Susana C. Arcos

Incidence of the pinewood nematode Bursaphelenchus xylophlius Steiner & Buhrer, 1934 (Nickle, 1970) in Spain

The pine wood nematode (PWN) Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Nickle, 1970), is a pathogenic species in the genus Bursaphelenchus Fuchs, 1937. The introduction and establishment of PWN in the Setubal Peninsula, Portugal (Mota et al., 1999), and the evidence that Monochamus galloprovincialis is its insect vector in this country (Sousa et al., 2001), has stimulated studies on its further distribution and its potential vector insects in Europe, including Spain. There is a clear risk of spread of pine wood nematode into Spanish territory from Portugal, both as a result of natural spread and due to the important timber trade between the two countries. The occurrence of Bursaphelenchus spp. in Spain has been previously studied (Abelleira et al., 2002, 2003; Escuer et al., 2003, 2004; Robertson et al., 2008). These studies have shown, in addition, the distribution of insects belonging to the Cerambycidae, Curculionidae and Scolytidae families (Cobos, 1986; Gil Sánchez & Pajares Alonso, 1986; Vives, 2000; Verdugo Paez, 2004), which are known to be vectors of Bursaphelenchus spp. other than B. xylophilus and are often found to be associated with stressed, dead or decaying conifers. Although it is generally accepted that B. xylophilus is the only truly pathogenic species (reviewed by Jones et al., 2008), other species may be involved in pine forest decline in some European regions or, alternatively, may simply be opportunistic infectors of stressed trees (Mamiya, 1999; Caroppo et al., 2000; Skarmoutsos et al., 2000). Pinus pinaster is the most representative

Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii.

The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI‐TOF/TOF analysis or de novo sequencing. Seventy‐five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty‐eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 (http://proteomecentral.proteomexchange.org/dataset/PXD000662).

Biosafety test for plant growth-promoting bacteria: proposed environmental and human safety index (EHSI) protocol

Plant growth-promoting bacteria (PGPB) colonize plants and enhance their growth by different mechanisms. Some of these microorganisms may represent a potential threat to human, animal or plant health; however, their use might be approved in parts of Europe if they have been recommended as plant growth enhancers. The current regulatory framework has resulted in a fragmented, contradictory system, and there is an urgent need to establish harmonized protocols for the predictability, efficiency, consistency and especially the safety of PGPB for human and animal health and for the environment. In response to current efforts to update biosafety policies and provide alternative methods to replace the use of vertebrate animals, we propose a panel of tests and an evaluation system to reliably determine the biosafety of bacterial strains used as PGPB. Based on the results of different tests, we propose a scoring system to evaluate the safety of candidates for PGPB within the limitations of the assays used.

Interplay among Resistance Profiles, High-Risk Clones, and Virulence in the Caenorhabditis elegans Pseudomonas aeruginosa Infection Model

ABSTRACT The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa is frequently linked to widespread international strains designated high-risk clones. In this work, we attempted to decipher the interplay between resistance profiles, high-risk clones, and virulence, testing a large (n = 140) collection of well-characterized P. aeruginosa isolates from different sources (bloodstream infections, nosocomial outbreaks, cystic fibrosis, and the environment) in a Caenorhabditis elegans infection model. Consistent with previous data, we documented a clear inverse correlation between antimicrobial resistance and virulence in the C. elegans model. Indeed, the lowest virulence was linked to XDR profiles, which were typically linked to defined high-risk clones. However, virulence varied broadly depending on the involved high-risk clone; it was high for sequence type 111 (ST111) and ST235 but very low for ST175. The highest virulence of ST235 could be attributed to its exoU+ type III secretion system (TTSS) genotype, which was found to be linked with higher virulence in our C. elegans model. Other markers, such as motility or pigment production, were not essential for virulence in the C. elegans model but seemed to be related with the higher values of the statistical normalized data. In contrast to ST235, the ST175 high-risk clone, which is widespread in Spain and France, seems to be associated with a particularly low virulence in the C. elegans model. Moreover, the previously described G154R AmpR mutation, prevalent in ST175, was found to contribute to the reduced virulence, although it was not the only factor involved. Altogether, our results provide a major step forward for understanding the interplay between P. aeruginosa resistance profiles, high-risk clones, and virulence.

Protein markers of Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Nickle, 1970) populations using quantitative proteomics and character compatibility.

