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This study assessed the effect of varying prenatal protein levels on the development of homing behavior in rat pups.Long-Evans rats were fed one of the four isocaloric diets containing 6% (n = 7 litters), 12% (n = 9), 18% (n = 9), or 25% (n = 10) casein prior to mating and throughout pregnancy. At birth, litters were fostered to well-nourished control mothers fed a 25% casein diet during pregnancy, and an adequate protein diet (25% casein) was provided to weaning. On postnatal days 5, 7, 9, 11, and 13, homing behaviors, including activity levels, rate of successful returns to the nest quadrant and latencies to reach the nest over a 3-minute test period were recorded from two starting positions in the home cage. Adult body and brain weights were obtained at sacrifice (postnatal day 130 or 200).Growth was impaired in pups whose mothers were fed a 6% or, to a lesser extent, a 12% casein diet relative to pups whose mothers were fed the 18 and 25% casein diets. The 6 and 12% prenatal protein levels resulted in lower activity levels, with the greatest reduction on postnatal day 13. However, only the 6% pups had reduced success and higher latencies in reaching the nest quadrant when compared with pups from the three other nutrition groups. Latency in reaching the nest quadrant was significantly and negatively associated with adult brain weight.Home orientation is a sensitive measure of developmental deficits associated with variations in prenatal protein levels, including levels of protein deficiency that do not lead to overt growth failure.

Fungi have been long used as model organisms to investigate genetic and cellular processes. An overview is provided of how fungi function at a genetic level, including ploidy, gene structure, and gene flow by sexual and asexual processes. The tools used to study fungal genetics are then described, such techniques having widespread applications in medical mycology research. Classical genetic analysis includes the use of gene mapping by sexual crossing and tetrad analysis, and forward genetic experimentation based on mutagenesis, for which various mutant screening approaches are described. Molecular genetic analysis includes gene manipulation by transformation; different methods for gene knockout and targeting, and their application for forward and reverse genetic approaches, are outlined. Finally, molecular genetic methods used to study gene expression and function are reviewed, including use of inducible or constitutive overexpression, real-time PCR, cellular localization of gene products by fluorescent tagging, and detection of protein–protein interactions.

Seasonal change in the temperate and polar regions of Earth determines how the world looks around us and, in fact, how we live our day-to-day lives. For biological organisms, seasonal change typically involves complex physiological and metabolic reorganization, the majority of which is regulated by photoperiodism. Photoperiodism is the ability of animals and plants to use day length or night length, resulting in life-historical transformations, including seasonal development, migration, reproduction, and dormancy. Seasonal timing determines not only survival and reproductive success but also the structure and organization of complex communities and, ultimately, the biomes of Earth. Herein, a small mosquito, Wyeomyia smithii, that lives only in the water-filled leaves of a carnivorous plant over a wide geographic range, is used to explore the genetic and evolutionary basis of photoperiodism. Photoperiodism in W. smithii is considered in the context of its historical biogeography in nature to examine the startling finding that recent rapid climate change can drive genetic change in plants and animals at break-neck speed, and to challenge the ponderous 80+ year search for connections between daily and seasonal time-keeping mechanisms. Finally, a model is proposed that reconciles the seemingly disparate 24-h daily clock driven by the invariant rotation of Earth about its axis with the evolutionarily flexible seasonal timer orchestrated by variable seasonality driven by the rotation of Earth about the Sun.

Genes required for fungal secondary metabolite production are usually clustered, co-regulated and expressed in stationary growth phase. Chromatin modification has an important role in co-regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP-qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin-level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta.

