Neuroplastin splice variants Np55 and Np65: Who is doing the job in macrophages?

Neuroplastin, a paralog of CD147/Basigin, is known as a neuronal cell adhesion molecule and as an auxiliary subunit of plasma membrane calcium ATPases in both neurons and adaptive immune cells. Recently, an interesting study by Ren et al. (2022) provided evidence for an important role of neuroplastin in macrophages during bacterial infection. Here, we critically discuss one aspect of this study, the assignment of this role to Np65 as one of two prominent splice variants of neuroplastin.

Heterozygosity is low where rare color variants in wild carnivores prevail.

Coat color and pattern are a distinguished feature in mammalian carnivores, shaped by climatic cycles and habitat type. It can be expressed in various ways, such as gradients, polymorphisms, and rare color variants. Although natural selection explains much of the phenotypic variation found in the wild, genetic drift and heterozygote deficiency, as prominent in small and fragmented populations, may also affect phenotypic variability through the fixation of recessive alleles. The aim of this study was to test whether rare color variants in the wild could relate to a deficiency of heterozygotes, resulting from habitat fragmentation and small population size. We present an overview of all rare color variants in the order Carnivora, and compiled demographic and genetic data of the populations where they did and did not occur, to test for significant correlations. We also tested how phylogeny and body weight influenced the presence of color variants with phylogenetic generalized linear mixed models (PGLMMs). We found 40 color-variable species and 59 rare color variants. In 17 variable phenotypic populations for which genetic diversity was available, the average A R was 4.18, H O = 0.59, and H E = 0.66, and F IS = 0.086. We found that variable populations displayed a significant reduction in heterozygosity and allelic richness compared to non-variable populations across species. We also found a significant negative correlation between population size and inbreeding coefficients. Therefore, it is possible that small effective size had phenotypic consequences on the extant populations. The high frequency of the rare color variants (averaging 20%) also implies that genetic drift is locally overruling natural selection in small effective populations. As such, rare color variants could be added to the list of phenotypic consequences of inbreeding in the wild.

Enhanced dispersal capacity in edge population individuals of a rapidly expanding butterfly.

Natural range shifts offer the opportunity to study the phenotypic and genetic changes contributing to colonization success. The recent range shift of the Southern small white butterfly (Pieris mannii) from the South to the North of Europe offers a prime example to examine a potential dispersal syndrome in range-expanding individuals. We compared butterflies from the core and edge populations using a multimodal approach addressing behavioral, physiological, and morphological traits related to dispersal capacity. Relative to individuals from the core range (France), individuals from the edge (Germany) showed a higher capacity and motivation to fly, and a higher flight metabolic rate. They were also smaller, which may enhance their flight maneuverability and help them cope with limited resource availability, thereby increasing their settlement success in novel environments. Altogether, the behavioral, physiological, and morphological differences observed between core and edge populations in P. mannii suggest the existence of a dispersal syndrome in range-expanding individuals. Whether these differences result from genetic and/or phenotypic responses remains, however, to be determined.

Evaluating hybrid speciation and swamping in wild carnivores with a decision-tree approach.

Hybridization is an important evolutionary force with a principal role in the origin of new species, known as hybrid speciation. However, ongoing hybridization can create hybrid swamping, in which parental genomes are completely lost. This can become a biodiversity threat if it involves species that have adapted to certain environmental conditions and occur nowhere else. Because conservation scientists commonly have a negative attitude toward hybrids, it is important to improve understanding of the influence of interspecific gene flow on the persistence of species. We reviewed the literature on species hybridization to build a list of all known cases in the order Carnivora. To examine the relative impact, we also noted level of introgression, whether fertile offspring were produced, and whether there was mention of negative or positive evolutionary effects (hybrid speciation and swamping). To evaluate the conservation implications of hybrids, we developed a decision-making tree with which to determine which actions should be taken to manage hybrid species. We found 53 hybrids involving 68 unique taxa, which is roughly 23% of all carnivore species. They mainly involved monophyletic (83%) and sympatric species (75%). For 2 species, the outcome of the assessment was to eliminate or restrict the hybrids: Ethiopian wolf (Canis simensis) and Scottish wildcat (Felis silvestris silvestris). Both species hybridize with their domestic conspecifics. For all other cases, we suggest hybrids be protected in the same manner as native species. We found no evidence of genomic extinction in Carnivora. To the contrary, some species appear to be of hybrid origin, such as the Asiatic black bear (Ursus thibetanus) and African golden wolf (Canis lupaster). Other positive outcomes of hybridization are novel genetic diversity, adaptation to extreme environments, and increased reproductive fitness. These outcomes are particularly valuable for counterbalancing genetic drift and enabling adaptive introgression in a human-dominated world.

