and then present an example to demonstrate its influence on the estimation of alternative reproductive strategies.

Estimating the prevalence of inbreeding from incomplete pedigrees.

A previous review of inbreeding in natural populations suggested that close inbreeding (inbreeding coefficient f = 0.25) is generally rare in wild birds and mammals. However, the review did not assess rates of moderate inbreeding (f = 0.125), which may make a rather larger contribution to overall inbreeding in a population. Furthermore, previous studies may have underestimated the prevalence of inbreeding in wild populations with incomplete pedigrees. By categorizing inbreeding events by the relationship of the parental pair, we suggest a simple method for estimating rates of close and moderate inbreeding from incomplete pedigree data. We applied this method to three wild populations of ruminants: red deer on Rum, Scotland, Soay sheep on Hirta, Scotland and reintroduced Arabian oryx on the Jiddat-al-Harasis, Oman. Although paternal half-sib pairs were the most common category of inbreeding in all three populations, there was considerable variation among populations in the frequencies of the various categories of inbreeding. This variation may be largely explained by differences in population size and dynamics, in maternal and paternal sibship size and in the overlap of reproductive lifespan of consecutive generations. Close and moderate inbreeding appear to be a routine part of breeding behaviour in these ruminant populations.

Energy-gain measurements from a microwave inverse free-electron-laser accelerator.

Experiments are reported on inverse free-electron-laser acceleration, including for the first time observations of the energy change as a function of relative injection phase of the electron bunches. The microwave accelerating structure consists of a uniform circular waveguide with a helical wiggler and an axial magnetic field. Acceleration of the entire beam by 6% is seen for 6 MeV electron bunches at optimum relative phase. Experimental results compare favorably, for accelerating phases, with predictions of a three-dimensional simulation that includes large-orbit effects.

Inbreeding depression influences lifetime breeding success in a wild population of red deer (Cervus elaphus).

Evolutionary and conservation biologists have a long-standing interest in the consequences of inbreeding. It is generally recognized that inbred individuals may experience reduced fitness or inbreeding depression. By the same token, relatively outbred individuals can have greater than average fitness, i.e. heterosis. However, nearly all of the empirical evidence for inbreeding depression comes from laboratory or domestic species. Inbreeding depression and heterosis are difficult to detect in natural populations due to the difficulties in establishing pedigrees. An alternative method is to correlate heterozygosity, which is measured using genetic markers, with a trait related to fitness. The typically studied traits, such as juvenile survival and growth rates, either cover only early life or are weakly correlated with lifetime breeding success (LBS). In this paper we show that heterozygosity is positively associated with male and female adult LBS in a wild population of red deer (Cervus elaphus) on the Isle of Rum, Scotland. To the authors' knowledge, this is the first time that inbreeding depression and/or heterosis have been detected for a trait highly correlated with overall fitness in both sexes in a wild population.

Statistical confidence for likelihood-based paternity inference in natural populations.

Paternity inference using highly polymorphic codominant markers is becoming common in the study of natural populations. However, multiple males are often found to be genetically compatible with each offspring tested, even when the probability of excluding an unrelated male is high. While various methods exist for evaluating the likelihood of paternity of each nonexcluded male, interpreting these likelihoods has hitherto been difficult, and no method takes account of the incomplete sampling and error-prone genetic data typical of large-scale studies of natural systems. We derive likelihood ratios for paternity inference with codominant markers taking account of typing error, and define a statistic delta for resolving paternity. Using allele frequencies from the study population in question, a simulation program generates criteria for delta that permit assignment of paternity to the most likely male with a known level of statistical confidence. The simulation takes account of the number of candidate males, the proportion of males that are sampled and gaps and errors in genetic data. We explore the potentially confounding effect of relatives and show that the method is robust to their presence under commonly encountered conditions. The method is demonstrated using genetic data from the intensively studied red deer (Cervus elaphus) population on the island of Rum, Scotland. The Windows-based computer program, CERVUS, described in this study is available from the authors. CERVUS can be used to calculate allele frequencies, run simulations and perform parentage analysis using data from all types of codominant markers.

Microsatellites reveal heterosis in red deer.