Background The role of clothing in the management of eczema (also called atopic dermatitis or atopic eczema) is poorly understood. This trial evaluated the effectiveness and cost-effectiveness of silk garments (in addition to standard care) for the management of eczema in children with moderate to severe disease. Methods and findings This was a parallel-group, randomised, controlled, observer-blind trial. Children aged 1 to 15 y with moderate to severe eczema were recruited from secondary care and the community at five UK medical centres. Participants were allocated using online randomisation (1:1) to standard care or to standard care plus silk garments, stratified by age and recruiting centre. Silk garments were worn for 6 mo. Primary outcome (eczema severity) was assessed at baseline, 2, 4, and 6 mo, by nurses blinded to treatment allocation, using the Eczema Area and Severity Index (EASI), which was log-transformed for analysis (intention-to-treat analysis). A safety outcome was number of skin infections. Three hundred children were randomised (26 November 2013 to 5 May 2015): 42% girls, 79% white, mean age 5 y. Primary analysis included 282/300 (94%) children (n = 141 in each group). The garments were worn more often at night than in the day (median of 81% of nights [25th to 75th centile 57% to 96%] and 34% of days [25th to 75th centile 10% to 76%]). Geometric mean EASI scores at baseline, 2, 4, and 6 mo were, respectively, 9.2, 6.4, 5.8, and 5.4 for silk clothing and 8.4, 6.6, 6.0, and 5.4 for standard care. There was no evidence of any difference between the groups in EASI score averaged over all follow-up visits adjusted for baseline EASI score, age, and centre: adjusted ratio of geometric means 0.95, 95% CI 0.85 to 1.07, (p = 0.43). This confidence interval is equivalent to a difference of −1.5 to 0.5 in the original EASI units, which is not clinically important. Skin infections occurred in 36/142 (25%) and 39/141 (28%) of children in the silk clothing and standard care groups, respectively. Even if the small observed treatment effect was genuine, the incremental cost per quality-adjusted life year was £56,811 in the base case analysis from a National Health Service perspective, suggesting that silk garments are unlikely to be cost-effective using currently accepted thresholds. The main limitation of the study is that use of an objective primary outcome, whilst minimising detection bias, may have underestimated treatment effects. Conclusions Silk clothing is unlikely to provide additional benefit over standard care in children with moderate to severe eczema. Trial registration Current Controlled Trials ISRCTN77261365, In a randomized controlled trial, Kim Suzanne Thomas and colleagues examine the effectiveness of silk garments vs. standard care for treating eczema in children., Author summary Why was this study done? Prior to this trial, evidence on the use of silk garments for the management of eczema was limited. Three randomised controlled trials (RCTs) had been conducted, but these were small (74 participants in total) and at risk of bias. The existing evidence was insufficient to guide clinical practice on the use of silk clothing in the management of eczema, and no cost-effectiveness analyses had been undertaken. What did the researchers do and find? We conducted a pragmatic, observer-blind RCT that recruited 300 children with moderate to severe eczema and followed them for six months. Participants were randomised to receive standard eczema care plus silk clothing (100% sericin-free silk garments; DermaSilk or DreamSkin) or standard care alone. After six months, there was no evidence of a difference between the groups in eczema severity (Eczema Area and Severity Index score) assessed by research nurses; the 95% confidence interval ranged from 1.5 points favouring silk clothing to 0.5 points favouring standard care, which is not a clinically important difference. Even if the potential small benefit of silk garments was genuine, our analysis suggests that they are unlikely to be cost-effective within currently accepted thresholds, with an incremental cost per quality-adjusted life year of £56,811. What do these findings mean? The CLOTHES Trial is the first large, independent RCT to have evaluated silk garments for the management of eczema. The results of this trial suggest that silk garments are unlikely to provide additional clinical or economic benefits over standard care for children with moderate to severe eczema. These results provide robust evidence for health commissioners and prescribers to make informed clinical decisions.

The nutrient base of aquatic tree-hole commu- nities is derived from leaf litter, benthic detritus, and wa- ter flowing down the tree trunk (stemflow water). Previ- ous studies in eastern North America with the mosquito, Aedes triseriatus, have identified leaf litter as a major and stemflow water as a minor source of mosquito nutri- tion, but did not consider the role of the benthic detritus or how the aggregate or relative contribution of these sources of mosquito nutrition changed during the year. We use the leaf litter, benthic detritus, and stemflow wa- ter from tree holes in western Oregon (USA) to deter- mine how these substrates affect mass at metamorphosis, biomass yield, and fitness (cohort replacement rate; R0) of the mosquito, Aedes sierrensis, through both natural and simulated winters, the normal growing season for larvae in tree holes. We found that fresh leaf litter consti- tutes the major determinant of mosquito fitness by a fac- tor of >15:1 over any other substrate taken directly from tree holes in nature. The other substrates, including the benthic detritus, individually make only a meager contri- bution to mosquito fitness but, when added to the leaf lit- ter, can sustain yield and improve fitness at high, limit- ing larval densities. Nutritional quality of tree-hole sub- strates declines by >90% from early (fall) to late (spring) in the larval growing season. At both times of year, the coarse or fine detritus provide minor resources, and stemflow water provides no detectable contribution to mosquito nutrition. The resources in the litter are not transported during the year to the benthic detritus; rather, these resources are either exploited by mosquitoes when they first become available, or they deteriorate and be- come progressively more unavailable to them. Growth and development of A. sierrensis feeding on dried and reconstituted tree-hole contents during a 6-month simu- lated winter in the laboratory showed: (1) the same rela- tive contributions of leaf litter, benthic detritus, and stemflow water to mosquito nutrition, (2) that the winter deterioration of substrate quality is a direct consequence of microbial decomposition, and (3) that pre-emptive competition from pre-existing A. sierrensis greatly in- creases substrate deterioration. We conclude that the pro- gressive winter deterioration of larval resources in com- bination with the dry summers of western North America are the most likely environmental factors that limit spe- cies diversity in tree holes and that have selected for ear- ly recruitment (autumnal hatching) of A. sierrensis and for its univoltine life cycle from Mexico to Canada.

