Evaluation of limited-sampling strategies to calculate AUC(0-24) and the role of CYP3A5 in Chilean pediatric kidney recipients using extended-release tacrolimus.

Background: Kidney transplantation (KTx) requires immunosuppressive drugs such as Tacrolimus (TAC) which is mainly metabolized by CYP3A5. TAC is routinely monitored by trough levels (C0) although it has not shown to be a reliable marker. The area-under-curve (AUC) is a more realistic measure of drug exposure, but sampling is challenging in pediatric patients. Limited-sampling strategies (LSS) have been developed to estimate AUC. Herein, we aimed to determine AUC(0-24) and CYP3A5 genotype in Chilean pediatric kidney recipients using extended-release TAC, to evaluate different LSS-AUC(0-24) formulas and dose requirements. Patients and methods: We analyzed pediatric kidney recipients using different extended-release TAC brands to determine their trapezoidal AUC(0-24) and CYP3A5 genotypes (SNP rs776746). Daily TAC dose (TAC-D mg/kg) and AUC(0-24) normalized by dose were compared between CYP3A5 expressors (*1/*1 and *1/*3) and non-expressors (*3/*3). We evaluated the single and combined time-points to identify the best LSS-AUC(0-24) model. We compared the performance of this model with two pediatric LSS-AUC(0-24) equations for clinical validation. Results: Fifty-one pharmacokinetic profiles were obtained from kidney recipients (age 13.1 ± 2.9 years). When normalizing AUC(0-24) by TAC-D significant differences were found between CYP3A5 expressors and non-expressors (1701.9 vs. 2718.1 ng*h/mL/mg/kg, p < 0.05). C0 had a poor fit with AUC(0-24) (r 2 = 0.5011). The model which included C0, C1 and C4, showed the best performance to predict LSS-AUC(0-24) (r 2 = 0.8765) and yielded the lowest precision error (7.1% ± 6.4%) with the lowest fraction (9.8%) of deviated AUC(0-24), in comparison to other LSS equations. Conclusion: Estimation of LSS-AUC(0-24) with 3 time-points is an advisable and clinically useful option for pediatric kidney recipients using extended-release TAC to provide better guidance of decisions if toxicity or drug inefficacy is suspected. The different CYP3A5 genotypes associated with variable dose requirements reinforce considering genotyping before KTx. Further multi-centric studies with admixed cohorts are needed to determine the short- and long-term clinical benefits.

Evolutionary responses of energy metabolism, development, and reproduction to artificial selection for increasing heat tolerance in Drosophila subobscura.

Adaptation to warming conditions involves increased heat tolerance and metabolic changes to reduce maintenance costs and maximize biological functions close to fitness. Evidence shows that energy metabolism evolves in response to warming conditions, but we know little about how heat stress intensity determines the evolutionary responses of metabolism and life history traits. Here, we evaluated the evolutionary responses of energy metabolism and life-history traits to artificial selection for increasing heat tolerance in Drosophila subobscura, using 2 protocols to measure and select heat tolerance: slow and fast ramping protocols. We found that the increase in heat tolerance was associated with reduced activity of the enzymes involved in the glucose-6-phosphate branchpoint but no changes of the metabolic rate in selected lines. We also found that the evolution of increased heat tolerance increased the early fecundity in selected lines and increased the egg-to-adult viability only in the slow-ramping selected lines. This work shows heat tolerance can evolve under different thermal scenarios but with different evolutionary outcomes on associated traits depending on the heat stress intensity. Therefore, spatial and temporal variability of thermal stress intensity should be taken into account to understand and predict the adaptive response to ongoing and future climatic conditions.

Effect of CYP3A4, CYP3A5, MDR1 and POR Genetic Polymorphisms in Immunosuppressive Treatment in Chilean Kidney Transplanted Patients.

