Resolution of a taxonomic conundrum: an ultrastructural and molecular description of the life cycle of Pleistophora mulleri (Pfeiffer 1895; Georgevitch 1929)

The classification of a microsporidian parasite observed in the abdominal muscles of amphipod hosts has been repeatedly revised but still remains inconclusive. This parasite has variable spore numbers within a sporophorous vesicle and has been assigned to the genera Glugea, Pleistophora, Stempellia, and Thelohania. We used electron microscopy and molecular evidence to resolve the previous taxonomic confusion and confirm its identification as Pleistophora mulleri. The life cycle of P. mulleri is described from the freshwater amphipod host Gammarus duebeni celticus. Infection appeared as white tubular masses within the abdominal muscle of the host. Light and transmission electron microscope examination revealed the presence of an active microsporidian infection that was diffuse within the muscle block with no evidence of xenoma formation. Paucinucleate merogonial plasmodia were surrounded by an amorphous coat immediately external to the plasmalemma. The amorphous coat developed into a merontogenetic sporophorous vesicle that was present throughout sporulation. Sporogony was polysporous resulting in uninucleate spores, with a bipartite polaroplast, an anisofilar polar filament and a large posterior vacuole. SSU rDNA analysis supported the ultrastructural evidence clearly placing this parasite within the genus Pleistophora. This paper indicates that Pleistophora species are not restricted to vertebrate hosts.