Photoperiodic time measurement regulating larval diapause in the pitcher-plant mosquito, Wyeomyia smithii, varies in a close relationship with latitude. The critical photoperiod mediating the maintenance and termination of diapause is positively correlated with latitude (r2 = 0.977) among six populations from southern (30–31° N), intermediate (40° N), and northern (46–49° N) latitudes in North America. The developmental response to unnaturally short and to unnaturally long photoperiods declines with increasing latitude, so that longer critical photoperiods are associated with a downward rather than a lateral shift in the photoperiodic response curve. Exotic light and dark cycles of varying period (T) with a short (10 h) photophase and a scotophase ranging from 14 (T = 24) to 62 (T = 72) h, reveal two geographic patterns: a decline in perturbability of the photoperiodic clock with increasing latitude, and no change with latitude in the 21-h period of rising and falling development with increasing T. These results show (1) that there is a rhythmic component to photoperiodic time measurement in W. smithii, (2) that the period of this rhythm is about 21 h in all populations, and (3) that more northern populations show decreasing responsiveness to photoperiod and increasing stability against perturbation by exotic period lengths (T > 24). Previous studies on W.␣smithii indicate that this single temperate species of a tropical and subtropical genus has evolved from south to north. We therefore conclude that the evolution of increasing critical photoperiod in W. smithii during its adaptive radiation into North America has more likely involved the amplitude and not the period of the underlying circadian pacemaker.

The mosquito, Wyeomyia smithii, enters a larval dormancy or diapause that is initiated, maintained, and terminated by photoperiod. The median or critical photoperiod regulating diapause increases from 12 h of light per day along the Gulf of Mexico, USA (30° N), to over 15 h in southern Canada (49° N). Photoperiodic time measurement in W. smithii comprises both rhythmic and hourglass (interval timer) components. Using interrupted-night and resonance experiments, we show that both the rhythmic and hourglass components are prominent in the southern (ancestral) populations and that the influence of the rhythmic component declines with increasing latitude, while the hourglass component remains strong in northern (derived) populations. Previously, it has been shown that the genetic differences in critical photoperiod between northern populations and their southern ancestors involve not only the additive (independent) effects of genes, but also gene-gene interaction (epistasis). We therefore conclude that adaptive evolution of W. smithii has probably involved the progressive epistatic masking of the ancestral rhythmic component resulting in photoperiodic time measurement in northern populations accomplished principally through a day-interval timer. A comparison of W. smithii with previous studies indicates that the decline in critical photoperiod with increasing latitude represents an overall decrease in response to light rather than a shift in the timing of photosensitivity among arthropods in general. We propose that the underlying functional components of photoperiodic time measurement, as well as the overt photoperiodic response, are either homologous or are themselves responding directly to selection over latitudinal gradients in seasonality.

Indirect military alliances suppress conflict between states that are up to three degrees removed in the alliance network., States form defensive military alliances to enhance their security in the face of potential or realized interstate conflict. The network of these international alliances is increasingly interconnected, now linking most of the states in a complex web of ties. These alliances can be used both as a tool for securing cooperation and to foster peace between direct partners. However, do indirect connections—such as the ally of an ally or even further out in the alliance network—result in lower probabilities of conflict? We investigate the extent to which military alliances produce peace between states that are not directly allied. We find that the peacemaking horizon of indirect alliances extends through the network up to three degrees of separation. Within this horizon of influence, a lack of decay in the effect of degrees of distance indicates that alliances do not diminish with respect to their ability to affect peace regardless of whether or not the states in question are directly allied. Beyond the three-degree horizon of influence, we observe a sharp decline in the effect of indirect alliances on bilateral peace. Further investigation reveals that the community structure of the alliance network plays a role in establishing this horizon, but the effects of indirect alliances are not spurious to the community structure.