The fitness consequences of inbreeding and outbreeding are poorly understood in natural populations. We explore two microsatellite-based variables, individual heterozygosity (likely to correlate with recent inbreeding) and a new individual-specific internal distance measure, mean d2 (focusing on events deeper in the pedigree), in relation to two measures of fitness expressed early in life, birth weight and neonatal survival, in 670 red deer calves (Cervus elaphus) born on the Isle of Rum between 1982 and 1996. For comparison, we also analyse inbreeding coefficients derived from pedigrees in which paternity was inferred by molecular methods. Only 14 out of 231 calves (6.1%) had non-zero inbreeding coefficients, and neither inbreeding coefficient nor individual heterozygosity was consistently related to birth weight or neonatal survival. However, mean d2 was consistently related to both fitness measures. Low mean d2 was associated with low birth weight, especially following cold Aprils, in which foetal growth is reduced. Low mean d2 was also associated with low neonatal survival, but this effect was probably mediated by birth weight because fitting birth weight to the neonatal survival model displaced mean d2 as an explanatory variable. We conclude that in the deer population fitness measures expressed early in life do not show evidence of inbreeding depression, but they do show evidence of heterosis, possibly as a result of population mixing. We also demonstrate the practical problems of estimating inbreeding via pedigrees compared with a direct marker-based estimate of individual heterozygosity. We suggest that, together, individual heterozygosity and mean d2, estimated using microsatellites, are useful tools for exploring inbreeding and outbreeding in natural population.

The maintenance of genetic polymorphism in small island populations: large mammals in the Hebrides.

Conventionally, small populations living on islands are expected to lose genetic variation by drift. Fluctuations in population size, combined with polygynous mating systems, are expected to contribute to the process by increasing sampling effects on genetic variation. However, in individually monitored populations of Red deer on Rum and Soay sheep on St. Kilda, which experience fluctuations in population size, two processes have been identified which mitigate loss of genetic variation. First, in a number of examples, population reductions are associated with selection. Selection may be in favour of heterozygotes, or, as we have documented in several cases, it may fluctuate in direction temporally. Second, in Soay sheep, in which mortality over population crashes is male-biased, ostensibly leading to low effective numbers of males, molecular studies show that there are systematic changes in the reproductive success of young males, and in variance in male success, that broaden genetic representation compared with expectation.

The inhalation toxicity of two commercial dyes: solvent yellow 33 and solvent green 3.

The quinoline dye 2-(2'-quinolyl)-1,3-indandione or Solvent Yellow 33 (SY) and the anthraquinone dye 1,4-di-p-toluidinoanthraquinone or Solvent Green 3 (SG) are used in many manufactured products including military smoke grenades. During manufacturing, SY or a combination of both SY and SG can be released into the air, exposing factory workers by inhalation to these dye compounds. The potential inhalation toxicity of these compounds was tested by exposing F344/N rats to different concentrations of SY or SY/SG dye mixture (30:70 w/w) for 6 hr/day, 5 days/week for 4 or 13 weeks. In the 4-week studies, rats were exposed to SY aerosols at average concentrations of 10 +/- 5, 51 +/- 10, or 230 +/- 30 mg/m3 (means +/- SD) or SY/SG aerosols at average concentrations of 11 +/- 5, 49 +/- 11, or 210 +/- 50 mg/m3 (means +/- SD). Rats exposed to the highest concentration of SY or SY/SG had body weights that were approximately 8% or 7% less, respectively, than their controls after exposure. Rats exposed to the highest concentration of SY/SG for 4 weeks also had reduced pulmonary gas exchange efficiency, airflow obstruction, mild pulmonary inflammation, slight Type II pulmonary epithelial cell hyperplasia, and proliferation of vacuolated alveolar macrophages. In the 13-week studies, rats were exposed to SY aerosols at average concentrations of 1.0 +/- 0.2, 10.8 +/- 1.8, or 100 +/- 17 mg/m3 (means +/- SD) or SY/SG aerosols at average concentrations of 1.1 +/- 0.5, 10.2 +/- 3.1, or 101 +/- 23 mg/m3 (means +/- SD). Animals exposed to the highest concentration of SY or SY/SG for 13 weeks had body weights that were approximately 5 or 9% less, respectively, than their controls after exposure and had accumulation of vacuolated alveolar macrophages in lungs. Rats exposed to the highest concentration of SY/SG dye mixture for 13 weeks also had indications of mild pulmonary inflammation and slight Type II pulmonary epithelial cell hyperplasia. Very little SY was found in lungs after any exposures, indicating its clearance from lungs was at a rapid rate. However, significant amounts of the SG component of the SY/SG mixture were detected in lungs after each exposure. Lung clearance half-times of SG from the 13-week exposure were estimated to be approximately 280 days. In summary, neither test material appeared to be highly toxic following inhalation. However, the slightly higher toxicity observed for SY/SG over SY alone is probably related to the longer lung retention of the SG component of the dye mixture.