Cyclosporine (CsA) and tacrolimus (TAC) are immunosuppressant drugs characterized by a narrow therapeutic range and high pharmacokinetic variability. The effect of polymorphisms in genes related to the metabolism and transport of these drugs, namely CYP3A4, CYP3A5, MDR1 and POR genes, has been evaluated in diverse populations. However, the impact of these polymorphisms on drug disposition is not well established in Latin American populations. Using TaqMan® probes, we determined the allelic frequency of seven variants in CYP3A4, CYP3A5, MDR1 and POR in 139 Chilean renal transplant recipients, of which 89 were treated with CsA and 50 with TAC. We tested associations between variants and trough and/or 2-hour concentrations, normalized by dose (C0/D and C2/D) at specific time points post-transplant. We found that CYP3A5*3/*3 carriers required lower doses of TAC. In TAC treated patients, most CYP3A5*3/*3 carriers presented higher C0/D and a high proportion of patients with C0 levels outside the therapeutic range relative to other genotypes. These results reinforce the value of considering CYP3A5 genotypes alongside therapeutic drug monitoring for TAC treated Chilean kidney recipients.

Gut Microbiota of Drosophila subobscura Contributes to Its Heat Tolerance and Is Sensitive to Transient Thermal Stress.

The gut microbiota can contribute to host physiology leading to an increase of resistance to abiotic stress conditions. For instance, temperature has profound effects on ectotherms, and the role of the gut microbiota on the thermal tolerance of ectotherms is a matter of recent research. However, most of these studies have been focused on single static temperatures instead of evaluating thermal tolerance in a wide range of stressful temperatures. Additionally, there is evidence supporting that the gut microbiota is sensitive to environmental temperature, which induces changes in its composition and diversity. These studies have evaluated the effects of thermal acclimation (>2 weeks) on the gut microbiota, but we know little about the impact of transient thermal stress on the composition and diversity of the gut microbiota. Thus, we investigated the role of the gut microbiota on the heat tolerance of Drosophila subobscura by measuring the heat tolerance of conventional and axenic flies exposed to different heat stressful temperatures (35, 36, 37, and 38°C) and estimating the heat tolerance landscape for both microbiota treatments. Conventional flies exposed to mild heat conditions exhibited higher thermal tolerance than axenic flies, whereas at higher stressful temperatures there were no differences between axenic and conventional flies. We also assessed the impact of transient heat stress on the taxonomical abundance, diversity, and community structure of the gut microbiota, comparing non-stressed flies (exposed to 21°C) and heat-stressed flies (exposed to 34°C) from both sexes. Bacterial diversity indices, bacterial abundances, and community structure changed between non-stressed and heat-stressed flies, and this response was sex-dependent. In general, our findings provide evidence that the gut microbiota influences heat tolerance and that heat stress modifies the gut microbiota at the taxonomical and structural levels. These results demonstrate that the gut microbiota contributes to heat tolerance and is also highly sensitive to transient heat stress, which could have important consequences on host fitness, population risk extinction, and the vulnerability of ectotherms to current and future climatic conditions.

CYP3A5 and UGT1A9 Polymorphisms Influence Immunosuppressive Therapy in Pediatric Kidney Transplant Recipients.

Background: Tacrolimus (TAC) and mycophenolic acid (MPA) are the main immunosuppressive drugs used in pediatric kidney transplantation. Single nucleotide polymorphisms (SNPs) in metabolizing enzymes and transporters might influence plasma levels of these drugs. Herein, we sought to determine the influence of SNPs on CYP3A5, MRP2 and UGT1A9 genes in Chilean pediatric kidney recipients using TAC and MPA. Patients and Methods: A prospective study was performed on 104 pediatric kidney recipients that used TAC and MPA for immunosuppression. The median age at the time of transplantation was 8.1 years [Q1-Q3 4.5-11.6 years] and the main clinical diagnosis was a structural anomaly. In a subgroup of patients, a complete steroid withdrawal was made at day 7. The CYP3A5 polymorphism (ancestral allele *1; variant allele *3) was determined in the entire cohort, while MRP2 -24G > A, UGT1A9 -275T > A, and UGT1A9 -2152C > T polymorphisms were determined in 53 patients. Genotypes were associated with trough drug concentrations (C0), dose requirements normalized by weight (TAC-D mg/kg) or body surface (MPA-D mg/m2), trough levels normalized by dose requirements (C0/D), and area under the curve in 12 h normalized by dose requirements (AUC0-12h/D). Results: The frequencies of the variant alleles CYP3A5*3, MRP2-24A, UGT1A9-275A, and UGT1A9-2152T were 76.9, 22.1, 6.6, and 2.9%, respectively. AUC0-12h/TAC-D were 1.6-fold higher in CYP3A5*3/*3 patients than in CYP3A5*1 carriers (CYP3A5*1/*3 and CYP3A5*1/*1). When analyzing patients with steroid withdrawal, CYP3A5*3/*3 patients had 1.7-fold higher AUC0-12h/TAC-D than the other genotypes. Patients carrying the CYP3A5*3/*3 genotype had higher TAC-C0, lower TAC-D and higher TAC-C0/D, consistently in a 6-months follow-up. Creatinine clearance was stable during the follow-up, regardless of the genotype. No significant differences between MRP2 and UGT1A9 genotypes were observed in MPA-C0, MPA-D or MPA-C0/D. However, patients carrying the UGT1A9-275A allele had lower AUC0-12h/MPA-D than those carrying the UGT1A9-275T ancestral allele. Conclusions: These results support that CYP3A5 and UGT1A9 genotyping in pediatric recipients might be useful and advisable to guide TAC and MPA dosing and monitoring in children that undergo kidney transplantation.

