Laboratory maintenance does not alter ecological and physiological patterns among species: a Drosophila case study.

Large comparative studies in animal ecology, physiology and evolution often use animals reared in the laboratory for many generations; however, the relevance of these studies hinges on the assumption that laboratory populations are still representative for their wild living conspecifics. In this study, we investigate whether laboratory-maintained and freshly collected animal populations are fundamentally different and whether data from laboratory-maintained animals are valid to use in large comparative investigations of ecological and physiological patterns. Here, we obtained nine species of Drosophila with paired populations of laboratory-maintained and freshly collected flies. These species, representing a range of ecotypes, were assayed for four stress-tolerance, two body-size traits and six life-history traits. For all of these traits, we observed small differences in species-specific comparisons between field and laboratory populations; however, these differences were unsystematic and laboratory maintenance did not eclipse fundamental species characteristics. To investigate whether laboratory maintenance influence the general patterns in comparative studies, we correlated stress tolerance and life-history traits with environmental traits for the laboratory-maintained and freshly collected populations. Based on this analysis, we found that the comparative physiological and ecological trait correlations are similar irrespective of provenience. This finding is important for comparative biology in general because it validates comparative meta-analyses based on laboratory-maintained populations.

Inbreeding depression across a nutritional stress continuum.

Many natural populations experience inbreeding and genetic drift as a consequence of nonrandom mating or low population size. Furthermore, they face environmental challenges that may interact synergistically with deleterious consequences of increased homozygosity and further decrease fitness. Most studies on inbreeding-environment (I-E) interactions use one or two stress levels, whereby the resolution of the possible stress and inbreeding depression interaction is low. Here we produced Drosophila melanogaster replicate populations, maintained at three different population sizes (10, 50 and a control size of 500) for 25 generations. A nutritional stress gradient was imposed on the replicate populations by exposing them to 11 different concentrations of yeast in the developmental medium. We assessed the consequences of nutritional stress by scoring egg-to-adult viability and body mass of emerged flies. We found: (1) unequivocal evidence for I-E interactions in egg-to-adult viability and to a lesser extent in dry body mass, with inbreeding depression being more severe under higher levels of nutritional stress; (2) a steeper increase in inbreeding depression for replicate populations of size 10 with increasing nutritional stress than for replicate populations of size 50; (3) a nonlinear norm of reaction between inbreeding depression and nutritional stress; and (4) a faster increase in number of lethal equivalents in replicate populations of size 10 compared with replicate populations of size 50 with increasing nutritional stress levels. Our data provide novel and strong evidence that deleterious fitness consequences of I-E interactions are more pronounced at higher nutritional stress and at higher inbreeding levels.

A Drosophila laboratory evolution experiment points to low evolutionary potential under increased temperatures likely to be experienced in the future.

The ability to respond evolutionarily to increasing temperatures is important for survival of ectotherms in a changing climate. Recent studies suggest that upper thermal limits may be evolutionary constrained. We address this hypothesis in a laboratory evolution experiment, encompassing ecologically relevant thermal regimes. To examine the potential for species to respond to climate change, we exposed replicate populations of Drosophila melanogaster to increasing temperatures (0.3 °C every generation) for 20 generations, whereas corresponding replicate control populations were held at benign thermal conditions throughout the experiment. We hypothesized that replicate populations exposed to increasing temperatures would show increased resistance to warm and dry environments compared with replicate control populations. Contrasting replicate populations held at the two thermal regimes showed (i) an increase in desiccation resistance and a decline in heat knock-down resistance in replicate populations exposed to increasing temperatures, (ii) similar egg-to-adult viability and fecundity in replicate populations from the two thermal regimes, when assessed at high stressful temperatures and (iii) no difference in nucleotide diversity between thermal regimes. The limited scope for adaptive evolutionary responses shown in this study highlights the challenges faced by ectotherms under climate change.

Genetic characterization of a herd of the endangered Danish Jutland cattle.

