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International audience ; Species specific detection and detection of groups of species such as ruminants is required according to European legislation dealing with the safe use of animal by-products in animal nutrition. Various methods are applied to the analysis of feed samples for the presence of banned processed animal proteins (PAPs) including meat and bone meal (MBM). Classical microscopy as described in the Commission Directive EC/2003/126 is the only official method to detect the presence of constituents of animal origin in feed, nevertheless some deviating protocols allowed under the old Directive (EC/88/1988) claim to gain comparable results. An inherent limitation of the official microscopic method is the lack of species specificity. Immnunoassays showed the most promising potential in research projects or intercomparison studies being able to detect ruminant PAPs at a concentration level of 0.5%. The aim of this paper is to present the results of the intercomparison study conducted on behalf of DG-Health and Consumer Protection (SANCO) in 2004 to establish whether the two-solvent Method would gain comparable results to the current European Method and to evaluate the current capability of immnuoassays of determining the species in PAPs present in feed.

Variation on bacterial communities living in the phyllosphere as epiphytes and endophytes has been attributed to plant host effects. However, there is contradictory or inconclusive evidence regarding the effect of plant genetics (below the species' level) and of plant tissue type on phyllosphere bacterial community assembly, in particular when epiphytes and endophytes are considered simultaneously. Here, both surface and internal bacterial communities of two olive (Olea europaea) cultivars were evaluated in twigs and leaves by molecular identification of cultivable isolates, with an attempt to answer these questions. Overall, Proteobacteria, Actinobacteria and Firmicutes were the dominant phyla, being epiphytes more diverse and abundant than endophytes. Host genotype (at cultivar level) had a structuring effect on the composition of bacterial communities and, in a similar way, for both epiphytes and endophytes. Plant organ (leaf vs. twig) control of the bacterial communities was less evident when compared with plant genotype and with a greater influence on epiphytic than on endophytic community structure. Each olive genotype/plant organ was apparently selective towards specific bacterial operational taxonomic units (OTUs), which may lead to specific feedbacks on fitness of plant genotypes. Bacterial recruitment was observed to happen mainly within epiphytes than in endophytes and in leaves as comparedwith twigs. Such host specificity suggested that the benefits derived fromthe plant– bacteria interaction should be considered at genetic levels below the species. ; The authors are grateful to the FEDER and Foundation for Science and Technology (FCT, Portugal) under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019) and BioISI (UID/MULTI/04046/2013), as well as the Horizon 2020, the European Union's Framework Programme for Research and Innovation, for financial support the project PRIMA/0002/2018 INTOMED—Innovative tools to combat crop pests in the Mediterranean. D. Mina thanks FCT, POPH-QREN and FSE forMSFRH-BD-105341/2014 grant. ; info:eu-repo/semantics/publishedVersion

BackgroundPesticide-environment interactions are bidirectional. The environment alters pesticides by metabolism and photodegradation, and pesticides in turn change the environment through nontarget or secondary effects.ObjectivesApproximately 900 currently used commercial pesticides of widely diverse structures act by nearly a hundred mechanisms to control insects, weeds, and fungi, usually with minimal disruption of nature's equilibrium. Here I consider some aspects of the discovery, development, and use of ecofriendly or green pesticides (i.e., pesticides that are safe, effective, and biodegradable with minimal adverse secondary effects on the environment). Emphasis is given to research in my laboratory.DiscussionThe need for understanding and improving pesticide-environment interactions began with production of the first major insecticide approximately 150 years ago: The arsenical poison Paris Green was green in color but definitely not ecofriendly. Development and use of other pesticides has led to a variety of problems. Topics considered here include the need for high purity [e.g., hexachlorocyclohexane and polychloroborane isomers and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)], environmental degradation and the bioactivity of resulting photoproducts and metabolites, pesticide photochemistry (including the use of structural optimization, photostabilizers, and photosensitizers to achieve suitable persistence), the presence of multiple active ingredients in botanical insecticides, the need to consider compounds with common mechanisms of action, issues related to primary and secondary targets, and chemically induced or genetically modified changes in plant biochemistry. Many insecticides are bird, fish, and honeybee toxicants, whereas herbicides and fungicides pose fewer environmental problems.ConclusionSix factors have contributed to the greening of pesticide-environment interactions: advances in pesticide chemistry and toxicology, banning of many chlorinated hydrocarbons, the development of new biochemical ...