The inhibition of rat and guinea pig cholinesterases by anionic hydrolysis products of methylphosphonic difluoride (difluoro).

Methylphosphonic difluoride (difluoro) and its hydrolysis products, methylphosphonofluoridate (MF) and fluoride, were examined for cholinesterase-inhibiting ability in rats and guinea pigs by both inhalation and intraperitoneal exposure routes. In vivo inhibition was compared to in vitro inhibition. In the whole animal, MF was the active chemical, but in vitro under special conditions, difluoro was more potent than MF and fluoride. Rat and guinea pig blood cholinesterase were equally sensitive to inhibition by MF, but only the guinea pig displayed cholinergic signs leading to death from MF toxicity. Data imply that MF is responsible for the cholinesterase inhibition resulting from exposure to DF vapor. MF may be the first example of a moderately strong acid shown to inhibit cholinesterase and cause death from cholinergic effects.

Use of a jet mill for dispersing dry powder for inhalation studies.

Compressed air-powered jet mills are used in the chemical and food industries for grinding and classifying powders. We adapted one type of these fluid energy mills as a powder generator for inhalation experiments. The generating system included a jet mill and a screw feeder, the jet mill consisting of an elongated channel, a feeding jet to deliver the material into the channel, and two high-speed air jets. High speed air circulating in the channel created turbulence and centrifugal forces to disperse powder. The jet mill used can be operated from 25 to 100 psig at flow rates of 300 to 900 L/min. Two test materials--a solvent yellow dye and a dye mixture of solvent green and solvent yellow--were used. Both dyes were soft and sticky and could not be dispersed with several other powder generators tested at the concentrations required for toxicity studies. Aerosol concentrations ranging from 10 to 1500 mg/m3 at a flow rate of 400 L/min were obtained by adjusting the feed rate to the jet mill. Stability of aerosol concentration during six-hour continuous generation was 15 to 20%. Comparisons of several generators for producing sticky organic powders are also discussed.

Interaction of formaldehyde with glutathione in the isolated/ventilated perfused lung and the isolated perfused liver.

The interaction of formaldehyde (CH2O) with reduced glutathione (GSH) was evaluated in aqueous solution and in isolated perfused lungs and livers. Addition of CH2O (0-4.9 mM) to a solution of 0.17 mM GSH in 2 mM EDTA, pH 7.4, resulted in a time- and concentration-dependent depletion of GSH. Perfusion of livers with fortified Krebs-Ringer bicarbonate buffer containing 0.3-4.9 mM CH2O resulted in a dose-dependent depletion of GSH. Perfusion of isolated ventilated lungs with perfusate containing 4.9 mM CH2O resulted in a depletion of GSH to 75% of controls. However, lower concentrations of CH2O in the lung perfusate did not result in depletion of GSH. These results demonstrate that exposure to CH2O in aqueous solution or in the perfused lung and liver is capable of depleting endogenous GSH. However, the concentrations of CH2O required to yield a significant depletion of endogenous GSH exceed those encountered in vivo. Thus, it is unlikely that depletion of GSH by CH2O is a causal factor in formaldehyde-induced toxicity.

Cytotoxicity and mutagenicity of vapor-phase pollutants in rat lung epithelial cells and Chinese hamster ovary cells grown on collagen gels.

Lung epithelial cell (cell line designated LEC) and Chinese hamster ovary (CHO) cells were grown on hydrated collagen gels and exposed directly to toxic vapor-phase pollutants. The cells were exposed to graded concentrations of phenol, formaldehyde, a volatile fraction of process stream material from an experimental coal gasifier and the nonparticulate, vapor phase of diesel engine exhaust. During exposures, the cells were maintained at an air/collagen interface by removing the medium overlying the hydrated collagen gel. Morphological changes indicative of cell retraction were found in LEC cell cultures exposed to phenol, formaldehyde, or diesel exhaust. Damage following exposure to the toxicants was quantitated in LEC and CHO cells by Trypan blue dye exclusion, a measure of plasma membrane integrity. Clone-forming ability was also used to measure cell survival in CHO cells. When measured by Trypan blue dye exclusion, phenol (EC50 = 2.1 mg/l) caused membrane damage to LEC cells but not CHO cells, while formaldehyde (EC50 = 31 and 42 micrograms/l for LEC and CHO, respectively) and diesel exhaust (EC50 = 11 and 29% of tailpipe exhaust in LEC and CHO cells, respectively) caused damage to both cell types. No cytotoxicity was observed in LEC or CHO cells exposed to the fraction from the coal gasifier. Essentially no mutagenic activity was associated with the exposure of CHO cells to formaldehyde or the vapor phase of diesel exhaust. Mutagenic activity was found in CHO cells exposed to ethylene oxide, the positive control. The results of this study indicate that mammalian cells grown on collagen gels can readily be exposed to vapors of chemicals and chemical mixtures. The cell exposure system may be generally useful in the analysis of toxic damage to mammalian cells resulting from gaseous or vapor-phase pollutants.