La especiación por hibridación es una fuerza evolutiva importante con un papel principal en el origen de una nueva especie. Sin embargo, la hibridación continua puede generar un estancamiento híbrido en el que se pierden por completo los genomas parentales. Esto puede convertirse en una amenaza para la biodiversidad si involucra a una especie que se ha adaptado a ciertas condiciones ambientales y sólo se encuentra en un lugar. Ya que los científicos de la conservación suelen tener una actitud negativa hacia los híbridos, es importante incrementar el entendimiento de la influencia que tiene el flujo interespecífico sobre la persistencia de las especies. Revisamos la literatura sobre la hibridación de especies para generar una lista de todos los casos conocidos en el orden Carnívora. También observamos el nivel de introgresión, si se produjo descendencia fértil y si hubo mención de los efectos evolutivos positivos o negativos (especiación híbrida y estancamiento) para analizar el impacto relativo. Desarrollamos un árbol de decisión con el cual determinar cuáles acciones deberían tomarse en el manejo de las especies híbridas para evaluar las implicaciones que tienen los híbridos para la conservación. Encontramos 53 híbridos de 68 taxones únicos, lo que representa aproximadamente el 23% de todos los carnívoros. Estos híbridos incluyen principalmente a especies monofiléticas (83%) y simpátricas (75%). Para dos especies, los resultados del análisis fueron la eliminación o restricción de los híbridos: el lobo etíope (Canis simensis) y el lince escocés (Felis silvestris silvestris). Ambas especies hibridan con sus coespecíficos domésticos. Para todos los demás casos sugerimos que se proteja a los híbridos de la misma manera que a las especies nativas. No encontramos evidencias de una extinción genómica en el orden Carnívora. Al contrario, algunas especies parecen tener un origen híbrido, como el oso negro asiático (Ursus thibetanus) y el lobo dorado africano (Canis lupaster). Otros resultados positivos de la hibridación son la diversidad genética novedosa, la adaptación a ambientes extremos y el incremento en la adaptabilidad reproductiva. Estos resultados son de valor particular para contrarrestar la deriva génica y permitir la introgresión adaptativa en un mundo dominado por humanos. Evaluación de la especiación y estancamiento en carnívoros silvestres con una estrategia de árbol de decisión.

Fluorimetric microplate assay for the determination of extracellular alkaline phosphatase kinetics and inhibition kinetics in activated sludge.

The microbial enzyme alkaline phosphatase contributes to the removal of organic phosphorus compounds from wastewaters. To cope with regulatory threshold values for permitted maximum phosphor concentrations in treated wastewaters, a high activity of this enzyme in the biological treatment stage, e.g., the activated sludge process, is required. To investigate the reaction dynamics of this enzyme, to analyze substrate selectivities, and to identify potential inhibitors, the determination of enzyme kinetics is necessary. A method based on the application of the synthetic fluorogenic substrate 4-methylumbelliferyl phosphate is proven for soils, but not for activated sludges. Here, we adapt this procedure to the latter. The adapted method offers the additional benefit to determine inhibition kinetics. In contrast to conventional photometric assays, no particle removal, e.g., of sludge pellets, is required enabling the analysis of the whole sludge suspension as well as of specific sludge fractions. The high sensitivity of fluorescence detection allows the selection of a wide substrate concentration range for sound modeling of kinetic functions.•Fluorescence array technique for fast and sensitive analysis of high sample numbers•No need for particle separation - analysis of the whole (diluted) sludge suspension•Simultaneous determination of standard and inhibition kinetics.

Real-time mechanisms of exacerbated synaptic remodeling by microglia in acute models of systemic inflammation and tauopathy.

