Association between the skin microbiome and MHC class II diversity in an amphibian.

Microbiomes play an important role in determining the ecology and behaviour of their hosts. However, questions remain pertaining to how host genetics shape microbiomes, and how microbiome composition influences host fitness. We explored the effects of geography, evolutionary history and host genetics on the skin microbiome diversity and structure in a widespread amphibian. More specifically, we examined the association between bacterial diversity and composition and the major histocompatibility complex class II exon 2 diversity in 12 moor frog (Rana arvalis) populations belonging to two geographical clusters that show signatures of past and ongoing differential selection. We found that while bacterial alpha diversity did not differ between the two clusters, MHC alleles/supertypes and genetic diversity varied considerably depending on geography and evolutionary history. Bacterial alpha diversity was positively correlated with expected MHC heterozygosity and negatively with MHC nucleotide diversity. Furthermore, bacterial community composition showed significant variation between the two geographical clusters and between specific MHC alleles/supertypes. Our findings emphasize the importance of historical demographic events on hologenomic variation and provide new insights into how immunogenetic host variability and microbial diversity may jointly influence host fitness with consequences for disease susceptibility and population persistence.

Signatures of historical selection on MHC reveal different selection patterns in the moor frog (Rana arvalis).

MHC genes are key components in disease resistance and an excellent system for studying selection acting on genetic variation in natural populations. Current patterns of variation in MHC genes are likely to be influenced by past and ongoing selection as well as demographic fluctuations in population size such as those imposed by post-glacial recolonization processes. Here, we investigated signatures of historical selection and demography on an MHC class II gene in 12 moor frog populations along a 1700-km latitudinal gradient. Sequences were obtained from 207 individuals and consecutively assigned into two different clusters (northern and southern clusters, respectively) in concordance with a previously described dual post-glacial colonization route. Selection analyses comparing the relative rates of non-synonymous to synonymous substitutions (dN/dS) suggested evidence of different selection patterns in the northern and the southern clusters, with divergent selection prevailing in the south but uniform positive selection predominating in the north. Also, models of codon evolution revealed considerable differences in the strength of selection: The southern cluster appeared to be under strong selection while the northern cluster showed moderate signs of selection. Our results indicate that the MHC alleles in the north diverged from southern MHC alleles as a result of differential selection patterns.

Warm vegetarians? Heat waves and diet shifts in tadpoles.

Temperature can play an important role in determining the feeding preferences of ectotherms. In light of the warmer temperatures arising with the current climatic changes, omnivorous ectotherms may perform diet shifts toward higher herbivory to optimize energetic intake. Such diet shifts may also occur during heat waves, which are projected to become more frequent, intense, and longer lasting in the future. Here, we investigated how heat waves of different duration affect feeding preferences in omnivorous anuran tadpoles and how these choices affect larval life history. In laboratory experiments, we fed tadpoles of three species on animal, plant, or mixed diet and exposed them to short heat waves (similar to the heat waves these species experience currently) or long heat waves (predicted to increase under climate change). We estimated the dietary choices of tadpoles fed on the mixed diet using stable isotopes and recorded tadpole survival and growth, larval period, and mass at metamorphosis. Tadpole feeding preferences were associated with their thermal background, with herbivory increasing with breeding temperature in nature. Patterns in survival, growth, and development generally support decreased efficiency of carnivorous diets and increased efficiency or higher relative quality of herbivorous diets at higher temperatures. All three species increased herbivory in at least one of the heat wave treatments, but the responses varied among species. Diet shifts toward higher herbivory were maladaptive in one species, but beneficial in the other two. Higher herbivory in omnivorous ectotherms under warmer temperatures may impact species differently and further contribute to changes in the structure and function of freshwater environments.

Divergence of gastropod life history in contrasting thermal environments in a geothermal lake.