Upgrading of suberin from cork and birch outer bark

O trabalho aqui apresentado teve como principal propósito o estudo do potencial da suberina como fonte de produtos de química fina e como precursor de novos materiais macromoleculares de origem renovável. O interesse na suberina reside, não só na sua ubiquidade e nas suas propriedades únicas em termos de composição química e hidrofobicidade, mas também no facto de ser um dos principais componentes macromoleculares dos subprodutos da indústria corticeira de Quercus suber L. no Sul da Europa, e da indústria de pasta de papel do Norte da Europa, que utiliza a Betula pendula Roth como matéria-prima. A primeira parte do presente trabalho consistiu no estudo detalhado da composição química da cortiça de Quercus suber L. e respectivos resíduos industriais bem como da casca de Betula pendula Roth recorrendo a diferentes técnicas de caracterização, nomeadamente GC-MS, IV, RMN de 1H e de 13C, DSC, termomicroscopia, TGA e difracção de raios-X. Os resultados mostraram que os produtos de despolimerização da suberina representam tipicamente uma fracção substancial de todas as amostras. Para além da suberina, foram também identificados nas diversas amostras quantidades variáveis de compostos triterpénicos, lenhina, polissacarídeos e matéria inorgânica. Os principais resultados da análise por GC-MS mostraram que todas as amostras de suberina despolimerizada são fontes abundantes de ω-hidroxiácidos e de ácidos dicarboxílicos, bem como dos correspondentes derivados epóxidados. No entanto, as quantidades relativas de cada componente identificado foram significativamente diferentes entre amostras. Por exemplo, em amostras de suberina da casca de Quercus suber L. isoladas por metanólise alcalina o composto maioritário encontrado foi o ácido 22-hidroxidocosanóico, enquanto que a suberina também proveniente da cortiça, mas isolada por hidrólise alcalina era composta maioritariamente pelo ácido 9,10-dihidroxioctadecanóico. Já no caso da amostra de suberina despolimerizada proveniente da casca externa da bétula o composto identificado como mais abundante foi o ácido 9,10-epoxi-18-hidroxioctadecanóico. A caracterização das diversas amostras de suberina despolimerizada por FTIR e RMN de 1H e de 13C foram concordantes com os resultados de GC-MS, evidenciando a sua natureza predominantemente lipofílica. Foi ainda determinada a razão entre os grupos CO2H/OH e CO2CH3/OH por RMN de 1H das amostras convenientemente derivatizadas com isocianato de tricloroacetilo, verificando-se que a suberina despolimerizada possuía quantidades não-estequiométricas destes grupos funcionais. A investigação do comportamento térmico das amostras de suberina despolimerizada, por DSC e termomicroscopia, bem como a análise por difracção de raios-X, permitiu concluir que algumas amostras de suberina despolimerizada possuíam importantes domínios cristalinos e pontos de fusão bem definidos, tipicamente próximos de 70 oC, enquanto outras amostras eram essencialmente amorfa. Factores como a fonte de suberina ou as condições de despolimerização estiveram na origem destas diferenças. iv Neste trabalho estudaram-se também os extractáveis lipofílicos da cortiça e dos seus resíduos industriais, em particular os do pó industrial de cortiça e dos condensados negros, mostrando que os extractáveis lipofílicos são uma fonte abundante de compostos triterpénicos, em particular de ácido betulínico e de friedelina. Foram ainda identificadas fracções abundantes de ω-hidroxiácidos e de ácidos dicarboxílicos no condensado negro. A segunda parte deste trabalho abordou a síntese e a caracterização de novos poliésteres alifáticos derivados de suberina. Estes materiais foram sintetizados utilizando, quer misturas de suberina despolimerizada, quer monómeros modelo estruturalmente análogos aos existentes na suberina. Recorreu-se para o efeito a duas aproximações distintas de polimerização por passos, a policondensação e a politransesterificação. Procurou-se em simultâneo maximizar a eficiência da polimerização em termos de peso molecular e de extensão da reacção e utilizar condições de reacção de química “verdes”. Neste sentido, utilizou-se a policondensação em emulsão utilizando um tensioactivo como catalisador e a policondensação em massa utilizando a lipase B de Candida antarctica. Adicionalmente foram também testado os catalisadores trifluorometanosulfonato de bismuto(III) no caso da policondensação, e ainda os catalisadores clássicos óxido de antimónio(III) e o carbonato de potássio no caso da politransesterificação. Os poliésteres resultantes foram caracterizados através de várias técnicas, tais como IV, RMN (de 1H e de 13C), DSC, DMA, TGA, difracção de raios-X e medidas dos ângulos de contacto. Verificou-se que os catalisadores trifluorometanosulfonato de bismuto (III), óxido de antimónio(III) e carbonato de potássio conduziram aos rendimentos de isolamento dos polímeros resultantes mais elevados. No caso dos poliésteres derivados da suberina os resultados em termos de rendimentos e pesos moleculares sofreram um incremento substancial quando a estequiometria da reacção de polimerização foi adequadamente balanceada (r=1) com a adição de uma quantidade extra de um comonómero. Verificou-se a predominância de diferentes estruturas consoante a amostra de suberina utilizada e as condições de síntese adoptada, predominando as cadeias lineares ou então quantidades substanciais de estruturas reticuladas. Globalmente, este primeiro estudo sistemático da utilização de suberina como um precursor de novos poliésteres alifáticos confirmou o elevado potencial deste recurso abundante e renovável como precursor para preparar materiais macromoleculares.

The Long Range Transport of Birch (Betula) Pollen from Poland and Germany Causes Significant Pre-season Concentrations in Denmark

Background Birch pollen is highly allergic and has the potential for episodically long range transport. Such episodes will in general occur out of the main pollen season. During that time allergy patients are unprotected and high pollen concentrations will therefore have a full allergenic impact. Objective To show that Denmark obtains significant quantities of birch pollen from Poland or Germany before the local trees start to flower. Methods Simultaneous observations of pollen concentrations and phenology in the potential source area in Poland as well as in Denmark were performed in 2006. The Danish pollen records from 2000-2006 were analysed for possible long range transport episodes and analysed with trajectories in combination with a birch tree source map. Results In 2006 high pollen concentrations were observed in Denmark with bi-hourly concentrations above 500 grains/ m3 before the local trees began to flower. Poland was identified as a source region. The analysis of the historical pollen record from Copenhagen shows significant pre-seasonal pollen episodes almost every year from 2000-2006. In all episodes trajectory analysis identified Germany or Poland as source regions. Conclusion Denmark obtains significant pre-seasonal quantities of birch pollen from either Poland or Germany almost every year. Forecasting of birch pollen quantities relevant to allergy patients must therefore take into account long-range transport. This cannot be based on measured concentrations in Denmark. The most effective way to improve the current Danish pollen forecasts is to extend the current forecasts with atmospheric transport models that take into account pollen emission and transport from countries such as Germany and Poland. Unless long range transport is taken into account pre-seasonal pollen episodes will have a full allergic impact, as the allergy patients in general will be unprotected during that time.