Remodeling of synapses by microglia is essential for synaptic plasticity in the brain. However, during neuroinflammation and neurodegenerative diseases, microglia can induce excessive synaptic loss, although the precise underlying mechanisms are unknown. To directly observe microglia-synapse interactions under inflammatory conditions, we performed in vivo two-photon time-lapse imaging of microglia-synapse interactions after bacterial lipopolysaccharide administration to model systemic inflammation, or after inoculation of Alzheimer's disease (AD) brain extracts to model disease-associated neuroinflammatory microglial response. Both treatments prolonged microglia-neuron contacts, decreased basal surveillance of synapses and promoted synaptic remodeling in response to synaptic stress induced by focal single-synapse photodamage. Spine elimination correlated with the expression of microglial complement system/phagocytic proteins and the occurrence of synaptic filopodia. Microglia were observed contacting spines, then stretching and phagocytosing spine head filopodia. Thus, in response to inflammatory stimuli microglia exacerbated spine remodeling through prolonged microglial contact and elimination of spines 'tagged' by synaptic filopodia.

Ca2+ Homeostasis by Plasma Membrane Ca2+ ATPase (PMCA) 1 Is Essential for the Development of DP Thymocytes.

The strength of Ca2+ signaling is a hallmark of T cell activation, yet the role of Ca2+ homeostasis in developing T cells before expressing a mature T cell receptor is poorly understood. We aimed to unveil specific functions of the two plasma membrane Ca2+ ATPases expressed in T cells, PMCA1 and PMCA4. On a transcriptional and protein level we found that PMCA4 was expressed at low levels in CD4-CD8- double negative (DN) thymocytes and was even downregulated in subsequent stages while PMCA1 was present throughout development and upregulated in CD4+CD8+ double positive (DP) thymocytes. Mice with a targeted deletion of Pmca1 in DN3 thymocytes had an almost complete block of DP thymocyte development with an accumulation of DN4 thymocytes but severely reduced numbers of CD8+ immature single positive (ISP) thymocytes. The DN4 thymocytes of these mice showed strongly elevated basal cytosolic Ca2+ levels and a pre-mature CD5 expression, but in contrast to the DP thymocytes they were only mildly prone to apoptosis. Surprisingly, mice with a germline deletion of Pmca4 did not show any signs of altered progression through the developmental thymocyte stages, nor altered Ca2+ homeostasis throughout this process. PMCA1 is, therefore, non-redundant in keeping cellular Ca2+ levels low in the early thymocyte development required for the DN to DP transition.

Genetic Variants in ARHGEF6 Cause Congenital Anomalies of the Kidneys and Urinary Tract in Humans, Mice, and Frogs.

BACKGROUND: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. METHODS: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. RESULTS: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. CONCLUSIONS: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

Adaptive neuro-fuzzy inference system (ANFIS) and multiple linear regression (MLR) modelling of Cu, Cd, and Pb adsorption onto tropical soils.

Soils interact in many ways with metal ions thereby modifying their mobility, phase distribution, plant availability, speciation, and so on. The most prominent of such interactions is sorption. In this study, we investigated the sorption of Pb, Cd, and Cu in five natural soils of Nigerian origin. A relatively sparsely used method of modelling soil-metal ion adsorption, i.e. adaptive neuro-fuzzy inference system (ANFIS), was applied comparatively with multiple linear regression (MLR) models. The isotherms were well described by Freundlich and Langmuir equations (R2 ≥ 0.95) and the kinetics by nonlinear two-stage kinetic model, TSKM (R2 ≥ 0.81). Based on the values delivered by the Langmuir equation, the maximum adsorption capacities (Qm*) were found to be in the ranges 10,000-20,000, 12,500-50,000, and 4929-35,037 µmol kg-1 for Cd, Cu, and Pb, respectively. The study revealed significant correlations between Qm* and routinely determined soil parameters such as soil organic carbon (Corg), cation exchange capacity (CEC), amorphous Fe and Mn oxides, and percentage clay content. These soil parameters, combined with operational variables (i.e. solution/soil pH, initial metal concentration (Co), and temperature), were used as input vectors in ANFIS and MLR models to predict the adsorption capacities (Qe) of the soil-metal ion systems. A total of 255 different ANFIS and 255 different MLR architectures/models were developed and compared based on three performance metrics: MAE (mean absolute error), RMSE (root mean square errors), and R2 (coefficient of determination). The best ANFIS returned MAEtest 0.134, RMSEtest 0.164, and R2test 0.76, while the best MLR returned MAEtest 0.158, RMSEtest 0.199, and R2test 0.66, indicating the predictive advantage of ANFIS over MLR. Thus, ANFIS can fairly accurately predict the adsorption capacity and/or distribution coefficient of a soil-metal ion system a priori. Nevertheless, more investigation is required to further confirm the robustness/generalisation of the proposed ANFIS.