The Pine Wood Nematode (PWN) Bursaphelenchus xylophilus is a severe forest pathogen in countries where it has been introduced and is considered a worldwide quarantine organism. In this study, protein markers for differentiating populations of this nematode were identified by studying differences among four selected Iberian and one American population. These populations were compared by quantitative proteomics (iTRAQ). From a total of 2860 proteins identified using the public database from the B. xylophilus genome project, 216 were unambiguous and significantly differentially regulated in the studied populations. Comparisons of their pairwise ratio were statistically treated and supported in order to convert them into discrete character states, suggesting that 141 proteins were not informative as population specific markers. Application of the Character Compatibility methodology on the remaining 75 proteins (belonging to families with different biological functions) excludes 27 which are incompatible among them. Considering only the compatible proteins, the method selects a subset of 30 specific unique protein markers which allowed the compared classification of the Iberian isolates. This approach makes it easier search for diagnostic tools and phylogenetic inference within species and populations of a pathogen exhibiting a high level of genetic diversity.

Potential Insect Vectors of Bursaphelenchus spp. (Nematoda: Parasitaphelenchidae) in Spanish Pine Forests

Potential insect vectors of Bursaphelenchus xylophilus (PWN) were studied. Pathways of introduction of PWN from Portugal to Europe, through Spain, were determined and traps were located in pine stands sites along the pathways. 19 Cerambycidae, 12 Scolytidae, 12 Buprestidae and 10 Curculionidae species have been found. Trapped insects were examined for the presence of nematodes under their elytra. Nematodes were found on Arhopalus ferus, Spondylis buprestoides, Hylastes ater, Hylurgus lingniperda, Orthotomicus erosus, Pityogenes bidentatus, Tomicus piniperda, Hylobius abietis and Pissodes validirrostris specimens. Monochamus galloprovincialis was the most important insect species, representing a risk for the introduction of the PWN in Spanish pine forests; Cerambycidae and Curculionidae species, were taken into account because they have been reported as vectors of other Bursaphelenchus spp.

Distribution of Xiphinema pyrenaicum Dalmasso, 1969 with notes on X. aceri Chizhov, Tiev & Turkina, 1986 (nematoda: Longidoridae)

A study of the distribution and morphometric characteristics of Xiphinema pyrenaicum and X. aceri was done because of their presence and distribution in Spain. More than 50 populations of X. pyrenaicum were studied and one population of X. aceri, of which the latter included the four juvenile stages not previously described. The study revealed both species to be typical of Mediterranean environments, X. pyrenaicum being widespread in the circum-mediterranean basin.

Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns

BackgroundAnisakis simplex sensu stricto and Anisakis pegreffii are sibling species of nematodes parasitic on marine mammals. Zoonotic human infection with third stage infective larvae causes anisakiasis, a debilitating and potentially fatal disease. These 2 species show evidence of hybridisation in geographical areas where they are sympatric. How the species and their hybrids differ is still poorly understood.ResultsThird stage larvae of Anisakis simplex s.s., Anisakis pegreffii and hybrids were sampled from Merluccius merluccius (Teleosti) hosts captured in waters of the FAO 27 geographical area. Specimens of each species and hybrids were distinguished with a diagnostic genetic marker (ITS). RNA was extracted from pools of 10 individuals of each taxon. Transcriptomes were generated using Illumina RNA-Seq, and assembled de novo. A joint assembly (here called merged transcriptome) of all 3 samples was also generated. The inferred transcript sets were functionally annotated and compared globally and also on subsets of secreted proteins and putative allergen families. While intermediary metabolism appeared to be typical for nematodes in the 3 evaluated taxa, their transcriptomes present strong levels of differential expression and enrichment, mainly of transcripts related to metabolic pathways and gene ontologies associated to energy metabolism and other pathways, with significant presence of excreted/secreted proteins, most of them allergens. The allergome of the 2 species and their hybrids has also been thoroughly studied; at least 74 different allergen families were identified in the transcriptomes.ConclusionsA. simplex s.s., A. pegreffi and their hybrids differ in gene expression patterns in the L3 stage. Strong parent-of-origin effects were observed: A. pegreffi alleles dominate in the expression patterns of hybrids albeit the latter, and A. pegreffii also display significant differences indicating that hybrids are intermediate biological entities among their parental species, and thus of outstanding interest in the study of speciation in nematodes. Analyses of differential expression based on genes coding for secreted proteins suggests that co-infections presents different repertoires of released protein to the host environment. Both species and their hybrids, share more allergen genes than previously thought and are likely to induce overlapping disease responses.