Developmental dysplasia of the hip screening programme in Chile.

PURPOSE: Developmental dysplasia of the hip (DDH) encompasses a wide pathological spectrum, from mild acetabular dysplasia to complete congenital hip dislocation at birth. Screening policies have been implemented in an effort to effectively identify and treat patients with DDH. Since 2009 there has been a national DDH programme in Chile. The current study evaluates the results of the programme in patients born between 2010 and 2015. METHODS: Records of patients hospitalized from 1st January 2010 to 31st December 2019 were retrieved from national databases. Those born from 1st January 2010 to 31st December 2015 who underwent a procedure for DDH under general anaesthesia during their first five years of life were selected. Sex, first surgical procedure and age at first surgical procedure were analyzed. The incidence of DDH that required major surgical treatment was calculated. RESULTS: A total of 961 children born from 1st January 2010 to 31st December 2015 underwent a procedure for DDH during their first five years of life. The number of major procedures was significantly lower than the number of minor procedures (269 vs 692). The incidence of major procedures was 0.18 per 1000 live births. Girls underwent a higher number of procedures than boys (831 vs 130), whereas 39.2% of the boys and 26.2% of the girls had major procedures. The mean age at the time of the first procedure was 15.35 months (sd 10.09; range 0.03 to 55.92 months). CONCLUSION: The present study suggests that the Chilean National DDH Screening Program is an appropriate programme with substantial benefits with respect to public health. LEVEL OF EVIDENCE: II.

Experimental evolution on heat tolerance and thermal performance curves under contrasting thermal selection in Drosophila subobscura.

Ectotherms can respond to global warming via evolutionary change of their upper thermal limits (CTmax ). Thus, the estimation of CTmax and its evolutionary potential is crucial to determine their vulnerability to global warming. However, CTmax estimations depend on the thermal stress intensity, and it is not completely clear whether its evolutionary capacity can be affected. Here, we performed an artificial selection experiment to increase heat tolerance using fast- and slow-ramping selection protocols in Drosophila subobscura. We found that heat tolerance evolved in both selection protocols, exhibiting similar evolutionary change rates and realized heritabilities. Additionally, we estimated the thermal performance curves (TPC) to evaluate correlated responses to selection on heat tolerance. We detected that thermal optimum increased in fast-ramping selection lines, but with a cost at the thermal performance breadth. Conversely, we did not detect changes in the TPC for the slow-ramping selection lines, indicating that thermal stress intensity has important effects on the evolution of thermal physiology of ectotherms. These findings, together with previous studies in D. subobscura reporting interpopulation variability and significant heritabilities for heat tolerance, suggest that evolutionary change can contribute to insect persistence in thermally changing environments and adaptation to global warming conditions.

Oxidative status and metabolic profile in a long-lived bird preparing for extreme endurance migration.