Experiments using natural populations have provided mixed support for thermal adaptation models, probably because the conditions are often confounded with additional environmental factors like seasonality. The contrasting geothermal environments within Lake Mývatn, northern Iceland, provide a unique opportunity to evaluate thermal adaptation models using closely located natural populations. We conducted laboratory common garden and field reciprocal transplant experiments to investigate how thermal origin influences the life history of Radix balthica snails originating from stable cold (6 °C), stable warm (23 °C) thermal environments or from areas with seasonal temperature variation. Supporting thermal optimality models, warm-origin snails survived poorly at 6 °C in the common garden experiment and better than cold-origin and seasonal-origin snails in the warm habitat in the reciprocal transplant experiment. Contrary to thermal adaptation models, growth rate in both experiments was highest in the warm populations irrespective of temperature, indicating cogradient variation. The optimal temperatures for growth and reproduction were similar irrespective of origin, but cold-origin snails always had the lowest performance, and seasonal-origin snails often performed at an intermediate level compared to snails originating in either stable environment. Our results indicate that central life-history traits can differ in their mode of evolution, with survival following the predictions of thermal optimality models, whereas ecological constraints have shaped the evolution of growth rates in local populations.

Genetic divergence and isolation by thermal environment in geothermal populations of an aquatic invertebrate.

Temperature is one of the most influential forces of natural selection impacting all biological levels. In the face of increasing global temperatures, studies over small geographic scales allowing investigations on the effects of gene flow are of great value for understanding thermal adaptation. Here, we investigated genetic population structure in the freshwater gastropod Radix balthica originating from contrasting thermal habitats in three areas of geothermal activity in Iceland. Snails from 32 sites were genotyped at 208 AFLP loci. Five AFLPs were identified as putatively under divergent selection in Lake Mývatn, a geothermal lake with an almost 20 °C difference in mean temperature across a distance of a few kilometres. In four of these loci, variation across all study populations was correlated with temperature. We found significant population structure in neutral markers both within and between the areas. Cluster analysis using neutral markers classified the sites mainly by geography, whereas analyses using markers under selection differentiated the sites based on temperature. Isolation by distance was stronger in the neutral than in the outlier loci. Pairwise differences based on outlier FST were significantly correlated with temperature at different spatial scales, even after correcting for geographic distance or neutral pairwise FST differences. In general, genetic variation decreased with increasing environmental temperature, possibly suggesting that natural selection had reduced the genetic diversity in the warm origin sites. Our results emphasize the influence of environmental temperature on the genetic structure of populations and suggest local thermal adaptation in these geothermal habitats.

Juvenile idiopathic arthritis patients with low inflammatory activity have increased adiposity.

OBJECTIVES: The aim of this study was to assess the effect of juvenile idiopathic arthritis (JIA), its subtypes and disease activity on anthropometric measurements, body composition, and nutritional parameters. METHOD: A cross-sectional cohort of 40 JIA patients, aged 3-10 years, was compared with 40 healthy children matched for age and gender. Concentrations of nutritional and inflammatory biomarkers in the blood, anthropometric measures, and clinical status were recorded and the parents filled in a 7-day food diary and completed the Childhood Health Assessment Questionnaire (CHAQ). RESULTS: The JIA patients had low disease activity: 60% had inactive disease, the median CHAQ score was 0.125, and the median erythrocyte sedimentation rate (ESR) was 6 mm/h. Significantly higher values for central and peripheral adiposity were found in JIA patients compared with in healthy controls [waist circumference mean (SD) 55.9 (4.9) vs. 53.4 (3.7) cm, p < 0.0001, and biceps skinfold thickness 6.2 (2.3) vs. 5.3 (1.7) cm, p = 0.035, respectively], and obesity/overweight was more common (30% vs. 12.5%, p = 0.056, respectively) despite no differences in weight-for-height. The intake of energy (kcal/day) was significantly higher in the JIA patients (p = 0.036). The nutritional biomarkers were comparable in both groups. The JIA subtype and disease activity did not affect body composition, energy intake, or the nutritional biomarkers. CONCLUSIONS: Even JIA patients with low disease activity have a higher central and peripheral adiposity and a higher energy intake than their healthy peers. Neither disease subtype nor disease activity had any association with changes in body composition.

The quantitative genetic basis of adaptive divergence in the moor frog (Rana arvalis) and its implications for gene flow.

