Morphometric discrimination of two sympatric sibling species in the Palaearctic region, Culicoides obsoletus Meigen and C-scoticus Downes & Kettle (Diptera: Ceratopogonidae), vectors of bluetongue and Schmallenberg viruses

Background Some Palaearctic biting midge species (subgenus Avaritia) have been implicated as vectors of bluetongue virus in northern Europe. Separation of two species (C. obsoletus and C. scoticus) is considered difficult morphologically and, often, these female specimens are grouped in entomological studies. However, species-specific identification is desirable to understand their life history characteristics, assess their roles in disease transmission or measure their abundance during arboviral outbreaks. This study aims to investigate whether morphometric identification techniques can be applied to female C. obsoletus and C. scoticus individuals trapped at different geographical regions and time periods during the vector season. Methods C. obsoletus and C. scoticus were collected using light-suction traps from the UK, France and Spain, with two geographical locations sampled per country. A total of 759 C. obsoletus/C. scoticus individuals were identified using a molecular assay based on the cytochrome c oxidase subunit I gene. Fifteen morphometric measurements were taken from the head, wings and abdomen of slide-mounted specimens, and ratios calculated between these measurements. Multivariate analyses explored whether a combination of morphometric variables could lead to accurate species identification. Finally, Culicoides spp. collected in France at the start, middle and end of the adult vector season were compared, to determine whether seasonal variation exists in any of the morphometric measurements. Results The principal component analyses revealed that abdominal characteristics: length and width of the smaller and larger spermathecae, and the length of the chitinous plates and width between them, are the most reliable morphometric characteristics to differentiate between the species. Seasonal variation in the size of each species was observed for head and wing measurements, but not abdominal measurements. Geographical variation in the size of Culicoides spp. was also observed and is likely to be related to temperature at the trapping sites, with smaller individuals trapped at more southern latitudes. Conclusions Our results suggest that female C. obsoletus and C. scoticus individuals can be separated under a stereomicroscope using abdominal measurements. Although we show the length and width of the spermathecae can be used to differentiate between the species, this can be time-consuming, so we recommend undertaking this using standardized subsampling of catches. (Résumé d'auteur

Modelling the effects of past and future climate on the risk of bluetongue emergence in Europe

Vector-borne diseases are among those most sensitive to climate because the ecology of vectors and the development rate of pathogens within them are highly dependent on environmental conditions. Bluetongue (BT), a recently emerged arboviral disease of ruminants in Europe, is often cited as an illustration of climate's impact on disease emergence, although no study has yet tested this association. Here, we develop a framework to quantitatively evaluate the effects of climate on BT's emergence in Europe by integrating high-resolution climate observations and model simulations within a mechanistic model of BT transmission risk. We demonstrate that a climate-driven model explains, in both space and time, many aspects of BT's recent emergence and spread, including the 2006 BT outbreak in northwest Europe which occurred in the year of highest projected risk since at least 1960. Furthermore, the model provides mechanistic insight into BT's emergence, suggesting that the drivers of emergence across Europe differ between the South and the North. Driven by simulated future climate from an ensemble of 11 regional climate models, the model projects increase in the future risk of BT emergence across most of Europe with uncertainty in rate but not in trend. The framework described here is adaptable and applicable to other diseases, where the link between climate and disease transmission risk can be quantified, permitting the evaluation of scale and uncertainty in climate change's impact on the future of such diseases

Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with Kms 10–20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit

Detection of Neptune-size planetary candidates with CoRoT data. Comparison with the planet occurrence rate derived from Kepler

[Abridged] Context. The CoRoT space mission has been searching for transiting planets since the end of December 2006. Aims. We aim to investigate the capability of CoRoT to detect small-size transiting planets in short-period orbits, and to compare the number of CoRoT planets with 2 \leq R_p \leq 4 Rearth with the occurrence rate of small-size planets provided by the distribution of Kepler planetary candidates (Howard et al. 2012). Methods. We performed a test that simulates transits of super-Earths and Neptunes in real CoRoT light curves and searches for them blindly by using the LAM transit detection pipeline. Results. The CoRoT detection rate of planets with radius between 2 and 4 Rearth and orbital period P \leq 20 days is 59% (31%) around stars brighter than r'=14.0 (15.5). By properly taking the CoRoT detection rate for Neptune-size planets and the transit probability into account, we found that according to the Kepler planet occurrence rate, CoRoT should have discovered 12 \pm 2 Neptunes orbiting G and K dwarfs with P \leq 17 days in six observational runs. This estimate must be compared with the validated Neptune CoRoT-24b and five CoRoT planetary candidates in the considered range of planetary radii. We thus found a disagreement with expectations from Kepler at 3 \sigma or 5 \sigma, assuming a blend fraction of 0% (six Neptunes) and 100% (one Neptune) for these candidates. Conclusions. This underabundance of CoRoT Neptunes with respect to Kepler may be due to several reasons. Regardless of the origin of the disagreement, which needs to be investigated in more detail, the noticeable deficiency of CoRoT Neptunes at short orbital periods seems to indirectly support the general trend found in Kepler data, i.e. that the frequency of small-size planets increases with increasing orbital periods and decreasing planet radii.Comment: 10 pages, 7 figures. Accepted for publication in A&

Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon

Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wallrelated genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and b-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening

Range expansion of the Bluetongue vector, Culicoides imicola, in continental France likely due to rare wind-transport events

The role of the northward expansion of Culicoides imicola Kieffer in recent and unprecedented outbreaks of Culicoides-borne arboviruses in southern Europe has been a significant point of contention. We combined entomological surveys, movement simulations of air-borne particles, and population genetics to reconstruct the chain of events that led to a newly colonized French area nestled at the northern foot of the Pyrenees. Simulating the movement of air-borne particles evidenced frequent wind-transport events allowing, within at most 36 hours, the immigration of midges from north-eastern Spain and Balearic Islands, and, as rare events, their immigration from Corsica. Completing the puzzle, population genetic analyses discriminated Corsica as the origin of the new population and identified two successive colonization events within west-Mediterranean basin. Our findings are of considerable importance when trying to understand the invasion of new territories by expanding species

Spatial abundance and clustering of Culicoides (Diptera: Ceratopogonidae) on a local scale

BACKGROUND: Biting midges, Culicoides, of the Obsoletus group and the Pulicaris group have been involved in recent outbreaks of bluetongue virus and the former was also involved in the Schmallenberg virus outbreak in northern Europe. METHODS: For the first time, here we investigate the local abundance pattern of these two species groups in the field by intensive sampling with a grid of light traps on 16 catch nights. Neighboring trap catches can be spatially dependent on each other, hence we developed a conditional autoregressive (CAR) model framework to test a number of spatial and non-spatial covariates expected to affect Culicoides abundance. RESULTS: The distance to sheep penned in the corner of the study field significantly increased the abundance level up to 200 meters away from the sheep. Spatial clustering was found to be significant but could not be explained by any known factors, and cluster locations shifted between catch nights. No significant temporal autocorrelation was detected. CAR models for both species groups identified a significant positive impact of humidity and significant negative impacts of precipitation and wind turbulence. Temperature was also found to be significant with a peak at just below 16 degrees Celcius. Surprisingly, there was a significant positive impact of wind speed. The CAR model for the Pulicaris group also identified a significant attraction to the smaller groups of sheep placed in the field. Furthermore, a large number of spatial covariates which were incorrectly found to be significant in ordinary regression models were not significant in the CAR models. The 95% C.I. on the prediction estimates ranged from 20.4% to 304.8%, underlining the difficulties of predicting the abundance of Culicoides. CONCLUSIONS: We found that significant spatial clusters of Culicoides moved around in a dynamic pattern varying between catch nights. This conforms with the modeling but was not explained by any of the tested covariates. The mean abundance within these clusters was up to 11 times higher for the Obsoletus group and 4 times higher for the Pulicaris group compared to the rest of the field

Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon

Methoderapport om Industriële emissies naar lucht te berekenen (actualisatie 2016) zoals gebruikt door de Emissieregistratie

In this technical report RIVM describes the updated methods that The Netherlands Pollutant Release and Transfer Register uses to calculate the emissions of contaminated substances into the air from the Industry, Energy Generating and Waste Processing sectors. Due to international treaties, such as the Kyoto protocol, the EU Emissions Ceiling (NEC Directive) and the Convention on Long-range Transboundary Air Pollution (CLRTAP), the Netherlands is obliged to always report in accordance with the most recent scientific insights regarding the emission of greenhouse gases, acidifying pollutants and substances related to large-scale air pollution. This description is used to substantiate the reported emissions. This report is targeted at the national and international reviewers that validate Dutch reports to the EU and UN.Het RIVM beschrijft in deze technische rapportage de geactualiseerde methoden waarmee de Nederlandse Emissieregistratie de uitstoot van verontreinigende stoffen naar de lucht berekent vanuit de sectoren Industrie, Energieopwekking en Afvalverwerking. Nederland is vanwege internationale verdragen, zoals het Kyoto-protocol, de EU-Emissieplafonds (NEC-Directive) en de Convention on Long-range Transboundary Air Pollution (CLRTAP), verplicht om steeds volgens de meest actuele wetenschappelijk inzichten te rapporteren over de uitstoot van broeikasgassen, verzurende stoffen en stoffen die gerelateerd zijn aan grootschalige luchtverontreiniging. Met deze beschrijving wordt de gerapporteerde uitstoot onderbouwd. Doelgroep voor deze rapportage zijn de (internationale) reviewers die de Nederlandse rapportages aan de EU en VN valideren.Ministerie van I&