The high metabolic activity associated with endurance flights and intense fuelling of migrant birds may produce large quantities of reactive oxygen species, which cause oxidative damage. Yet it remains unknown how long-lived birds prepare for oxidative challenges prior to extreme flights. We combined blood measurements of oxidative status and enzyme and fat metabolism in Hudsonian godwits (Limosa haemastica, a long-lived shorebird) before they embarked on non-stop flights longer than 10,000 km during their northbound migrations. We found that godwits increased total antioxidant capacity (TAC) and reduced oxidative damage (TBARS) as the pre-migratory season progressed, despite higher basal metabolic rates before departure. Elevations in plasma ß-hydroxybutyrate and uric acid suggest that lipid and protein breakdown supports energetic requirements prior to migration. Significant associations between blood mitochondrial cytochrome-c oxidase and plasma TAC (negative) and TBARS (positive) during winter indicate that greater enzyme activity can result in greater oxidative damage and antioxidant responses. However enzyme activity remained unchanged between winter and premigratory stages, so birds may be unable to adjust metabolic enzyme activity in anticipation of future demands. These results indicate that godwits enhance their oxidative status during migratory preparation, which might represent an adaptation to diminish the physiological costs of long-distance migration.

Evolutionary potential of thermal preference and heat tolerance in Drosophila subobscura.

Evolutionary change of thermal traits (i.e., heat tolerance and behavioural thermoregulation) is one of the most important mechanisms exhibited by organisms to respond to global warming. However, the evolutionary potential of heat tolerance, estimated as narrow-sense heritability, depends on the methodology employed. An alternative adaptive mechanism to buffer extreme temperatures is behavioural thermoregulation, although the association between heat tolerance and thermal preference is not clearly understood. We suspect that methodological effects associated with the duration of heat stress during thermal tolerance assays are responsible for missing this genetic association. To test this hypothesis, we estimated the heritabilities and genetic correlations for thermal traits in Drosophila subobscura, using high-temperature static and slow ramping assays. We found that heritability for heat tolerance was higher in static assays (h2  = 0.134) than in slow ramping assays (h2  = 0.084), suggesting that fast assays may provide a more precise estimation of the genetic variation of heat tolerance. In addition, thermal preference exhibited a low heritability (h2  = 0.066), suggesting a reduced evolutionary response for this trait. We also found that the different estimates of heat tolerance and thermal preference were not genetically correlated, regardless of how heat tolerance was estimated. In conclusion, our data suggest that these thermal traits can evolve independently in this species. In agreement with previous evidence, these results indicate that methodology may have an important impact on genetic estimates of heat tolerance and that fast assays are more likely to detect the genetic component of heat tolerance.

Shared and unique features of bacterial communities in native forest and vineyard phyllosphere.

Phyllosphere bacteria have received little attention despite their important roles in shaping plant performance traits. In this study, we characterize the bacterial communities on leaves of native trees inhabiting sclerophyllous forests in central Chile, one of the world's biodiversity hotspots. Additionally, we provide profiles of bacterial communities on grape leaves and berries of organic and conventional vineyards. Results of 16S rRNA gene amplicon sequence analysis showed that 45% of OTUs were shared across forest leaves, grape leaves, and grape berries. Conventional management had higher number of OTUs shared with forest leaves than organic management. In addition, grape leaves subjected to conventional management had higher alpha diversity than those with organic management, while no significant effect of agricultural management was observed in grape berries. Indicator analysis showed that Bdellovibrio, Beijerinckia, and Spirosoma were typical for forest leaves, whereas Enhydrobacter, Delftia, Proteiniclasticum, Arsenicicoccus, and Alkaliphilus were typical for the vineyard phyllosphere. Regarding agricultural managements, Beijerinckia, Sedimentibacter, Nesterenkonia, Gluconobacter, Conexibacter, and Anaeromyxobacter were typical for conventional grape leaves, whereas no genus-level indicator was found for organic vineyard leaves. These results provide new insights of the diversity patterns of the phyllosphere microbiome in native and cultivated lands and suggest that both of these microbiomes are connected and integrated systems.

Assessing the effects of human activities on the foraging opportunities of migratory shorebirds in Austral high-latitude bays.