Are the Birch Trees in Southern England a Source of Betula Pollen for North London?

Birch pollen is highly allergenic. Knowledge of daily variations, atmospheric transport and source areas of birch pollen is important for exposure studies and for warnings to the public, especially for large cities such as London. Our results show that broad-leaved forests with high birch tree densities are located to the south and west of London. Bi-hourly Betula pollen concentrations for all the days included in the study, and for all available days with high birch pollen counts (daily average birch pollen counts >80 grains/m3), show that, on average, there is a peak between 1400 hours and 1600 hours. Back-trajectory analysis showed that, on days with high birch pollen counts (n=60), 80% of air masses arriving at the time of peak diurnal birch pollen count approached North London from the south in a 180 degree arc from due east to due west. Detailed investigations of three Betula pollen episodes, with distinctly different diurnal patterns compared to the mean daily cycle, were used to illustrate how night-time maxima (2200–0400 hours) in Betula pollen counts could be the result of transport from distant sources or long transport times caused by slow moving air masses. We conclude that the Betula pollen recorded in North London could originate from sources found to the west and south of the city and not just trees within London itself. Possible sources outside the city include Continental Europe and the Betula trees within the broad-leaved forests of Southern England.

Release of Bet v 1 from Birch Pollen 1 from 5 European 2 Countries. Results from the HIALINE Study

Exposure to allergens is pivotal in determining sensitization and allergic symptoms in individuals. Pollen grain counts in ambient air have traditionally been assessed to estimate airborne allergen exposure. However, the exact allergen content of ambient air is unknown. We therefore monitored atmospheric concentrations of birch pollen grain and the matched major birch pollen allergen Bet v 1 simultaneously across Europe within the EU-funded project HIALINE (Health Impacts of Airborne Allergen Information Network). Pollen count was assessed with Hirst type pollen traps at 10 l/min at sites in France, United Kingdom, Germany, Italy and Finland. Allergen concentrations in ambient air were sampled at 800l/min with a Chemvol high-volume cascade impactor equipped with stages PM>10μm, 10 μm>PM>2.5μm, and in Germany also 2.5 μm>PM>0.12μm. The major birch pollen allergen Bet v 1 was determined with an allergen specific ELISA. Bet v 1 isoform patterns were analyzed by 2D-SDS-PAGE blots and mass spectrometric identification. Basophil activation was tested in an FcεR1-humanized rat basophil cell line passively sensitized with serum of a birch pollen lmptomatic patient. Compared to 10 previous years, 2009 was a representative birch pollen season for all stations. About 90% of the allergen was found in the PM>10μm fraction at all stations. Bet v 1 isoforms pattern did not varied substantially neither during ripening of pollen nor between different geographical locations. The average European allergen release from birch pollen was 3.2 pg Bet v 1/pollen and did not vary much between the European countries. However, in all countries a >10-fold difference in daily allergen release per pollen was measured which could be explained by long range transport of pollen with a deviating allergen release. Basophil activation by ambient air extracts correlated better with airborne allergen than with pollen concentration. Although Bet v 1 is a mixture of different isoforms, its fingerprint is constant across Europe. Bet v 1 was also exclusively linked to pollen. Pollen from different days varied >10-fold in allergen release. Thus exposure to allergen is inaccurately monitored by only monitoring birch pollen grains. Indeed, a humanized basophil activation test correlated much better with allergen concentrations in ambient air than with pollen count. Monitoring the allergens themselves together with pollen in ambient air might be an improvement in allergen exposure assessment.

