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A recent electrical penetration graph (EPG) study identified a unique intracellular puncture that is associated with inoculation of the semipersistently transmitted, phloem-limited Beet yellows virus (BYV, Closterovirus ) by the aphid Myzus persicae . This new aphid EPG pattern (named phloem-pd) always occurs shortly before phloem sieve element phase (PSEP) and has a similar voltage drop as PSEP, both of which are less than the voltage drop of standard-pds. Structure of pd subphase II-2 differs between phloem-pds and standard-pds. The objective of this study was to determine the type of phloem cell penetrated during phloem-pds. Stylets of M. persicae feeding on sugar beet ( Beta vulgaris ) were fixed in situ by cryofixation during phloem-pds, standard-pds and PSEP waveform E1. The cell penetrated by the stylet tips was then identified by confocal laser-scanning microscopy and micro-computed tomography. Sieve elements (SEs) or companion cells (CCs) were penetrated during phloem-pds, whereas cells other than SEs or CCs (mesophyll, bundle sheath cells and possibly phloem parenchyma) were penetrated during standard-pds. SEs were penetrated during waveform E1. The implications of these findings for inoculation of other phloem-limited viruses that currently are thought to be inoculated primarily during waveform E1 are discussed.

We monitored two high mountain streams from the Sierra Nevada (Southern Spain) monthly throughout an annual cycle, measuring and analyzing physical and chemical parameters. Temperature was hourly registered. We studied life cycles and larval feeding of four mayfly species [Serratella ignita (Poda, 1761), Serratella spinosa nevadensis (Alba-Tercedor, 1981), Alainites muticus (Linnaeus, 1758), and Baetis alpinus Pictet, 1843] and two stonefly species [Amphinemura triangularis (Ris, 1902) and Capnioneura mitis Despax, 1932]. Some species changed their strategies (growth rate, life cycle duration, and/or voltinism) in accordance with different conditions (depending on the altitude and temperature). The gut content data showed that species fed mainly on detritus (as collector-gatherers or as shredders), except B. alpinus that behaved as a scraper in one of the streams. Differences in the larval diet of the same species in different streams, and even through its life, support the idea of not using general models to extrapolate the function of a species from a higher taxonomical level.

The European Water Framework Directive establishes the need to define stream type-specific reference conditions to identify “high ecological status”. Methods for selecting reference sites using a priori criteria have been proposed by many authors. A review of these criteria revealed that the most relevant criteria for streams and rivers were those related to riparian vegetation, diffuse and point sources of pollution, river morphology and hydrological conditions and regulation. In this work, we propose 20 criteria that reflect the characteristics of Mediterranean streams and their most frequent disturbances for the selection of reference sites in Mediterranean streams in Spain. We studied 162 sites located in 33 Mediterranean basins belonging to five stream types. Of the locations, 57% were selected as a priori reference sites by having applied the proposed criteria. Reference sites were identified for all stream types except for “large watercourses” which includes the lower reaches of some rivers in this study area. This a priori selection of reference sites was subjected to validation using the macroinvertebrate community by applying of an IBMWP threshold, which is considered to be an indicator of undisturbed sites in Mediterranean streams. This approach determined that whole of this selection (100%) could be considered valid reference sites. Furthermore, we identified differences in the reference conditions for each stream type on the basis of macroinvertebrate assemblage composition.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

In Spain, a national project known as GUADALMED, focusing on Mediterranean streams, has been carried out from 1998 to 2005 to implement the European water framework directive (WFD) requirements. One of the main objectives of the second phase of the project (2002-2005) was to develop a predictive system for the Spanish Mediterranean aquatic macroinvertebrate communities. A combined-season (spring, summer, and autumn) predictive model was developed by using the latest improvements on the selection of best predictor variables. Overall model performance measures were used to select the best discriminant function (DF) models, and also to evaluate their biases and precision. The final predictive model was based on the best five DF models. Each one of these models involved eight environmental variables. Final observed (O), expected (E), and O/E values for the number of macroinvertebrate families (NFAM) and two biotic indices (IBMWP and IASPT) were calculated by averaging their values, previously weighted by the quality of each DF model. Regression analyses among the final O and E values for the calibration dataset showed a high proximity to the ideal theoretical model, where the final E values explained 73-84% of the variation present in the macroinvertebrate communities of the Spanish Mediterranean watercourses. The ANOVA performed among the reference (calibration and validation) and test datasets showed clear differences for the O/E values. Finally, the assessments carried out by the predictive model were sensitive to anthropogenic pressure present in the study area and allowed the definition of five ecological status classes according to the WFD requirements.