Human presence at intertidal areas could impact coastal biodiversity, including migratory waterbird species and the ecosystem services they provide. Assessing this impact is therefore essential to develop management measures compatible with migratory processes and associated biodiversity. Here, we assess the effects of human presence on the foraging opportunities of Hudsonian godwits (Limosa haemastica, a trans-hemispheric migratory shorebird) during their non-breeding season on Chiloé Island, southern Chile. We compared bird density and time spent foraging in two similar bays with contrasting disturbance levels: human presence (mostly seaweed harvesters accompanied by dogs) was on average 0.9±0.4 people per 10 ha in the disturbed bay, whereas it was negligible (95% days absent) in the non-disturbed bay. Although overall abundances were similar between bays, godwit density was higher in the non-disturbed bay throughout the low tide period. Both days after the start of the non-breeding season and tidal height significantly affected godwit density, with different effects in either bay. Time spent foraging was significantly higher in the non-disturbed bay (86.5±1.1%) than in the disturbed one (81.3±1.4%). As expected, godwit density significantly decreased with the number of people and accompanying dogs in the disturbed bay. Our results indicate that even a low density of people and dogs can significantly reduce the foraging opportunities of shorebirds. These constraints, coupled with additional flushing costs, may negatively affect godwits' pre-migratory fattening. Hence, as a first step we suggest limiting human presence within bays on Chiloé to 1 person per 10 ha and banning the presence of accompanying dogs in sensitive conservation areas.

Effects of agricultural management on phyllosphere fungal diversity in vineyards and the association with adjacent native forests.

Agriculture is one of the main drivers of land conversion, and agriculture practices can impact on microbial diversity. Here we characterized the phyllosphere fungal diversity associated with Carménère grapevines under conventional and organic agricultural management. We also explored the fungal diversity present in the adjacent sclerophyllous forests to explore the potential role of native forest on vineyard phyllosphere. After conducting D2 and ITS2 amplicon sequencing, we found that fungal diversity indices did not change between conventional and organic vineyards, but community structure was sensitive to the agricultural management. On the other hand, we found a high proportion of shared fungal OTUs between vineyards and native forests. In addition, both habitats had similar levels of fungal diversity despite forest samples were derived from multiple plant species. In contrast, the community structure was different in both habitats. Interestingly, the native forest had more unidentified species and unique OTUs than vineyards. Forest dominant species were Aureobasidium pullulans and Endoconidioma populi, whereas Davidiella tassiana, Didymella sp., and Alternaria eichhorniae were more abundant in vineyards. Overall, this study argues that a better understanding of the relationship native forests and agroecosystems is needed for maintaining and enhancing ecosystem services provided by natural ecosystems. Finally, knowledge of microbial communities living in the Chilean Mediterranean biome is needed for appropriate conservation management of these biomes and their classification as biodiversity hotspots.

Is microbial terroir related to geographic distance between vineyards?

While there are substantial studies suggesting that characteristics of wine are related to regional microbial community composition (microbial terroir), there has been little discussion about what factors affect variation in regional microbial community composition. In this study, we compared the microbial community composition of leaves and berries of a grape variety (Carmenere) from six different Chilean vineyards within 35 km of each other. In order to determine relationships between spatial proximity and microbial compositional dissimilarity, we sequenced amplicons of the internal transcribed spacer (ITS) region for fungi and 16S rRNA gene for bacteria. Results showed that both the fungal and the bacterial community compositions of the studied vineyards differed, but this difference was much clearer in fungi than in bacteria. In addition, while bacterial community dissimilarity was not correlated with geographic distance, the leaf and berry fungal community dissimilarities between locations increased with geographic distance. This indicates that spatial processes play an important role in structuring the biogeographic pattern of grape-associated fungal communities at local scales, which might in turn contribute to the local identity of wine.

Metagenomic analysis exploring taxonomic and functional diversity of soil microbial communities in Chilean vineyards and surrounding native forests.

Mediterranean biomes are biodiversity hotspots, and vineyards are important components of the Mediterranean landscape. Over the last few decades, the amount of land occupied by vineyards has augmented rapidly, thereby increasing threats to Mediterranean ecosystems. Land use change and agricultural management have important effects on soil biodiversity, because they change the physical and chemical properties of soil. These changes may also have consequences on wine production considering that soil is a key component of terroir. Here, we describe the taxonomic diversity and metabolic functions of bacterial and fungal communities present in forest and vineyard soils in Chile. To accomplish this goal, we collected soil samples from organic vineyards in central Chile and employed a shotgun metagenomic approach to sequence the microbial DNA. Additionally, we studied the surrounding native forest to obtain a baseline of the soil conditions in the area prior to the establishment of the vineyard. Our metagenomic analyses revealed that both habitats shared most of the soil microbial species. The most abundant genera in the two habitats were the bacteria Candidatus Solibacter and Bradyrhizobium and the fungus Gibberella. Our results suggest that the soil microbial communities are similar in these forests and vineyards. Therefore, we hypothesize that native forests surrounding the vineyards may be acting as a microbial reservoir buffering the effects of the land conversion. Regarding the metabolic diversity, we found that genes pertaining to the metabolism of amino acids, fatty acids, and nucleotides as well as genes involved in secondary metabolism were enriched in forest soils. On the other hand, genes related to miscellaneous functions were more abundant in vineyard soils. These results suggest that the metabolic function of microbes found in these habitats differs, though differences are not related to taxonomy. Finally, we propose that the implementation of environmentally friendly practices by the wine industry may help to maintain the microbial diversity and ecosystem functions associated with natural habitats.