Knowledge on the relative contribution of direct genetic, maternal and environmental effects to adaptive divergence is important for understanding the drivers of biological diversification. The moor frog (Rana arvalis) shows adaptive divergence in embryonic and larval fitness traits along an acidification gradient in south-western Sweden. To understand the quantitative genetic basis of this divergence, we performed reciprocal crosses between three divergent population pairs and reared embryos and larvae at acid and neutral pH in the laboratory. Divergence in embryonic acid tolerance (survival) was mainly determined by maternal effects, whereas the relative contributions of maternal, additive and nonadditive genetic effects in larval life-history traits differed between traits, population pairs and rearing environments. These results emphasize the need to investigate the quantitative genetic basis of adaptive divergence in multiple populations and traits, as well as different environments. We discuss the implications of our findings for maintenance of local adaptation in the context of migrant and hybrid fitness.

Geographic variation in corticosterone response to chronic predator stress in tadpoles.

Chronic stress often affects growth and development negatively, and these effects are often mediated via glucocorticoid hormones, which elevate during stress. We investigated latitudinal variation in corticosterone (CORT) response to chronic predator stress in Rana temporaria tadpoles along a 1500-km latitudinal cline in Sweden tadpoles, in a laboratory experiment. We hypothesized that more time-constrained high-latitude populations have evolved a lower CORT response to chronic stress to maintain higher growth under stressful conditions. Southern tadpoles had higher CORT content in response to predators after 1 day of exposure, whereas there was no increase in CORT in the northern populations. Two weeks later, there were no predator-induced CORT elevations. Artificially elevated CORT levels strongly decreased growth, development and survival in both northern and southern tadpoles. We suggest that the lower CORT response in high-latitude populations can be connected with avoidance of CORT-mediated reduction in growth and development, but also discuss other possible explanations.

Costs and benefits of defences induced by predators differing in dangerousness.

While theoretical studies predict that inducible defences should be fine-tuned according to the qualities of the predator, very few studies have investigated how dangerousness of predators, i.e. the rate at which predators kill prey individuals, affects the strength of phenotypic responses and resulting benefits and costs of induced defences. We performed a comprehensive study on fitness consequences of predator-induced responses by involving four predators (leech, water scorpion, dragonfly larva and newt), evaluating costs and benefits of responses, testing differences in dangerousness between predators and measuring responses in several life history traits of prey. We raised Rana dalmatina tadpoles in the presence of free-ranging predators, in the presence of caged predators, and exposed naive and experienced tadpoles to free-ranging predators. Tadpoles adjusted the intensities of their behavioural and morphological defences to predator dangerousness. Survival was lower in the nonlethal presence of the most dangerous predator, while we could not detect costs of induced defences at or after metamorphosis. When exposed to free-ranging predators, small, but not large, tadpoles benefited from exhibiting an induced phenotype in terms of elevated survival when compared to naive tadpoles, but we did not observe higher survival either in tadpoles exhibiting more extreme phenotypes or in tadpoles exposed to the type of predator they were raised with. These results indicate that while predator-induced defences can mirror dangerousness of predators, costs and benefits do not necessarily scale to the magnitude of plastic responses.

Brain plasticity over the metamorphic boundary: carry-over effect of larval environment on froglet brain development.

Brain development shows high plasticity in response to environmental heterogeneity. However, it is unknown how environmental variation during development may affect brain architecture across life history switch points in species with complex life cycles. Previously, we showed that predation and competition affect brain development in common frog (Rana temporaria) tadpoles. Here, we studied whether larval environment had carry-over effects in brains of metamorphs. Tadpoles grown at high density had large optic tecta at metamorphosis, whereas tadpoles grown under predation risk had small diencephala. We found that larval density had a carry-over effect on froglet optic tectum size, whereas the effect of larval predation risk had vanished by metamorphosis. We discuss the possibility that the observed changes may be adaptive, reflecting the needs of an organism in given environmental and developmental contexts.

Allen's rule revisited: quantitative genetics of extremity length in the common frog along a latitudinal gradient.