In this paper we present results from a genetic characterization of a herd of the Danish Jutland cattle breed named the Kortegaard herd (n = 135; 57 males and 78 females). The herd is genotyped on the Bovine HD BeadChip microarray with 697,548 evenly spaced SNP across the bovine genome. The aim of the study was to characterize the genetic profile of the Kortegaard herd, which has been closed for several generations, by quantifying the degree of genetic homogeneity within the herd and to compare its genetic profile to that of other cattle breeds. A total of 868 animals from the Angus, Belgian Blue, Charolais, Friesian, Hereford, Holstein, Holstein-Friesian crosses, Limousin, and Simmental breeds was used for genetic profile comparisons. The level of genetic variation within the breeds were quantified by the expected heterozygosity (H(E)), observed heterozygosity (H(O)), average minor allele frequency (MAF), the degree of polymorphism, and runs of homozygosity (ROH), which are contiguous lengths of homozygous genotypes of varying length. Interestingly, the Kortegaard herd had the lowest within-breed genetic variation (lowest H(E), H(O), and MAF), showed moderate levels of short ROH (<5 Mb), and had the highest mean long ROH (>5 Mb) compared to all the other breeds. This is possibly due to recent consanguineous matings, a strong founder effect, and a lack of gene flow from other herds and breeds. We further examined whether the observed genetic patterns in the Kortegaard herd can be used to design breeding strategies for the preservation of the genetic pool by focusing on a subset of SNP outside homozygote regions. By calculating the pairwise identical-by-state between all possible matings, we designed a breeding plan that maximized heterozygosity in the short term. The benefits and limitations of such a breeding strategy are discussed.

Characterization of the genetic profile of five Danish dog breeds.

This investigation presents results from a genetic characterization of 5 Danish dog breeds genotyped on the CanineHD BeadChip microarray with 170,000 SNP. The breeds investigated were 1) Danish Spitz (DS; n=8), 2) Danish-Swedish Farm Dog (DSF; n=18), 3) Broholmer (BR; n=22), 4) Old Danish Pointing Dog (ODP; n=24), and 5) Greenland Dog (GD; n=23). The aims of the investigation were to characterize the genetic profile of the abovementioned dog breeds by quantifying the genetic differentiation among them and the degree of genetic homogeneity within breeds. The genetic profile was determined by means of principal component analysis (PCA) and through a Bayesian clustering method. Both the PCA and the Bayesian clustering method revealed a clear genetic separation of the 5 breeds. The level of genetic variation within the breeds varied. The expected heterozygosity (HE) as well as the degree of polymorphism (P%) ranked the dog breeds in the order DS>DSF>BR>ODP>GD. Interestingly, the breed with a tenfold higher census population size compared to the other breeds, the Greenland Dog, had the lowest within-breed genetic variation, emphasizing that census size is a poor predictor of genetic variation. The observed differences in variation among and within dog breeds may be related to factors such as genetic drift, founder effects, genetic admixture, and population bottlenecks. We further examined whether the observed genetic patterns in the 5 dog breeds can be used to design breeding strategies for the preservation of the genetic pool of these dog breeds.

Humidity affects genetic architecture of heat resistance in Drosophila melanogaster.

Laboratory experiments on Drosophila have often demonstrated increased heritability for morphological and life-history traits under environmental stress. We used parent-offspring comparisons to examine the impact of humidity levels on the heritability of a physiological trait, resistance to heat, measured as knockdown time at constant temperature. Drosophila melanogaster were reared under standard nonstressful conditions and heat-shocked as adults at extreme high or low humidity. Mean knockdown time was decreased in the stressful dry environment, but there was a significant sex-by-treatment interaction: at low humidity, females were more heat resistant than males, whereas at high humidity, the situation was reversed. Phenotypic variability of knockdown time was also lower in the dry environment. The magnitude of genetic correlation between the sexes at high humidity indicated genetic variation for sexual dimorphism in heat resistance. Heritability estimates based on one-parent-offspring regressions tended to be higher under desiccation stress, and this could be explained by decreased environmental variance of heat resistance at low humidity. There was no indication that the additive genetic variance and evolvability of heat resistance differed between the environments. The pattern of heritability estimates suggests that populations of D. melanogaster may have a greater potential for evolving higher thermal tolerance under arid conditions.