Acute and genetic toxicity of 1-nitropyrene and its fate after single oral doses to rats.

The mammalian acute and genetic toxicity of 1-nitropyrene (NP) was studied because this and other nitroarenes are highly mutagenic toward bacteria and have been identified in emissions from combustion processes. A suspension of NP did not cause observable signs of acute toxicity and was not lethal when administered to male and female rats at single oral doses as high as 5.0 g/kg. Histological examination of stomach, intestine, lung, heart, spleen, pancreas, adrenal, and kidney from rats euthanized at 4 and 14 d after treatment revealed no detectable differences from control rats. Urine and feces were collected for 4 d after treatment with 5.0 g/kg. About 70% of the dose was present in the feces as NP, and about 2% was present as the reduced metabolite 1-aminopyrene (AP). Sulfate and glucuronide conjugates of AP were present in small amounts (less than 1%) in the urine, showing that at least some of the dose was absorbed. Bone marrow cells from female rats given NP orally at 0.5, 1.5, and 5.0 g/kg showed a slight dose-related increase in the frequency of sister chromatid exchanges. Both NP and AP showed low mutagenicity in Chinese hamster ovary cells in vitro. Evidence of reductive metabolism of NP in rats raises concern about the potential exposure of humans to this compound. However, the weak in vivo and in vitro genetic toxicity of NP at high dose levels in mammalian systems suggests that the potential hazard may not be as high as predicted from bacterial mutagenicity data.

Dose-dependent disposition of ethylene glycol in the rat after intravenous administration.

A dose-dependent change was observed in the disposition of 14C-labeled ethylene glycol (EG) after iv administration of 20, 200, 1000, and 2000 mg/kg to Fischer 344 rats. The part of the dose expired as CO2 decreased from 39% at 20 and 200 mg/kg to 26% at 1000 and 2000 mg/kg, while urinary excretion of radiocarbon increased from 35 to 56%. The increase in urinary 14C was almost entirely attributable to [14C] glycolate, which comprised 20% of the dose in 24 h at the two higher dose levels and only 2% at the lower doses. High doses of EG limited the processes responsible for glycolate metabolism, supporting the idea that this acid is a major contributing factor to the acute toxicity of EG. Compensatory urinary excretion of glycolate resulted in minimal dose-dependent effects on 14C blood clearance. Blood clearance of 14C occurred in an initial rapid phase (half-life, 3-5 h), when plasma was comprised predominantly of ethylene glycol, that persisted for 12 h at 20 mg/kg EG and 30 h at 2000 mg/kg. The dose-dependent profile of EG metabolism argues against the use of very high chronic doses in studies intended to estimate health risks of long-term, low-level exposure to EG.

Ocular toxicity of 2,2,2-trifluoroethanol in rabbits.

Three commercial formulations of 2,2,2-trifluoroethanol (TFE) were applied to eyes of rabbits and evaluated for their potential to cause ocular damage. All three products were severe eye irritants and water irrigation shortly after exposure was not effective in reducing ocular damage. Gross and histological observations revealed marked conjunctivitis, moderate to severe ulcerative keratitis, and hyperemia and edema of the iris, all of which showed evidence of resolution over the three-week course of investigation. However, complete reversal of all effects was not attained. TFE and its commercial formulations should be viewed as potentially severe human ocular irritants.

Thermal sensitive paper as a diagnostic for intense relativistic electron beam dynamics.

Thermal sensitive paper has been used as a diagnostic for an intense relativistic electron beam propagating in a rippled magnetic field. The E(r)xB(z) rotation of the beam has been measured from the exposed pattern on the thermal paper and used to calculate the electrostatic field of the beam E(r), and the corresponding values of electron density and beam current. Exposed strips of thermal paper show longitudinal modulation of the radial electron velocity with a period corresponding to that of a rippled magnetic field; modulation of the radial electron velocity at the cyclotron frequency has also been observed.