Pollen Season and Climate: Is the Timing of Birch Pollen Release in the UK Approaching its Limit?

In light of heightened interest in the response of pollen phenology to temperature, we investigated recent changes to the onset of Betula (birch) pollen seasons in central and southern England, including a test of predicted advancement of the Betula pollen season for London. We calculated onset of birch pollen seasons using daily airborne pollen data obtained at London, Plymouth and Worcester, determined trends in the start of the pollen season and compared timing of the birch pollen season with observed temperature patterns for the period 1995–2010. We found no overall change in the onset of birch pollen in the study period although there was evidence that the response to temperature was nonlinear and that a lower asymptotic start of the pollen season may exist. The start of the birch pollen season was strongly correlated with March mean temperature. These results reinforce previous findings showing that the timing of the birch pollen season in the UK is particularly sensitive to spring temperatures. The climate relationship shown here persists over both longer decadal-scale trends and shorter, seasonal trends as well as during periods of ‘sign-switching’ when cooler spring temperatures result in later start dates. These attributes, combined with the wide geographical coverage of airborne pollen monitoring sites, some with records extending back several decades, provide a powerful tool for the detection of climate change impacts, although local site factors and the requirement for winter chilling may be confounding factors.

Lessons Learnt From Ragweed and Birch Studies

Here we review some of the most important aspects of recent work on Ragweed (Ambrosia) and birch (Betula) concerning: 1) sources, 2) trends & phenology and 3) dispersion and transformation. Sources: At Northern latitudes the birch fraction in forests usually exceeds 50% of all broadleaved trees and the abundance of birch decreases with latitude from 5%-20% in many mid-latitude regions and down to 0%-2% in more southern areas. Birches are also commonly found in small woodlands or planted as ornamental trees in urban areas. Ragweeds are herbaceous weed species that are associated with areas of disturbance. Ragweed is native to North America, but considered an invasive species in Europe, Australia and China. In Europe, the four main centres are: The Pannonian Plain, Ukraine, The Po Valley (Italy) and the Rhone Valley (France). Trends & Phenology: Birch pollen seasons have started earlier during the last decades. This trend appears have decreased during recent years despite increasing spring temperatures. Ragweed tends to experience less change in flowering date as ragweed flowering depends on photoperiod. Ragweed is increasing its distribution in Europe, but airborne concentrations of ragweed pollen are not universally increasing, e.g. due to control measures or pest attacks. Dispersion & transformation: The beginning of the birch pollen season is often heralded by episodes of Long Distance Transport (LDT) from the south. Similar LDT episodes are intermittently seen for ragweed, which can reach as far north as Scandinavia. Humidity and air pollution can modify pollen grains during atmospheric transport. This can cause a change in allergenic potential of the pollen grain and is a direction for future research including the effect of co-exposure of air pollution and the transformation of aeroallergens.

Pollen From Alder (Alnus sp.), Birch (Betula sp.) and Oak (Quercus sp.) in the UK Originate From Small Woodlands

We have analysed the pollen seasons in Worcester for the period 2005–12 for alder (Alnus), birch (Betula) and oak (Quercus) by using back trajectory calculations and produced the first detailed source maps for these three pollen types. The study shows considerable variations in the source–receptor relationship of three of the most important tree pollen types in England with respect to allergy. Long Distance Transport is observed for Quercus and Betula but not for Alnus. The new source maps show a number of high emitting areas for Betula and Quercus, mainly near London, in the Midlands and in Wales. The production of source maps is sensitive to the used type of land cover data and how well they incorporate small woodlands. Two satellite products, Corine Land Cover and Globcover, are compared with the detailed national land cover product Land Cover Map 2007. The broad scale satellite products show either up to 50% less woody coverage or a direct misplacement of woodlands. The Lagrangian back trajectory model, the pollen count observations and the source maps altogether suggest that small woodlands (below 25 ha) play a major role in the overall pollen load in urban areas in England.