Constant, cycling, hot and cold thermal environments: strong effects on mean viability but not on genetic estimates.

It has frequently been suggested that trait heritabilities are environmentally sensitive, and there are genetic trade-offs between tolerating different environments such as hot and cold or constant and fluctuating temperatures. Future climate predictions suggest an increase in both temperatures and their fluctuations. How species will respond to these changes is uncertain, particularly as there is a lack of studies which compare genetic performances in constant vs. fluctuating environments. In this study, we used a nested full-sib/half-sib breeding design to examine how the genetic variances and heritabilities of egg-to-adult viability differ at high and low temperatures with and without daily fluctuations in temperatures using Drosophila melanogaster as a model organism. Although egg-to-adult viability was clearly sensitive to developmental temperatures, heritabilities were not particularly sensitive to developmental temperatures. Moreover, we found that egg-to-adult viabilities at different developmental temperatures were positively correlated, suggesting a common genetic background for egg-to-adult viability at different temperatures. Finding both a uniform genetic background coupled with rather low heritabilities insensitive to temperatures, our results suggest evolutionary responses are unlikely to be limited by temperature effects on genetic parameters or negative genetic correlations, but by the direct effects of stressful temperatures on egg-to-adult viability accompanied with low heritabilities.

Can evolution of sexual dimorphism be triggered by developmental temperatures?

Genetic prerequisites for the evolution of sexual dimorphism, sex-specific heritabilities and low or negative genetic correlations between homologous traits in males and females are rarely found. However, sexual dimorphism is evolving rapidly following environmental change, suggesting that sexual dimorphism and its genetic background could be environmentally sensitive. Yet few studies have explored the sensitivity of the genetic background of sexual dimorphism on environmental variation. In this study, on Drosophila melanogaster, we used a large nested full-sib-half-sib breeding design where families were split into four different developmental temperatures: two constant temperature treatments of 25 and 30 °C and two cycling temperatures with means of 25 and 30 °C, respectively. After emergence, we tested heat shock tolerance of adult flies. We found that sexual dimorphism was strongly affected by temperature during development. Moreover, we found that female heritability was significantly lower in flies developing at hot temperature and more so under hot and cycling temperatures. Interestingly, most of the genetic variation for heat shock tolerance was orthogonal (i.e. noncorrelated) between sexes, allowing independent evolution of heat shock tolerance in males and females. These findings give support to the hypothesis that the evolution of sexual dimorphism can be influenced by the environments experienced during development.

A comprehensive assessment of geographic variation in heat tolerance and hardening capacity in populations of Drosophila melanogaster from eastern Australia.

We examined latitudinal variation in adult and larval heat tolerance in Drosophila melanogaster from eastern Australia. Adults were assessed using static and ramping assays. Basal and hardened static heat knockdown time showed significant linear clines; heat tolerance increased towards the tropics, particularly for hardened flies, suggesting that tropical populations have a greater hardening response. A similar pattern was evident for ramping heat knockdown time at 0.06°C min(-1) increase. There was no cline for ramping heat knockdown temperature (CT(max) ) at 0.1°C min(-1) increase. Acute (static) heat knockdown temperature increased towards temperate latitudes, probably reflecting a greater capacity of temperate flies to withstand sudden temperature increases during summer in temperate Australia. Larval viability showed a quadratic association with latitude under heat stress. Thus, patterns of heat resistance depend on assay methods. Genetic correlations in thermotolerance across life stages and evolutionary potential for critical thermal limits should be the focus of future studies.