Molecular tracking and prevalence of the red colour morph restricted to a harvested leopard population in South Africa.

The red leopard (Panthera pardus) colour morph is a colour variant that occurs only in South Africa, where it is confined to the Central Bushveld bioregion. Red leopards have been spreading over the past 40 years, which raises the speculation that the prevalence of this phenotype is related to low dispersal of young individuals owing to high off-take in the region. Intensive selective hunting tends to remove large resident male leopards from the breeding population, which gives young male leopards the chance to mate with resident female leopards that are more likely to be their relatives, eventually increasing the frequency of rare genetic variants. To investigate the genetic mechanisms underlying the red coat colour morph in leopards, and whether its prevalence in South Africa relates to an increase in genetic relatedness in the population, we sequenced exons of six coat colour-associated genes and 20 microsatellite loci in twenty Wild-type and four red leopards. The results were combined with demographic data available from our study sites. We found that red leopards own a haplotype in homozygosity identified by two SNPs and a 1 bp deletion that causes a frameshift in the tyrosinase-related protein 1 (TYRP1), a gene known to be involved in the biosynthesis of melanin. Microsatellite analyses indicate clear signs of a population bottleneck and a relatedness of 0.11 among all pairwise relationships, eventually supporting our hypothesis that a rare colour morph in the wild has increased its local frequency due to low natal dispersal, while subject to high human-induced mortality rate.

IKKγ/NEMO Localization into Multivesicular Bodies.

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3ß (GSK-3ß) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3ß leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3ß and V-ATPase in NF-κB signaling activation.

Differential Requirement of Vav Proteins for Btk-dependent and -Independent Signaling During B Cell Development.

Btk and Vav proteins are all components of the signalosome that builds upon B cell receptor (BCR) activation. However, the role of Vav proteins within the signalosome is quite complex and not yet fully understood. Until now, studies of these have focused predominantly on a deficiency of Vav proteins alone or in combination with other Vav protein family members. Since a physical association of Btk with Vav was shown previously, we asked whether these molecules lie in the same or independent signaling pathways. By analyzing Vav1 and Vav3 single knock-out mice and generating double-knock-out animals deficient for either Vav1 or Vav3 and Btk, we observed, in line with previous publications, no severe B cell developmental defects when either Vav1 or Vav3 alone are not expressed. However, a simultaneous deficiency of Btk together with either Vav1 or Vav3 leads to a severe reduction of splenic B cells, which exhibit an immature phenotype. B cell developmental defects of Btk/Vav1-double deficient mice in the periphery were more severe than those observed in Btk-single-deficient animals. Additionally, morphological changes in splenic microarchitecture were observed in double- but also in single-knock-out mutants. These observations were accompanied by reduced BCR-induced Ca2+ mobilization, proliferation, germinal center formation and immunoglobulin secretion. Although deletion of Btk alone impaired Ca2+ mobilization upon BCR activation, the defect was even more severe when Vav1 or Vav3 were also mutated, indicating that Btk and the Vav proteins act in separate pathways that converge on Ca2+ signaling. In vitro ASC differentiation suggests that both B and T cells contribute to the observed phenotype of a Btk/Vav-double deficiency. Our results show that Vav proteins and Btk are both components of the BCR-activated signalosome but control separate signaling pathways important for B cell development.

Irreversible impact of early thermal conditions: an integrative study of developmental plasticity linked to mobility in a butterfly species.

Within populations, phenotypic plasticity may allow adaptive phenotypic variation in response to selection generated by environmental heterogeneity. For instance, in multivoltine species, seasonal changes between and within generations may trigger morphological and physiological variation enhancing fitness under different environmental conditions. These seasonal changes may irreversibly affect adult phenotypes when experienced during development. Yet, the irreversible effects of developmental plasticity on adult morphology have rarely been linked to life-history traits even though they may affect different fitness components such as reproduction, mobility and self-maintenance. To address this issue, we raised larvae of Pieris napi butterflies under warm or cool conditions to subsequently compare adult performance in terms of reproduction performance (as assessed through fecundity), displacement capacity (as assessed through flight propensity and endurance) and self-maintenance (as assessed through the measurement of oxidative markers). As expected in ectotherms, individuals developed faster under warm conditions and were smaller than individuals developing under cool conditions. They also had more slender wings and showed a higher wing surface ratio. These morphological differences were associated with changes in the reproductive and flight performance of adults, as individuals developing under warm conditions laid fewer eggs and flew larger distances. Accordingly, the examination of their oxidative status suggested that individuals developing under warm conditions invested more strongly into self-maintenance than individuals developing under cool conditions (possibly at the expense of reproduction). Overall, our results indicate that developmental conditions have long-term consequences on several adult traits in butterflies. This plasticity probably acts on life-history strategies for each generation to keep pace with seasonal variations and may facilitate acclimation processes in the context of climate change.