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being X. arboricola pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in X. arboricola pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 102 CFU ml−1, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for X. arboricola pv. pruni strains from different origins as well as for closely related Xanthomonas species, non-Xanthomonas species, saprophytic bacteria, and healthy Prunus samples. The efficiency of the developed protocol was evaluated with field samples of 14 Prunus species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and X. arboricola pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for X. arboricola pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being X. arboricola pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in X. arboricola pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 10(2) CFU ml(-1), thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for X. arboricola pv. pruni strains from different origins as well as for closely related Xanthomonas species, non-Xanthomonas species, saprophytic bacteria, and healthy Prunus samples. The efficiency of the developed protocol was evaluated with field samples of 14 Prunus species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and X. arboricola pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for X. arboricola pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

Macrophage colony‐stimulating factor (CSF1 or M‐CSF) and interleukin 34 (IL34) are secreted cytokines that control macrophage survival and differentiation. Both act through the CSF1 receptor (CSF1R), a type III transmembrane receptor tyrosine kinase. The functions of CSF1R and both ligands are conserved in birds. We have analyzed protein‐coding sequence divergence among avian species. The intracellular tyrosine kinase domain of CSF1R was highly conserved in bird species as in mammals but the extracellular domain of avian CSF1R was more divergent in birds with multiple positively selected amino acids. Based upon crystal structures of the mammalian CSF1/IL34 receptor‐ligand interfaces and structure‐based alignments, we identified amino acids involved in avian receptor‐ligand interactions. The contact amino acids in both CSF1 and CSF1R diverged among avian species. Ligand‐binding domain swaps between chicken and zebra finch CSF1 confirmed the function of variants that confer species specificity on the interaction of CSF1 with CSF1R. Based upon genomic sequence analysis, we identified prevalent amino acid changes in the extracellular domain of CSF1R even within the chicken species that distinguished commercial broilers and layers and tropically adapted breeds. The rapid evolution in the extracellular domain of avian CSF1R suggests that at least in birds this ligand‐receptor interaction is subjected to pathogen selection. We discuss this finding in the context of expression of CSF1R in antigen‐sampling and antigen‐presenting cells. ; UK Government's Department for International Development. Grant Number: OPP1127286

19 páginas, 6 figuras, 4 tablas. ; Microbial symbionts form abundant and diverse components of marine sponge holobionts, yet the ecological and evolutionary factors that dictate their community structure are unresolved. Here, we characterized the bacterial symbiont communities of three sympatric host species in the genus Ircinia from the NW Mediterranean Sea, using electron microscopy and replicated 16S rRNA gene sequence clone libraries. All Ircinia host species harbored abundant and phylogenetically diverse symbiont consortia, comprised primarily of sequences related to other sponge-derived microorganisms. Community-level analyses of bacterial symbionts revealed host species-specific genetic differentiation and structuring of Ircinia-associated microbiota. Phylogenetic analyses of host sponges showed a close evolutionary relationship between Ircinia fasciculata and Ircinia variabilis, the two host species exhibiting more similar symbiont communities. In addition, several bacterial operational taxonomic units were shared between I. variabilis and Ircinia oros, the two host species inhabiting semi-sciophilous communities in more cryptic benthic habitats, and absent in I. fasciculata, which occurs in exposed, high-irradiance habitats. The generalist nature of individual symbionts and host-specific structure of entire communities suggest that: (1) a 'specific mix of generalists' framework applies to bacterial symbionts in Ircinia hosts and (2) factors specific to each host species contribute to the distinct symbiont mix observed in Ircinia hosts. ; This research was supported by the Spanish Government projects CTM2010-17755 and CTM2010-22218 and by the US National Science Foundation under grant 0853089. ; Peer reviewed