Effectiveness of microsatellite and SNP markers for parentage and identity analysis in species with low genetic diversity: the case of European bison.

The European bison (Bison bonasus) has recovered successfully after a severe bottleneck about 90 years ago but has been left with low genetic variability that may substantially hinder parentage and identity analysis. According to pedigree analysis, over 80% of the genes in the contemporary population descend from just two founder animals and inbreeding coefficients averaged almost 0.5, whereas microsatellite heterozygosity does not exceed 0.3. We present a comparison of the effectiveness of 17 microsatellite and 960 single nucleotide polymorphism (SNP) markers for paternity and identity analysis in the European bison. Microsatellite-based paternity and identity analysis was unsuccessful because of low marker heterozygosity and is not a practical approach in this species. Simulations using SNP markers suggest that 80-90 randomly selected loci, or just 50-60 of the most heterozygous loci, would be sufficient to ensure successful paternity and identity analysis in this species. For the purpose of standardizing future analysis, a panel of 50-60 bovine SNPs characterized by high heterozygosity and an even distribution in the genome could be selected. This panel of markers could be typed using VeraCode (Illumina) or similar SNP genotyping systems. The low cost of these SNP genotyping methods compared with a 16 locus microsatellite survey means that off-the-shelf SNP genotyping systems developed for domestic species represent powerful tools for genetic analysis in related species, and can be effective even in bottlenecked species in which heterozygosity of other markers such as microsatellites may be very low.

Sex specific effects of heat induced hormesis in Hsf-deficient Drosophila melanogaster.

In insects mild heat stress early in life has been reported to increase life span and heat resistance later in life, a phenomenon termed hormesis. Here, we test if the induction of the heat shock response by mild heat stress is mediating hormesis in longevity and heat resistance at older age. To test this hypothesis we used two heat shock transcription factor (Hsf) mutant stocks. One stock harbours a mutation giving rise to a heat sensitive Hsf which inactivates the heat shock response at high temperature and the other is a rescued mutant giving rise to a wild-type phenotype. We measured longevity, heat resistance and expression level of a heat shock protein, Hsp70, in controls and mildly heat treated flies. We found a marked difference between males and females with males showing a beneficial effect of the early heat treatment on longevity and heat resistance later in life in the rescued line, seemingly mediated by the production of heat shock proteins (Hsps). The results indicate that heat inducible Hsps are important for heat induced hormesis in longevity and heat stress resistance. However, the results also suggest that other processes are involved and that different mechanisms might have marked sex specific impact.

Developmental instability as an estimator of genetic stress.

To set conservation priorities, scientists should be able to assess the relative threats posed by the effects of loss of genetic variability, inbreeding and outbreeding as these can generate 'genetic stress'. Developmental instability (DI) has been suggested as an indicator of stress, possibly being more sensitive than other measures. However, there is controversy as to whether DI is an accurate and reliable tool for assessing the degree of genetic stress. After 50 years of the presentation of Lerner's conjecture, there are still several unresolved questions about the relationship between DI and genetic stress. Here, we review studies on mechanisms behind DI. The current status on the use of DI as an indicator of genetic stress is discussed, and suggestions are presented on how to obtain more knowledge on the potential of DI in an evolutionary context.

Effects of inbreeding and rate of inbreeding in Drosophila melanogaster- Hsp70 expression and fitness.

Induction of heat shock proteins (Hsp) is a well-known mechanism through which cells cope with stressful conditions. Hsp are induced by a variety of extrinsic stressors. However, recently intrinsic stressors (aging and inbreeding) have been shown to affect expression of Hsp. Increased homozygosity due to inbreeding may disrupt cellular homeostasis by causing increased expression of recessive deleterious mutations and breakdown of epistatic interactions. We investigated the effect of inbreeding and the rate of inbreeding on the expression of Hsp70, larval heat resistance and fecundity. In Drosophila melanogaster we found that inbred lines (F approximately 0.67) had significantly up-regulated expression of Hsp70, and reduced heat resistance and fecundity as compared with outbred control lines. A significant negative correlation was observed between Hsp70 expression and resistance to an extreme heat stress in inbred lines. We interpreted this as an increased requirement for Hsp70 in the lines suffering most from inbreeding depression. Inbreeding depression for fecundity was reduced with a slower rate of inbreeding compared with a fast rate of inbreeding. Thus, the effectiveness of purging seems to be improved with a slower rate of inbreeding.