PLCG1 is required for AML1-ETO leukemia stem cell self-renewal.

In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

Maternal B cell signaling orchestrates fetal development in mice.

B cell participation in early embryo/fetal development and the underlying molecular pathways have not been explored. To understand whether maternal B cell absence or impaired signaling interferes with placental and fetal growth, we paired CD19-deficient (CD19-/-) mice, females with B cell-specific MyD88 (BMyD88-/-) or IL10 (BIL10-/-) deficiency as well as wild-type and MyD88-/- controls on C57Bl/6 background with BALB/c males. Pregnancies were followed by ultrasound and Doppler measurements. Implantation number was reduced in BMyD88-/- and MyD88-/- mice. Loss of MyD88 or B cell-specific deletion of MyD88 or IL10 resulted in decreased implantation areas at gestational day (gd) 5, gd8 and gd10, accompanied by reduced placental thickness, diameter and areas at gd10. Uterine artery resistance was enhanced in BIL10-/- dams at gd10. Challenge with 0.4 mg lipopolysaccharide/kg bodyweight at gd16 revealed that BMyD88-/-, BIL10-/- and CD19-/- mothers delivered preterm, whereas controls maintained their pregnancy. B cell-specific MyD88 and IL10 expression is essential for appropriate in utero development. IL10+B cells are involved in uterine blood flow regulation during pregnancy. Finally, B cell-specific CD19, MyD88 and IL10 expression influences susceptibility towards preterm birth.

Non-destructive investigation of extracellular enzyme activities and kinetics in intact freshwater biofilms with mineral beads as carriers.

Current procedures for fluorometric detection of extracellular hydrolytic enzyme activities in intact aquatic biofilms are very laborious and insufficiently standardized. To facilitate the direct determination of a multitude of enzymatic parameters without biofilm disintegration, a new approach was followed. Beads made of different mineral materials were subjected to biofilm growth in various aquatic environments. After biofilm coating, the beads were singly placed in microplate wells, containing the required liquid analytical medium and a fluorogenic substrate. Based on fluorometric detection of the enzymatically generated reaction products, enzyme activities and kinetics were determined. Mean enzymatic activities of ceramic bead-attached biofilms grown in a natural stream followed the decreasing sequence L-alanine aminopeptidase > L-leucine aminopeptidase > phosphomonoesterase > ß-glucosidase > phosphodiesterase > α-glucosidase > sulfatase. After one week of exposure, the relative standard deviations of enzyme activities ranged from 21 to 67%. Sintered glass bead-associated biofilms displayed the lowest standard deviations ranging from 19 to 34% in all experiments. This material proved to be suitable for short-time experiments in stagnant media. Ceramic beads were stable during more than three weeks of exposure in a natural stream. Biofilm formation was inhomogeneous or poorly visible on glass and lava beads accompanied by high variations of enzyme activities. The applicability of the method to study enzyme inhibition reactions was successfully proven by the determination of inhibition effects of caffeine on biofilm-associated phosphodiesterase.Key points• Optimized method to determine enzymatic parameters in aquatic biofilms• Direct investigation of bead-bound biofilms without biofilm disintegration• Fluorometric detection offers high sensitivity and sample throughput.

Indications for rapid evolution of trait means and thermal plasticity in range-expanding populations of a butterfly.

Currently, poleward range expansions are observed in many taxa, often in response to anthropogenic climate change. At the expanding front, populations likely face cooler and more variable temperature conditions, imposing thermal selection. This may result in changes in trait means or plasticity, the relative contribution of which is not well understood. We, here, investigate evolutionary change in range-expanding populations of the butterfly Pieris mannii, by comparing populations from the core and the newly established northern range under laboratory conditions. We observed both changes in trait means and in thermal reaction norms. Range-expanding populations showed a more rapid development, potentially indicative of counter-gradient variation and an increased cold tolerance compared with core populations. Genotype-environment interactions prevailed in all associated traits, such that the above differences were restricted to cooler environmental conditions. In range-expanding populations, plasticity was decreased in developmental traits enabling relatively rapid growth even under cooler conditions but increased in cold tolerance arguably promoting higher activity under thermally challenging conditions. Notably, these changes must have occurred within a time period of ca. 10 years only. Our results suggest, in line with contemporary theory, that the evolution of plasticity may play a hitherto underestimated role for adaptation to climatic variation. However, rather than generally increased or decreased levels of plasticity, our results indicate fine-tuned, trait-specific evolutionary responses to increase fitness in novel environments.