AbstractThe role of environmental factors and landscape heterogeneity on species distribution on different spatial scales is one of the most important questions in community ecology. Variations in the environmental gradient characteristics, host attributes and spatial scales may influence the parasites distribution. The helminth metacommunity of 12 small mammal species was investigated in an Atlantic Forest reserve located in the State of Rio de Janeiro, southeast Brazil. We evaluated the influence of environmental variables, host attributes and spatial factors on the helminth metacommunity of small mammals, considering infracommunity and component community levels. Twenty-nine helminth morphospecies were recovered. The host attributes and spatial variables influenced the abundance of helminth species in the metacommunities for rodents and marsupials together, and for rodents alone at the infracommunity level. Host body mass, host diet and spatial variables at broad spatial scale (among localities) were the most important variables to explain the variation in helminth abundance. Parasite species richness influenced this variation only for the marsupial helminth metacommunity at the infracommunity level. The metacommunity showed larger turnover (parasite replacement) than nestedness (parasite loss) for their helminth species at both infracommunity and component community levels, which is associated with a high host specificity, and low helminth sharing among hosts for most species, resulting in a structured metacommunity.

Variation on bacterial communities living in the phyllosphere as epiphytes and endophytes has been attributed to plant host effects. However, there is contradictory or inconclusive evidence regarding the effect of plant genetics (below the species' level) and of plant tissue type on phyllosphere bacterial community assembly, in particular when epiphytes and endophytes are considered simultaneously. Here, both surface and internal bacterial communities of two olive (Olea europaea) cultivars were evaluated in twigs and leaves by molecular identification of cultivable isolates, with an attempt to answer these questions. Overall,Proteobacteria,ActinobacteriaandFirmicuteswere the dominant phyla, being epiphytes more diverse and abundant than endophytes. Host genotype (at cultivar level) had a structuring effect on the composition of bacterial communities and, in a similar way, for both epiphytes and endophytes. Plant organ (leaf vs. twig) control of the bacterial communities was less evident when compared with plant genotype and with a greater influence on epiphytic than on endophytic community structure. Each olive genotype/plant organ was apparently selective towards specific bacterial operational taxonomic units (OTUs), which may lead to specific feedbacks on fitness of plant genotypes. Bacterial recruitment was observed to happen mainly within epiphytes than in endophytes and in leaves as compared with twigs. Such host specificity suggested that the benefits derived from the plant-bacteria interaction should be considered at genetic levels below the species. ; The authors are grateful to the FEDER and Foundation for Science and Technology (FCT, Portugal) under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019) and BioISI (UID/MULTI/04046/2013), as well as the Horizon 2020, the European Union's Framework Programme for Research and Innovation, for financial support the project PRIMA/0002/2018 INTOMED—Innovative tools to combat crop pests in the Mediterranean. D. Mina thanks FCT, POPH-QREN and FSE ...

Statistical analysis of 10 quantitative and 9 qualitative diagnostic characters of leaves and shoots of three Russian (Populus laurifolia Ledeb., P. nigra L., P. suaveolens Fisch.) and two Central Asian (P. usbekistanica Komarov, P. macrocarpa (Schrenk.) N. Pavl. et Lipsch.) poplar species of the Tacamahaca (Spach) Penjkovsky subgenus was performed. It was shown that according to studied quantitative characters P. nigra (Aegirus section) and P. suaveolens (Tacamahaca section) belong to opposite "poles". This could be possibly due to the fact that the first species is the most common for lowlands poplar in said group, and the second species is the most mountainous and confined to the most severe climatic conditions. The natural areas of these two species are also geographically extremely far from one another. The other three species occupy an intermediate position both in their characters, in terms of growth, and geographically. In addition, they all hybridize in nature with Populus nigra, and Populus laurifolia also with P. suaveolens, which can also explain the intermediate nature of their characters. It was shown that the leaf characters, such as leaf length and width, leaf length and width ratio etc., reflect the specificity of the studied species somewhat worse than a number of specific characters (leaf maximum width position, leaf teeth height and lateral edge radius of curvature, leaf top length and width).