Introduced Drosophila subobscura populations perform better than native populations during an oviposition choice task due to increased fecundity but similar learning ability.

The success of invasive species is tightly linked to their fitness in a putatively novel environment. While quantitative components of fitness have been studied extensively in the context of invasive species, fewer studies have looked at qualitative components of fitness, such as behavioral plasticity, and their interaction with quantitative components, despite intuitive benefits over the course of an invasion. In particular, learning is a form of behavioral plasticity that makes it possible to finely tune behavior according to environmental conditions. Learning can be crucial for survival and reproduction of introduced organisms in novel areas, for example, for detecting new predators, or finding mates or oviposition sites. Here we explored how oviposition performance evolved in relation to both fecundity and learning during an invasion, using native and introduced Drosophila subobscura populations performing an ecologically relevant task. Our results indicated that, under comparable conditions, invasive populations performed better during our oviposition task than did native populations. This was because invasive populations had higher fecundity, together with similar cognitive performance when compared to native populations, and that there was no interaction between learning and fecundity. Unexpectedly, our study did not reveal an allocation trade-off (i.e., a negative relationship) between learning and fecundity. On the contrary, the pattern we observed was more consistent with an acquisition trade-off, meaning that fecundity could be limited by availability of resources, unlike cognitive ability. This pattern might be the consequence of escaping natural enemies and/or competitors during the introduction. The apparent lack of evolution of learning may indicate that the introduced population did not face novel cognitive challenges in the new environment (i.e., cognitive "pre-adaptation"). Alternatively, the evolution of learning may have been transient and therefore not detected.

Comparison of soil microbial communities inhabiting vineyards and native sclerophyllous forests in central Chile.

Natural ecosystems provide services to agriculture such as pest control, soil nutrients, and key microbial components. These services and others in turn provide essential elements that fuel biomass productivity. Responsible agricultural management and conservation of natural habitats can enhance these ecosystem services. Vineyards are currently driving land-use changes in many Mediterranean ecosystems. These land-use changes could have important effects on the supporting ecosystems services related to the soil properties and the microbial communities associated with forests and vineyard soils. Here, we explore soil bacterial and fungal communities present in sclerophyllous forests and organic vineyards from three different wine growing areas in central Chile. We employed terminal restriction fragment length polymorphisms (T-RFLP) to describe the soil microbial communities inhabiting native forests and vineyards in central Chile. We found that the bacterial community changed between the sampled growing areas; however, the fungal community did not differ. At the local scale, our findings show that fungal communities differed between habitats because fungi species might be more sensitive to land-use change compared to bacterial species, as bacterial communities did not change between forests and vineyards. We discuss these findings based on the sensitivity of microbial communities to soil properties and land-use change. Finally, we focus our conclusions on the importance of naturally derived ecosystem services to vineyards.

Heat tolerance in Drosophila subobscura along a latitudinal gradient: Contrasting patterns between plastic and genetic responses.