Ecogeographical rules linking climate to morphology have gained renewed interest because of climate change. Yet few studies have evaluated to what extent geographical trends ascribed to these rules have a genetic, rather than environmentally determined, basis. This applies especially to Allen's rule, which states that the relative extremity length decreases with increasing latitude. We studied leg length in the common frog (Rana temporaria) along a 1500 km latitudinal gradient utilizing wild and common garden data. In the wild, the body size-corrected femur and tibia lengths did not conform to Allen's rule but peaked at mid-latitudes. However, the ratio of femur to tibia length increased in the north, and the common garden data revealed a genetic cline consistent with Allen's rule in some trait and treatment combinations. While selection may have shortened the leg length in the north, the genetic trend seems to be partially masked by environmental effects.

First-generation linkage map for the common frog Rana temporaria reveals sex-linkage group.

The common frog (Rana temporaria) has become a model species in the fields of ecology and evolutionary biology. However, lack of genomic resources has been limiting utility of this species for detailed evolutionary genetic studies. Using a set of 107 informative microsatellite markers genotyped in a large full-sib family (800 F1 offspring), we created the first linkage map for this species. This partial map-distributed over 15 linkage groups-has a total length of 1698.8 cM. In line with the fact that males are the heterogametic sex in this species and a reduction of recombination is expected, we observed a lower recombination rate in the males (map length: 1371.5 cM) as compared with females (2089.8 cM). Furthermore, three loci previously documented to be sex-linked (that is, carrying male-specific alleles) in adults from the wild mapped to the same linkage group. The linkage map described in this study is one of the densest ones available for amphibians. The discovery of a sex linkage group in Rana temporaria, as well as other regions with strongly reduced male recombination rates, should help to uncover the genetic underpinnings of the sex-determination system in this species. As the number of linkage groups found (n=15) is quite close to the actual number of chromosomes (n=13), the map should provide a useful resource for further evolutionary, ecological and conservation genetic work in this and other closely related species.

Predation- and competition-mediated brain plasticity in Rana temporaria tadpoles.

An increasing number of studies have demonstrated phenotypic plasticity in brain size and architecture in response to environmental variation. However, our knowledge on how brain architecture is affected by commonplace ecological interactions is rudimentary. For example, while intraspecific competition and risk of predation are known to induce adaptive plastic modifications in morphology and behaviour in a wide variety of organisms, their effects on brain development have not been studied. We studied experimentally the influence of density and predation risk on brain development in common frog (Rana temporaria) tadpoles. Tadpoles grown at low density and under predation risk developed smaller brains than tadpoles at the other treatment combinations. Further, at high densities, tadpoles developed larger optic tecta and smaller medulla oblongata than those grown at low densities. These results demonstrate that ecological interactions - like intraspecific competition and predation risk - can have strong effects on brain development in lower vertebrates.

The interaction of multiple environmental stressors affects adaptation to a novel habitat in the natterjack toad Bufo calamita.

The potential to adapt to novel environmental conditions is a key area of interest for evolutionary biology. However, the role of multiple selection pressures on adaptive responses has rarely been investigated in natural populations. In Sweden, the natterjack toad Bufo calamita inhabits two separate distribution areas, one in southernmost Sweden and one on the west coast. We characterized the larval habitat in terms of pond size and salinity in the two areas, and found that the western populations are more affected by both desiccation risk and pond salinity than the southern populations. In a common garden experiment manipulating salinity and temperature, we found that toads from the west coast populations were locally adapted to shorter pond duration as indicated by their higher development and growth rates. However, despite being subjected to higher salinity stress in nature, west coast toads had a poorer performance in saline treatments. We found that survival in the saline treatments in the west coast populations was positively affected by larger body mass and longer larval period. Furthermore, we found negative genetic correlations between body mass and growth rate and their plastic responses to salinity. These results implicate that the occurrence of multiple environmental stressors needs to be accounted for when assessing the adaptive potential of organisms and suggest that genetic correlations may play a role in constraining adaptation of natural populations.

Short-term dominance: stability and consequences for subsequent growth.