We thank the government of Costa Rica for permission to collect butterflies, BGI for sequencing, R. Hudson for assistance with ms, and reviewers for comments on the manuscript. Computational infrastructure, data storage, and resources were provided to individual laboratories by Boston University, Temple University, University of Chicago, the University of Hawaii, and the California Academy of Sciences. Additional high-performance computing was facilitated by an NSF XCEDE start-up allocation (TG-MCB120130) to J.R.G. and funding from NSF EPSCoR (0554657 PI J. Gaines) at the University of Hawaii. Funding for this project was provided by National Science Foundation grants DEB-1316037 (to M.R.K.) and DEB-1021036 (to S.P.M.). (0554657 - NSF EPSCoR at the University of Hawaii; DEB-1316037 - National Science Foundation; DEB-1021036 - National Science Foundation)

The African citrus psyllid, Trioza erytreae, vectors citrus greening or huanglongbing (HLB) disease. The psyllid has been reported from mainland Europe, where it is rapidly spreading from the northwest to the southwest of the Iberian Peninsula. In order to reduce its spread and population levels, a classical biological control program with the parasitoid Tamarixia dryi is under development in Spain. We evaluated the host specificity of T. dryi using 11 non-target psyllid (NTP) species, including five species of the genus Trioza. The psyllids were selected based on phylogenetic and ecological criteria. Tamarixia dryi exhibited a high host specificity. Females did not parasitize any of the 11 NTPs tested, except for one nymph of a gall-forming Trioza species closely related to Trioza montanetana. Tamarixia dryi only laid one egg on a nymph when it was removed from the gall on Convolvulus canariensis and exposed directly to the parasitoid. However, the immature parasitoid died before emerging. We further confirmed that T. dryi did not parasitize a representative triozid species, Trioza laurisilvae, of the endemic Canarian fauna after long time exposure. Our results demonstrate that T. dryi is a highly specific parasitoid and its introduction, release and establishment in Europe within the classical biological control program of T. erytreae should not affect other psyllid species. Therefore, no significant environmental impact is expected. ; The research leading to these results was partially funded by European Union's Horizon 2020 research and innovation program under grant agreement No. 727459 "Insect-borne prokaryote-associated diseases in tropical and subtropical perennial crops" – TROPICSAFE, the Spanish Ministry of Economy and Competitiveness (INIA E-RTA RTA2015-00005-C06), the Conselleria d'Agricultura, Pesca i Alimentació de la Generalitat Valenciana and the Dirección General de Agricultura del Gobierno de Canarias. J. Pérez-Rodríguez was supported by a predoctoral grant to from Generalitat Valenciana. ...

Endo-β-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. Here, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycan (Dns-SG) as a substrate, and discovered a strain that exhibits ENGase activity in the culture supernatant; this strain, named here as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. Recombinant proteins, purified from Escherichia coli expressing candidate genes ORF1152, ORF1188, ORF3046 and ORF3750 exhibited fucose-containing oligosaccharides-specific ENGase activity. These ENGases exhibited optimum activities at very acidic pHs (between pH 2.3–2.5). BLAST searches using sequences of these candidate genes identified two fungal homologs of ORF1188, one in Beauveria bassiana and the other in Cordyceps militaris. Recombinant ORF1188, Beauveria and Cordyceps ENGases released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of these novel ENGases but also suggested that natural glycoproteins could be their substrates.