The detrimental effect of fungal pathogens on forest trees is an increasingly important problem that has implications for the health of our planet. Despite this, the study of molecular plant-microbe interactions in forest trees is in its infancy, and very little is known about the roles of effector molecules from forest pathogens. Dothistroma septosporum causes a devastating needle blight disease of pines, and intriguingly, is closely related to Cladosporium fulvum, a tomato pathogen in which pioneering effector biology studies have been carried out. Here, we studied D. septosporum effectors that are shared with C. fulvum, by comparing gene sequences from global isolates of D. septosporum and assessing effector function in both host and non-host plants. Many of the effectors were predicted to be non-functional in D. septosporum due to their pseudogenization or low expression in planta, suggesting adaptation to lifestyle and host. Effector sequences were polymorphic among a global collection of D. septosporum isolates, but there was no evidence for positive selection. The DsEcp2-1 effector elicited cell death in the non-host plant Nicotiana tabacum, whilst D. septosporum DsEcp2-1 mutants showed increased colonization of pine needles. Together these results suggest that DsEcp2-1 might be recognized by an immune receptor in both angiosperm and gymnosperm plants. This work may lead to the identification of plant targets for DsEcp2-1 that will provide much needed information on the molecular basis of gymnosperm-pathogen interactions in forests, and may also lead to novel methods of disease control.

This part of our series includes about a third of the large and varied subgenus Carex. The sections included are related both in terms of general anatomy and morphology (including leaf-surface characters), and in DNA sequence data. Most are plants of wet habitats. Generally, the sections appear natural on leaf characters except for Carex where C. hirta and C. lasiocarpa have very different leaves. However, all of the species are distinctive in leaf-surface or leaf-section characters. In section Vesicariae, C. vesicaria and C. saxatilis are hypostomous and have smooth surfaces but differ in leaf section, and C. rostrata differs from both in having the stomata predominantly on the upper, papillose surface. In section Limosae, C. limosa has prominent papillae on both surfaces; in C. magellanica, the upper surface is smoother and the lower surface has shorter papillae; C. rariflora differs from both in having amphistomous leaves. These three species all have distinctive stomata. Section Phacocystis is variabl...

Summary Dothistroma needle blight is a forest disease of increasing international importance due to its ability to kill as well as to retard the growth of pines. It is caused by fungi in the genus Dothistroma that produce dothistromin, a non-host selective toxin and virulence factor that is involved in necrosis of pine tissue. Recent studies of the genome of one of the main pathogenic species, Dothistroma septosporum, showed that it contains many similarities to that of the biotroph Cladosporium fulvum, including the presence of candidate biotrophic effector genes, which supported the hypothesis that D. septosporum has a hemi-biotrophic lifestyle. Using Pinus radiata as a host, we used a combination of microscopy, histological and molecular tools to further test this hypothesis and to determine the stage of the disease cycle in which dothistromin toxin is produced. The results showed a biotrophic-type phase in which the fungus grew over the needle surface, penetrated through stomatal pores and colonized epistomatal chambers. The subsequent necrotrophic phase was characterized by colonization of the mesophyll and production of dothistromin, with a >100-fold increase in dothistromin levels from early necrotic lesion to sporulating lesion stages. This is consistent with the role of dothistromin as a virulence factor that is involved in lesion expansion.

Key message Dense linkage maps derived by analysing SNP dosage in autotetraploids provide detailed information about the location of, and genetic model at, quantitative trait loci. Abstract Recent developments in sequencing and genotyping technologies enable researchers to generate high-density single nucleotide polymorphism (SNP) genotype data for mapping studies. For polyploid species, the SNP genotypes are informative about allele dosage, and Hackett et al. (PLoS ONE 8:e63939, 2013) presented theory about how dosage information can be used in linkage map construction and quantitative trait locus (QTL) mapping for an F1 population in an autotetraploid species. Here, QTL mapping using dosage information is explored for simulated phenotypic traits of moderate heritability and possibly non-additive effects. Different mapping strategies are compared, looking at additive and more complicated models, and model fitting as a single step or by iteratively re-weighted modelling. We recommend fitting an additive model without iterative re-weighting, and then exploring non-additive models for the genotype means estimated at the most likely position. We apply this strategy to re-analyse traits of high heritability from a potato population of 190 F1 individuals: flower colour, maturity, height and resistance to late blight (Phytophthora infestans (Mont.) de Bary) and potato cyst nematode (Globodera pallida), using a map of 3839 SNPs. The approximate confidence intervals for QTL locations have been improved by the detailed linkage map, and more information about the genetic model at each QTL has been revealed. For several of the reported QTLs, candidate SNPs can be identified, and used to propose candidate trait genes. We conclude that the high marker density is informative about the genetic model at loci of large effects, but that larger populations are needed to detect smaller QTLs. Electronic supplementary material The online version of this article (doi:10.1007/s00122-014-2347-2) contains supplementary material, which is available to authorized users.