Dominance status was determined among groups of four fish by using individuals from eight brown trout Salmo trutta populations. Subsequent growth of the fish was later recorded in larger groups. Seven months after the first set of trials, an additional set of dominance trials was performed by using the same fish. Social status affected subsequent growth; individuals having the lowest ranks grew less when compared to the higher ranking fish. Furthermore, the short term dominance hierarchy was rather stable between the two trials. This was especially the case with the lowest ranking fish, which tended to remain in the lowest position also in the second trial. The results suggest that the short term dominance trials done among few conspecifics reflect relatively well not only the subordinates' relative but also absolute social status.

A randomized, controlled trial of tonsillectomy in periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome.

OBJECTIVE: We carried out a prospective, randomized, controlled trial to clarify the effect of tonsillectomy on the clinical course of periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome. STUDY DESIGN: Twenty-six consecutive children (mean age 4.1 years) with at least 5 PFAPA attacks were recruited from 3 tertiary care pediatric hospitals during 1999-2003 and randomly allocated to tonsillectomy or follow-up alone. They were all followed up with symptom diaries for 12 months. Tonsillectomy was allowed after 6 months in the control group if the attacks recurred. RESULTS: Six months after randomization all 14 children in the tonsillectomy group and 6/12 children in the control group (50%) were free of symptoms (difference 50%, 95% confidence interval 23% to 75%, P < .001). Tonsillectomy was performed on 5/6 of the patients in the control group who still had symptoms after 6 months. The remaining unoperated child in the control group had recurrences of the fever episodes throughout the follow-up, but the symptoms became less severe, and the parents did not choose tonsillectomy. CONCLUSION: Tonsillectomy appeared to be effective for treating PFAPA syndrome. The fever episodes ceased without any intervention in half of the control subjects. We conclude that although the mechanisms behind this syndrome are unknown, tonsillectomy can be offered as an effective intervention for children with PFAPA.

T-cell responses to enterovirus antigens in children with type 1 diabetes.

Enterovirus infections, implicated in the pathogenesis of type 1 diabetes in a number of studies, may precipitate the symptoms of clinical diabetes and play a role in the initiation of the beta-cell damaging process. The aim of this study was to evaluate whether cellular immune responses to enterovirus antigens are abnormal in children with type 1 diabetes. Lymphocyte proliferation responses to enterovirus antigens were analyzed in 41 children with new-onset type 1 diabetes, 23 children with type 1 diabetes for 4-72 months, and healthy control children in subgroups matched for HLA-DQB1 risk alleles, sex, and age. Children with diabetes for 4-72 months more often had T-cell responses to the Coxsackievirus B4-infected cell lysate antigen than children with new-onset diabetes (P < 0.01) or control children (P < 0.01). Responses to recombinant nonstructural protein 2C of Coxsackievirus B4 were also more frequent in children with type 1 diabetes for 4-72 months when compared with control subjects (P = 0.03), whereas the responses to purified Coxsackievirus B4 and recombinant VP0 protein, which did not contain nonstructural proteins, did not differ. These data suggest that T-cell responses to Coxsackievirus B4 proteins and particularly to the antigens containing the nonstructural proteins of the virus are increased in children with type 1 diabetes after the onset of the disease. However, in children with new-onset diabetes, responses were normal or even decreased. This phenomenon was specific for enteroviruses and could be caused by trapping of enterovirus-specific T-cells in the pancreas.

Detection of Chlamydia pneumoniae in human nonrheumatic stenotic aortic valves.