A test of quantitative genetic theory using Drosophila- effects of inbreeding and rate of inbreeding on heritabilities and variance components.

Inbreeding is expected to decrease the heritability within populations. However, results from empirical studies are inconclusive. In this study, we investigated the effects of three breeding treatments (fast and slow rate of inbreeding - inbred to the same absolute level - and a control) on heritability, phenotypic, genetic and environmental variances of sternopleural bristle number in Drosophila melanogaster. Heritability, and phenotypic, genetic and environmental variances were estimated in 10 replicate lines within each of the three treatments. Standard least squares regression models and Bayesian methods were used to analyse the data. Heritability and additive genetic variance within lines were higher in the control compared with both inbreeding treatments. Heritabilities and additive genetic variances within lines were higher in slow compared with fast inbred lines, indicating that slow inbred lines retain more evolutionary potential despite the same expected absolute level of inbreeding. The between line variance was larger with inbreeding and more than twice as large in the fast than in the slow inbred lines. The different pattern of redistribution of genetic variance within and between lines in the two inbred treatments cannot be explained invoking the standard model based on selective neutrality and additive gene action. Environmental variances were higher with inbreeding, and more so with fast inbreeding, indicating that inbreeding and the rate of inbreeding affect environmental sensitivity. The phenotypic variance decreased with inbreeding, but was not affected by the rate of inbreeding. No inbreeding depression for mean sternopleural bristle number was observed in this study. Considerable variance between lines in additive genetic variance within lines was observed, illustrating between line variation in evolutionary potential.

Sustained-release Madopar HBS compared with standard Madopar in the long-term treatment of de novo parkinsonian patients.

In this Danish-Norwegian randomized double-blind parallel-group multicentre study, we compared the therapeutic response of slow-release Madopar HBS to standard Madopar in 134 de novo patients with idiopathic Parkinson's disease during a 5-year period. The drugs were dosed according to the individual need of the patients. The Webster, NUDS, UPDRS and Hoehn & Yahr scales were used for evaluation of symptoms. Addition of a morning dose of standard Madopar 62.5 mg was allowed after 6 months. Bromocriptine could be administered but not Selegiline. Sixty-five patients got Madopar HBS and 69 standard Madopar. Surprisingly, no differences were found as to the mean daily levodopa dose, the mean number of daily doses or the use of the doses of bromocriptine. Unexpectedly, we found a trend towards a more frequent use of a morning dose of standard Madopar in the group treated with the standard formulation. No differences were observed in the occurrence of motor fluctuations or dyskinesia, the incidence of which was relatively low. Sustained-release Madopar (HBS) thus proved to be as effective as standard Madopar in the long-term treatment of de novo parkinsonian patients, but the drug showed no advantage in postponing or reducing the long-term levodopa treatment problems.

Prophylactic treatment of classical and non-classical migraine with metoprolol--a comparison with placebo.

A double-blind investigation with parallel groups was carried out in three Danish neurological clinics to evaluate the effect of metoprolol (Beloc, Betaloc, Seloken) versus placebo in migraine patients. 71 patients were included; 62 completed the study. The following parameters were used in the evaluation: frequency of headache attacks, days with migraine, severity score (days X intensity), and the consumption of pain-relieving tablets. The results of the study show that metoprolol 200 mg in Durules (a controlled release formulation) once daily is more effective regarding all evaluated parameters than placebo and that metoprolol is well tolerated.