Microbial impacts on plant-herbivore interactions: the indirect effects of a birch pathogen on a birch aphid

The role of indirect interactions in structuring communities is becoming increasingly recognised. Plant fungi can bring about changes in plant chemistry which may affect insect herbivores that share the same plant, and hence the two may interact indirectly. This study investigated the indirect effects of a fungal pathogen (Marssonina betulae) of silver birch (Betula pendula) on an aphid (Euceraphis betulae), and the processes underpinning the interaction. There was a strong positive association between natural populations of the aphid and leaves bearing high fungal infection. In choice tests, significantly more aphids settled on leaves inoculated with the fungus than on asymptomatic leaves. Individual aphids reared on inoculated leaves were heavier, possessed longer hind tibiae and displayed enhanced embryo development compared with aphids reared on asymptomatic leaves; population growth rate was also positively correlated with fungal infection when groups of aphids were reared on inoculated branches. Changes in leaf chemistry were associated with fungal infection with inoculated leaves containing higher concentrations of free-amino acids. This may reflect a plant-initiated response to fungal attack in which free amino acids from the degradation of mesophyll cells are translocated out of infected leaves via the phloem. These changes in plant chemistry are similar to those occurring during leaf senescence, and are proposed as the mechanistic basis for the positive interaction between the fungus and aphid.

Influence of host plant heterogeneity on the distribution of a birch aphid

1. The spatial and temporal abundance of the aphid Euceraphis betulae was investigated in relation to heterogeneity in host plant ( Betula pendula) vigour and pathogenic stress. The performance of aphids feeding on vigorous and stressed foliage was also examined. 2. The plant stress and plant vigour hypotheses have been suggested as opposing ways in which foliage quality influences herbivore abundance. In many plants, however, vigorous growing foliage co-exists with stressed or damaged foliage. 3. There was a negative correlation between branch growth ( vigour) and branch stress ( leaf chlorosis), with the most vigorous branches displaying little or no stress, and the most stressed branches achieving poor growth. There was a similar negative correlation between vigour and stress at the level of individual trees, which themselves represented a continuum in quality. 4. At the beginning of the season, E. betulae were intermittently more abundant on vigorous branches than on branches destined to become stressed, but aphids became significantly more abundant on stressed branches later in the season, when symptoms of stress became apparent. Similar patterns of aphid abundance were seen on vigorous and stressed trees in the following year. 5. Euceraphis betulae performance was generally enhanced when feeding on naturally stressed B. pendula leaves, but there was some evidence for elevated potential reproduction when feeding on vigorous leaves too. 6. Overall, plant stress probably influences E. betulae distribution more than plant vigour, but the temporal and spatial variability in plant quality suggests that plant vigour could play a role in aphid distribution early in the season.

Changing management in Scottish birch woodlands: a potential threat to local invertebrate biodiversity

The silvicultural management of Scottish birch woodlands for timber production is replacing traditional low intensity management practices, such as domesticated livestock grazing. These new management practices involve thinning of existing woodlands to prescribed densities to maximize biomass and timber quality. Although presently infrequent, the wide scale adoption of this practice could affect invertebrate community diversity. The impact of these changes in management on Staphylinidae and Carabidae (Coleoptera) in 19 woodlands in Aberdeenshire, north-east Scotland was investigated. Grazing and logging practices were important determinants of beetle community structure. Woodland area had no effect on any measure of beetle community structure, although isolation did influence the abundance of one carabid species. Changes towards timber production forestry will influence the structure of invertebrate communities, although the scale at which this occurs will determine its effect.