OBJECTIVES: We sought to study the possible presence of Chlamydia pneumoniae in aortic valve stenosis (AVS). BACKGROUND: Inflammation and immune mechanisms are considered important for the pathogenesis of nonrheumatic AVS. All chlamydial species are able to cause heart infections, and seroepidemiologic studies have indicated an association between chronic C. pneumoniae infection and coronary artery disease. Furthermore, the organism has been demonstrated in atherosclerotic lesions. METHODS: Aortic valve specimens with varying degrees of macroscopic disease were obtained from 35 subjects--17 consecutive patients undergoing aortic valve replacement for treatment of nonrheumatic AVS and 18 age-matched subjects at autopsy. The possible presence of C. pneumoniae in aortic valves was studied by immunohistochemical analysis, polymerase chain reaction or transmission electron microscopy, or a combination of these. RESULTS: Positive immunohistochemical staining with C. pneumoniae specific antibody was found in 9 (53%) of 17 patients with advanced aortic valve disease requiring surgical treatment (group A), 8 (80%) of 10 cadavers with clearly macroscopic aortic valve pathology (group B) and 1 (12%) of 8 grossly normal cadaver control subjects (group C). Statistical significance with regard to the presence of C. pneumoniae was found when combined diseased subjects (groups A and B: total 17 of 27 subjects) were compared with group C (p = 0.018). However, when group A was compared with group C, there was only marginal statistical significance (p = 0.088). Finally, there was a strong statistical significance (p = 0.015) when groups B and C were compared. Chlamydia pneumoniae DNA was also found in three stenotic valves, and in two of the three tested valve specimens chlamydia-like particles were seen by electron microscopy. CONCLUSIONS: Chlamydia pneumoniae is frequently present in nonrheumatic AVS. Similarly, the high number of C. pneumoniae infections detected in the early lesions of "degenerative" AVS suggest that this pathogen may play an etiologic role in the development of this disease. The validity of this relation requires additional study.

High-fat, high-cholesterol diet increases the incidence of gastritis in LDL receptor-negative mice.

Transgenic and knockout mice are widely used as models for atherogenesis studies. While developing a Helicobacter infection model in LDL receptor-negative (LDLR(-/-)) mice, we noticed that mice fed a high-fat, high-cholesterol diet often contracted gastritis independent of infection. To further investigate this finding, we studied 27 male and 18 female LDLR(-/-) mice fed high-fat, 1% or 1.25% cholesterol diets for 3 to 4 months. The extent of atherosclerosis was morphometrically analyzed in the whole aorta, and the degree of gastric inflammation was scored histologically in hematoxylin-eosin-stained stomach sections. The autoantibody titers to epitopes of oxidized LDL were also measured. Mice fed high-fat, high-cholesterol diets had a significantly higher incidence of gastritis than mice fed normal chow, 62% versus 5%, respectively (P<0.0001). This effect was specific for LDLR(-/-) mice, because no difference in gastritis was found in wild-type mice fed either diet. Animals with gastritis showed slightly more atherosclerosis than animals without gastritis: 16.3+/-6.4% versus 12.8+/-3.4% in males and 9.4+/-3.5% versus 6.5+/-3.3% in females. Cholesterol-fed mice also had significantly higher IgG autoantibody titers against modified LDL than normal chow-fed animals, but no difference was seen between the gastritis and nongastritis groups. We conclude that the standard high-fat, high-cholesterol diet commonly used in many murine models to induce atherosclerosis increased the incidence of gastritis significantly in LDLR(-/-) mice.

Association of Helicobacter pylori infection with elevated serum lipids.

Helicobacter pylori causes a chronic gastric infection, which has been associated with coronary heart disease. To evaluate the mechanisms of this association, we studied whether the infection affects serum lipid levels as previously shown in acute infections. We analysed the serum samples of 880 males who participated in a reindeer herders' health survey in Northern Finland in 1989. H. pylori IgG and IgA antibodies were measured by enzyme-linked immunosorbent assay and triglyceride, total cholesterol and high-density lipoprotein cholesterol concentrations by routine enzymatic methods. A total of 52% of the subjects were positive for both H. pylori specific IgG and IgA and 31% were antibody-negative. The serum triglyceride and total cholesterol concentrations were significantly higher in the males with positive IgG and IgA antibody titres for H. pylori than in the males with no signs of infection (1.20 vs. 1.03 mmol/l, P < 0.001 and 6.59 vs. 6.11 mmol/l, P < 0.001, respectively). The associations remained statistically significant in non-smokers after the adjustment for age, body mass index (BMI) and social class. The finding supports the hypothesis that chronic infections may modify the serum lipid profile in a way that increases the risk of atherosclerosis.