Susceptibility to global warming relies on how thermal tolerances respond to increasing temperatures through plasticity or evolution. Climatic adaptation can be assessed by examining the geographic variation in thermal-related traits. We studied latitudinal patterns in heat tolerance in Drosophila subobscura reared at two temperatures. We used four static stressful temperatures to estimate the thermal death time (TDT) curves, and two ramping assays with fast and slow heating rates. Thermal death time curves allow estimation of the critical thermal maximum (CT(max)), by extrapolating to the temperature that would knock down the flies almost "instantaneously," and the thermal sensitivity to increasing stressful temperatures. We found a positive latitudinal cline for CT(max), but no clinal pattern for knockdown temperatures estimated from the ramping assays. Although high-latitude populations were more tolerant to an acute heat stress, they were also more sensitive to prolonged exposure to less stressful temperatures, supporting a trade-off between acute and chronic heat tolerances. Conversely, developmental plasticity did not affect CT(max) but increased the tolerance to chronic heat exposition. The patterns observed from the TDT curves help to understand why the relationship between heat tolerance and latitude depends on the methodology used and, therefore, these curves provide a more complete and reliable measurement of heat tolerance.

Vanishing chromosomal inversion clines in Drosophila subobscura from Chile: is behavioral thermoregulation to blame?

Chromosomal inversion clines paralleling the long-standing ones in native Palearctic populations of Drosophila subobscura evolved swiftly after this species invaded the Americas in the late 1970s and early 1980s. However, the new clines did not consistently continue to converge on the Old World baseline. Our recent survey of Chilean populations of D. subobscura shows that inversion clines have faded or even changed sign with latitude. Here, we investigate the hypothesis that this fading of inversion clines might be due to the Bogert effect, namely, that flies' thermoregulatory behavior has eventually compensated for environmental variation in temperature, thus buffering selection on thermal-related traits. We show that latitudinal divergence in thermal preference (Tp) has evolved in Chile for females, with higher-latitude flies having a lower mean Tp. Plastic responses in Tp also lessen latitudinal thermal variation because flies developed at colder temperatures prefer warmer microclimates. Our results are consistent with the idea that active behavioral thermoregulation might buffer environmental variation and reduce the potential effect of thermal selection on other traits as chromosomal arrangements.

Phenotypic and genetic effects of contrasting ethanol environments on physiological and developmental traits in Drosophila melanogaster.

A central problem in evolutionary physiology is to understand the relationship between energy metabolism and fitness-related traits. Most attempts to do so have been based on phenotypic correlations that are not informative for the evolutionary potential of natural populations. Here, we explored the effect of contrasting ethanol environments on physiological and developmental traits, their genetic (co)variances and genetic architecture in Drosophila melanogaster. Phenotypic and genetic parameters were estimated in two populations (San Fernando and Valdivia, Chile), using a half-sib family design where broods were split into ethanol-free and ethanol-supplemented conditions. Our findings show that metabolic rate, body mass and development times were sensitive (i.e., phenotypic plasticity) to ethanol conditions and dependent on population origin. Significant heritabilities were found for all traits, while significant genetic correlations were only found between larval and total development time and between development time and metabolic rate for flies of the San Fernando population developed in ethanol-free conditions. Posterior analyses indicated that the G matrices differed between ethanol conditions for the San Fernando population (mainly explained by differences in genetic (co)variances of developmental traits), whereas the Valdivia population exhibited similar G matrices between ethanol conditions. Our findings suggest that ethanol-free environment increases the energy available to reduce development time. Therefore, our results indicate that environmental ethanol could modify the process of energy allocation, which could have consequences on the evolutionary response of natural populations of D. melanogaster.

Keeping pace with climate change: what is wrong with the evolutionary potential of upper thermal limits?

The potential of populations to evolve in response to ongoing climate change is partly conditioned by the presence of heritable genetic variation in relevant physiological traits. Recent research suggests that Drosophila melanogaster exhibits negligible heritability, hence little evolutionary potential in heat tolerance when measured under slow heating rates that presumably mimic conditions in nature. Here, we study the effects of directional selection for increased heat tolerance using Drosophila as a model system. We combine a physiological model to simulate thermal tolerance assays with multilocus models for quantitative traits. Our simulations show that, whereas the evolutionary response of the genetically determined upper thermal limit (CTmax) is independent of methodological context, the response in knockdown temperatures varies with measurement protocol and is substantially (up to 50%) lower than for CTmax. Realized heritabilities of knockdown temperature may grossly underestimate the true heritability of CTmax. For instance, assuming that the true heritability of CTmax in the base population is h(2) = 0.25, realized heritabilities of knockdown temperature are around 0.08-0.16 depending on heating rate. These effects are higher in slow heating assays, suggesting that flawed methodology might explain the apparently limited evolutionary potential of cosmopolitan D. melanogaster.