Large- and small-scale geographic structures affecting genetic patterns across populations of an Alpine butterfly.

Understanding factors influencing patterns of genetic diversity and the population genetic structure of species is of particular importance in the current era of global climate change and habitat loss. These factors include the evolutionary history of a species as well as heterogeneity in the environment it occupies, which in turn can change across time. Most studies investigating spatio-temporal genetic patterns have focused on patterns across wide geographic areas rather than local variation, but the latter can nevertheless be important particularly in topographically complex areas. Here, we consider these issues in the Sooty Copper butterfly (Lycaena tityrus) from the European Alps, using genome-wide SNPs identified through RADseq. We found strong genetic differentiation within the Alps with four genetic clusters, indicating western, central, and eastern refuges, and a strong reduction of genetic diversity from west to east. This reduction in diversity may suggest that the southwestern refuge was the largest one in comparison to other refuges. Also, the high genetic diversity in the west may result from (a) admixture of different western refuges, (b) more recent demographic changes, or (c) introgression of lowland L. tityrus populations. At small spatial scales, populations were structured by several landscape features and especially by high mountain ridges and large river valleys. We detected 36 outlier loci likely under altitudinal selection, including several loci related to membranes and cellular processes. We suggest that efforts to preserve alpine L. tityrus should focus on the genetically diverse populations in the western Alps, and that the dolomite populations should be treated as genetically distinct management units, since they appear to be currently more threatened than others. This study demonstrates the usefulness of SNP-based approaches for understanding patterns of genetic diversity, gene flow, and selection in a region that is expected to be particularly vulnerable to climate change.

An advanced method for the immunohistochemical detection of nitric oxide synthase (NOS) in the female genital tract.

The expression of nitric oxide synthase (NOS) in male and female urogenital tissues has been investigated by using conventional light microscopical immunoperoxidase staining. We present an improved immunohistochemical method for the specific and simultaneous detection of endothelial and neuronal NOS (eNOS/nNOS) in vaginal tissue. Specific antibodies have been used in combination with the tyramide signal amplification method. We found a subepithelial meshwork of varicose nerve fibers. A subpopulation of fibers presented immunoreactivity specific for nNOS. Epithelial cells also showed cytoplasmatic labeling for nNOS. Arteries presenting signals for eNOS in their endothelial layer were found in close proximity to nNOS-positive nerve fibers.

Birds of different morphs use slightly different strategies to achieve similar reproductive performance following heatwave exposure.

Responses to extreme climatic events may differ between individuals of distinct morphs which differ in life-history strategies, resulting in climate change 'winners' and 'losers' within species. We examined the reproductive performance and carry-over effects on offspring of black- and red-headed Gouldian finches Erythrura gouldiae after exposure to simulated heatwaves of moderate or severe intensity. We expected black-headed pairs' reproductive performance to decline after the severe heatwave because only the condition of black-headed females deteriorates during such a heatwave. Supporting the fact that Gouldian finches of different morphs use alternative reproductive strategies, we found that black-headed females initiated egg-laying a month earlier than red-headed females after experiencing a severe heatwave. We also found that this severe heatwave resulted in shorter spermatozoa in males irrespective of their morph. Despite these effects associated with heatwave intensity, the overall reproductive performance of both morphs was not affected by this factor, which was possibly due to an increased nestling provisioning rate by parents after exposure to the severe heatwave. However, offspring still bore the cost of parental exposure to the severe heatwave, as they showed a reduced condition (lower plasma antioxidant capacity and transient lower breathing rate) and higher oxidative damage (at least in fledglings with black-headed parents). These results suggest that inter-morph phenotypic variability in the Gouldian finch does not result in clear differences in reproductive performance following heatwave exposure, despite basal phenotypic differences between morphs. Whether animals using alternative reproductive strategies are, in the end, differently affected by climate changes will likely depend on the capacity of their offspring to recover from altered developmental conditions.