9 páginas, 3 tablas, 3 figuras. ; Dispersal limitation and environmental selection are the main processes shaping free-living microbial communities, but host-related factors may also play a major role in structuring symbiotic communities. Here, we aimed to determine the effects of isolation-by-distance and host species on the spatial structure of sponge-associated bacterial communities using as a model the abundant demosponge genus Ircinia. We targeted three co-occurring Ircinia species and used terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene sequences to explore the differentiation of their bacterial communities across a scale of hundreds of kilometres in the Western Mediterranean Sea. Multivariate analysis and nonmetric multidimensional scaling plots of T-RFLP profiles showed that bacterial communities in Ircinia sponges were structured by host species and remained stable across sampling locations, despite geographic distances (80–800 km) and diverse local conditions. While significant differences among some locations were observed in Ircinia variabilis-derived communities, no correlation between geographic distance and community similarity was consistently detected for symbiotic bacteria in any host sponge species. Our results indicate that bacterial communities are mostly shaped by host species-specific factors and suggest that evolutionary processes acting on longterm symbiotic relationships have favored spatial stability of sponge-associated bacterial communities. ; This research was funded by the Spanish Government projects CTM2010-17755 and CTM2010- 22218, the Catalan Government grant 2009SGR-484 for Consolidated Research Groups, the US National Science Foundation under grant 0853089, and a FI-DGR fellowship to L.P. ; Peer reviewed

13 pages, 3 figures, 2 tables, 57 references. I am most indebted to Regino Zamora and José María Gómez for their invaluable intellectual and logistical support during this study. I thank the Consejería de Medio Ambiente (Junta de Andalucía) and the Directors of the Sierra Nevada National Park and the Sierra de Baza Natural Park for permission to do fieldwork. Many thanks also to Sergio de Haro, Elena Baraza and José Antonio Hódar for field assistance. David Nesbitt improved the English version. This research is part of REDBOME (www.ugr.es/~redbome/) and GLOBIMED (www.globimed.net) networks on forest ecology. ; 1. Plant recruitment is a multiphase process that takes place in environments that are heterogeneous in space and time. In this work, I analyse how environmental heterogeneity in Mediterranean forests affects dynamics of early recruitment at different scales, using the wind-dispersed tree Acer opalus subsp. granatense as a case study. 2. Seed dispersal and viability, post-dispersal predation, seedling emergence and seedling and sapling survival were evaluated in different habitats (regional scale) and microhabitats (local scale). Simultaneously, a review of the literature on spatial dynamics of plant recruitment in Mediterranean systems was conducted to look for general patterns and investigate their fit to the Acer system. Nineteen woody and herbaceous species were included in the review. 3. At the regional scale, Acer recruitment dynamics strongly converged among sites of the same habitat. This was mainly due to large seedling emergence and survival differences among habitats. Although most of the studies reviewed analyzed only one site per habitat type, they also support strong regional variation (either site- or habitat-specific) in recruitment patterns. 4. At the local scale, Acer recruitment was microhabitat-specific, a result shared by almost all the reviewed species independently of their life form and dispersal syndrome. This was mostly due to spatial differences in seed arrival (higher under conspecifics) and seedling survival (higher under nurse shrubs). 5. Spatial discordance among seed rain and recruitment was found in 60% of the reviewed species at the regional scale, and in 67% at the local scale. Acer results supported this predominant lack of concordance. Discordance among seed rain and recruitment suggests that regeneration is largely limited by safe sites than by seed availability. Because seedling survival was the limiting process with a larger impact on the magnitude and spatial pattern of recruitment, safe sites might be defined as those where seedlings have a higher survival probability. 6. Synthesis : This study indicates that the influence of seed dispersal on the spatial patterns and demography of plant species could be limited in heterogeneous and stressful environments (as are found in the Mediterranean), where recruitment is restricted to a small fraction of the landscape. If we are to preserve the distribution and abundance of Mediterranean species in the face of environmental changes, we need to explicitly consider the strong patch-specificity that characterizes their recruitment process at all scales. ; This study was supported by a grant PFPU-MECD to L.G.A., the Andalusian government grant (PAI) rnm-220 and by the coordinated Spanish MEC projects HETEROMED (REN2002-04041-CO2-01/GLO) and DINAMED (CGL2005-05830-C03-03). ; Peer reviewed