Dothistromin is a broad-spectrum mycotoxin produced by the Dothideomycete pine needle pathogen Dothistroma septosporum. It accumulates in lesions, causing characteristic red bands on needles infected with this fungus. Dothistromin is similar in structure to the aflatoxin precursor versicolorin B and the biosynthetic pathways of these toxins share many common gene products. Although dothistromin is not essential for pathogenicity in dothistroma needle blight, its presence in infected needles suggests it might have a role in the disease process. The hypothesis that dothistromin is a virulence factor was tested by studying Pinus radiata infected with dothistromin-deficient mutants of D. septosporum. The mutants were able to infect pine needles and complete their life cycle as previously shown, and were unaffected in spore germination, epiphytic growth or needle penetration. However, colonization of the mesophyll by the mutants was restricted compared to the wild type. Correspondingly, lesions produced by the mutants were smaller and produced significantly fewer spores than lesions produced by wildtype strains. Interestingly, ‘green islands’, in which chlorophyll was maintained at a higher level than in adjacent chlorotic and necrotic regions, surrounded early-appearing lesions caused by both wildtype and mutant strains. At a later stage of disease development green islands were still present in the mutant but appeared to be masked by the extended dothistromin-containing lesions in the wild type. Overall the results support a role for dothistromin in virulence in dothistroma needle blight.

Most species of Carex subgenus Vignea have several similar hermaphrodite spikes, channelled or keeled leaves, smooth below and smooth to roughened above with low papillae at the apices of the epidermal cells, and are hypostomous with superficial stomata. Most of the variation is quantitative, in growth-habit, in the breadth and folding of the leaf, in the roughness of the upper leaf surface, and in the form and degree of the toothing of the margins and keel of the leaf. The main exceptions to this pattern are the section Heleoglochin, where the stomata are in distinctive pits or grooves in the lower surface, the section Physoglochin (C. dioica and related species), which is dioecious (in Europe), single-spiked and has linear leaves, and the section Heleonastes (C. lachenalii) which has characteristically papillose leaf surfaces and the similar C. canescens (section Canescentes) which is amphistomous. The approximate size and distribution of stomata, papillae and teeth (‘prickles’) of the species are liste...

Pathogen incursions are a major impediment for global forest health. How pathogens and forest trees coexist over time, without pathogens simply killing their long-lived hosts, is a critical but unanswered question. The Dothistroma Needle Blight pathogen Dothistroma septosporum was introduced into New Zealand in the 1960s and remains a low-diversity, asexual population, providing a unique opportunity to analyze the evolution of a forest pathogen. Isolates of D. septosporum collected from commercial pine forests over 50 years were compared at whole-genome and phenotype levels. Limited genome diversity and increased diversification among recent isolates support the premise of a single introduction event. Isolates from the 1960s show significantly elevated virulence against Pinus radiata seedlings and produce higher levels of the virulence factor dothistromin compared to isolates collected in the 1990s and 2000s. However, later isolates have no increased tolerance to copper, used in fungicide treatments of infested forests and traditionally assumed to be a strong selection pressure. The isolated New Zealand population of this forest pathogen therefore appears to have become less virulent over time, likely in part to maintain the viability of its long-lived host. This finding has broad implications for forest health and highlights the benefits of long-term pathogen surveys.

Scanning electron micrographs are presented of ten species of Carex, illustrating the range of leaf characters displayed by this technique. Leaf-surface characters include the distribution and form of the stomata on the two surfaces, whether they are flush with the leaf-surface or sunk in pits or hidden amongst papillae, and the size, texture and ornamentation of the epidermal cells on the two surfaces of the leaf. Most Carices have their stomata mainly on the underside of the leaf (hypostomous); C. nigra and C. rostrata have most of their stomata on the upperside (epistomous); and C. canescens has stomata on both leaf surfaces (amphistomous). Stomatal distribution can be seen with a hand lens and is a useful field character. The stomata in many species are flush with the smooth leaf surface but, in C. flacca and C. panacea, they are hidden among the closely-set papillae covering the lower leaf surface. In C. rostrata, they are surrounded by papillae on the upper surface in contrast to C. vesicari...

Lecanosticta acicola is an ascomycete that causes brown spot needle blight of pine species in many regions of the world. This pathogen is responsible for a major disease of Pinus palustris in the USA and is a quarantine organism in Europe. In order to study the genetic diversity and patterns of spread of L. acicola, eleven microsatellite markers and two mating type markers were developed. An enrichment protocol was used to isolate microsatellite-rich DNA regions, and 18 primer pairs were designed to flank these regions, of which eleven were polymorphic. A total of 93 alleles were obtained across all loci from forty isolates of L. acicola from the USA with an allelic diversity range of 0.095 to 0.931 per locus. Cross-species amplification with some of the markers was obtained with L. gloeospora, L. guatemalensis and Dothistroma septosporum, but not with D. pini. Mating type (MAT) markers amplifying both idiomorphs were also developed to determine mating type distribution in populations. These markers were designed based on alignments of both idiomorphs of nine closely related plant pathogens, and a protocol for multiplex PCR amplification of the MAT loci was optimised. The MAT markers are not species specific and also amplify the MAT loci in Dothistroma septosporum, D. pini, L. gloeospora and L. guatemalensis. Both types of genetic markers developed in this study will be valuable for future investigations of the population structure, genetic diversity and invasion history of L. acicola on a global scale.

Dothistroma septosporum causes Dothistroma needle blight (DNB) on pines throughout the world. An increase in incidence and severity of DNB, associated with climatic factors, raises an urgent need to screen for resistance in plant hosts and to detect changes in virulence of the pathogen. The availability of the genome sequence for this fungal pathogen also opens up possibilities for biochemical and molecular studies leading to new methods of disease control. All these activities require a reliable and reproducible DNB pathogenicity assay, but this has been notoriously difficult to achieve in controlled glasshouse conditions. We therefore aimed to improve DNB assays by investigating the effects of various pathogen, environment and host factors, using Pinus radiata. The production of spores in sufficient quantity and quality for infection was optimised by using pine needle minimal medium with glucose (PMMG). The critical environmental parameter for achieving high levels of DNB (>80 % of needles with disease symptoms) was leaf wetness; use of individual covers for 4–7 days post inoculation, followed by incubation in covered chambers containing water foggers for the remainder of the infection cycle, enabled reliably high rates of disease incidence. Using this assay, it was possible to distinguish between trees from families with different levels of field resistance, using clones taken as cuttings from young trees (

Controlled laboratory experiments were used to show that Oregon and Alaskan three-spined stickleback Gasterosteus aculeatus, collected from locations differing by 18° of latitude, exhibited no significant variation in length of the polyglutamine domain of the clock protein or in photoperiodic response within or between latitudes despite the fact that male and female G. aculeatus are photoperiodic at both latitudes. Hence, caution is urged when interpreting variation in the polyglutamine repeat (PolyQ) domain of the gene clock in the context of seasonal activities or in relationship to photoperiodism along geographical gradients.

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Seasonal change in the temperate and polar regions of Earth determines how the world looks around us and, in fact, how we live our day-to-day lives. For biological organisms, seasonal change typically involves complex physiological and metabolic reorganization, the majority of which is regulated by photoperiodism. Photoperiodism is the ability of animals and plants to use day length or night length, resulting in life-historical transformations, including seasonal development, migration, reproduction, and dormancy. Seasonal timing determines not only survival and reproductive success but also the structure and organization of complex communities and, ultimately, the biomes of Earth. Herein, a small mosquito, Wyeomyia smithii, that lives only in the water-filled leaves of a carnivorous plant over a wide geographic range, is used to explore the genetic and evolutionary basis of photoperiodism. Photoperiodism in W. smithii is considered in the context of its historical biogeography in nature to examine the startling finding that recent rapid climate change can drive genetic change in plants and animals at break-neck speed, and to challenge the ponderous 80+ year search for connections between daily and seasonal time-keeping mechanisms. Finally, a model is proposed that reconciles the seemingly disparate 24-h daily clock driven by the invariant rotation of Earth about its axis with the evolutionarily flexible seasonal timer orchestrated by variable seasonality driven by the rotation of Earth about the Sun.

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