The southeastern mountains of Spain represent the southernmost limit of the genus Austropotamobius and the species A. pallipes (Lereboullet). The taxonomic position of this isolated crayfish in southern Spain is not clear, being genetically close to A. italicus, but morphologically distinct. A severe decline occurred during the 1980s, especially due to expansion of the alien species Procambarus clarkii, a North American freshwater crayfish and a vector of the aphanomycosis disease. In order to design a strategy for native crayfish conservation, recent trends in native crayfish populations, influence of isolation and habitat variables on their survival and possibilities for their recovery through restocking were studied. A decline in populations was observed between 1991 and 2002 (from 26 to 14 populations), and a total extinction could be predicted within the next 13 years. Two well-defined periods of rapid extinction rates were detected, 1991-1994 (1.25 populations/year) and 1999-2002 (1.50 populations/year). Main causes of extinction for these two periods were invasion by P. clarkii and mortality by unknown causes, respectively. The isolation variables had some positive effects on survival of populations but these cannot offer a sufficient guarantee, since several cases of extinction can be affected by a large component of stochastic factors, including random catastrophes. On the other hand, survival trials and restocking experiments showed that it was possible to recover lost habitats, when P. clarkii was absent and environmental conditions were good. It is concluded that it is possible to avoid extinction of the native crayfish populations; however, the conservation strategies must be based on an urgent stocking/restocking program.[PUBLICATION ABSTRACT]

We studied aspects of the nymphal biology and ecology of 3 Perlodidae species (Guadalgenus franzi, Hemimelaena flaviventris, and Isoperla curtata) in a Mediterranean seasonal stream in the southern Iberian Peninsula. Their life-history strategies were greatly influenced by the characteristics of their environment, i.e., a summer dry period with relatively warm temperatures, but strategies differed among species. Guadalgenus franzi was semivoltine and probably underwent nymphal quiescence when the stream was dry. Hemimelaena flaviventris and I. curtata had relatively short univoltine life cycles that overlapped, but I. curtata was slightly ahead of H. flaviventris. Both species passed the dry period in the egg stage, probably with a diapause phase. Growth rates of H. flaviventris and I. curtata peaked before emergence, whereas growth rate of G. franzi peaked immediately before and after the dry period. The 3 species had relatively short flight periods compared with other species from seasonal streams. Guadalgenus franzi and I. curtata were mainly scrapers that fed on diatoms, whereas H. flaviventris was mainly predatory. Diet changed somewhat in relation to size, and prey electivity patterns differed among species. All 3 species preferred Chironomidae, but G. franzi preferred Chironomidae principally when smaller. All 3 species had high secondary production relative to other stonefly species from both temporary and permanent waters.

In this study we compared water-quality results obtained by a modified version of the Biological Monitoring Working Party Score System (BMWP′, a multimetric method) with those from multivariate methods--TWINSPAN and Canonical Correspondence Analysis (CCA)--in a river basin in southern Spain affected by organic pollution. Main environmental variables correlated to macroinvertebrate distributions were identified by CCA. Biological quality of the water was assessed by the BMWP′ biotic index, which classified the sites according to the tolerance of taxa to organic pollution. TWINSPAN was used to classify the sites according to benthic communities, and CCA was performed to establish the relationships among groups of sites, taxa, and abiotic variables. The results showed a clear separation of river sites according to their water composition and degree of pollution; nutrient content and water hardness were the main factors influencing the faunal distribution. Most of the sites in the study area were polluted, and consequently the most frequently encountered taxa were tolerant species. Moreover, the worsening of the water quality downstream was accompanied by a substitution of species, even within the same genus, from less to more pollution tolerant. The results obtained by the multivariate analyses used were highly satisfactory for correctly interpreting the assemblages and substitutions of macroinvertebrates, according to the main environmental variables measured in the basin. Moreover, the classification of sites according to macroinvertebrate requirements, provided by TWINSPAN analysis, was closely related to the classification according to tolerance to water pollution, provided by the biotic index. In addition, a significant part of the variance of each taxon as a pollution bioindicator, in the sites where they were found, was explained by the main canonical axis. Thus, the BMWP′ biotic index, of easy applicability in river basins, proved to be an easy tool for water-quality assessment.

Mayflies (Ephemeroptera) are among the oldest pterygote insects, with the earliest fossils dating back to the Late Carboniferous. Within mayflies, Leptophlebiidae are a highly diverse and widespread group, with approximately 140 genera and 640 species. Whereas taxonomy, systematics, and phylogeny of extant Leptophlebiidae are in the focus of extensive studies, little is known about leptophlebiid fossil taxa. Because fossil remains of Ephemeroptera in sedimentary rocks are relatively rare, inclusions of mayflies in amber are a unique source of information on their evolution and diversity in the past. Leptophlebiidae found in Cenozoic resins mostly belong to the subfamilies Leptophlebiinae (in Eocene Baltic amber) and Atalophlebiinae (in Miocene Dominican and Mexican ambers). In the present contribution, we confirm the first finding of the genus Calliarcys from Eocene Baltic amber by using Micro-CT, which allowed confirming its generic placement by visualizing diagnostic key characters otherwise hidden by a cloud of turbidity. Additionally, we present first molecular data on the extant species Calliarcys humilis Eaton, 1881 from the Iberian Peninsula and the barcode gap analysis for Leptophlebiinae and Habrophlebiinae.

A new Italian earthworm morphologically close to the similarly large and anecic Eophila tellinii (Rosa, 1888) is described. Distribution of Eophila crodabepis sp. nov. extends over 750 km2 from East to West on the Asiago Plateau and Vittorio Veneto Hills, from North to South on mounts Belluno Prealps (Praderadego and Cesen), Asiago, Grappa and onto the Montello foothills. This range abuts that of Eophila tellinii in northern Friuli Venezia Giulia region. Known localities of both E. tellinii and E. crodabepis sp. nov. are mapped. mtDNA barcoding definitively separates the new western species from classical Eophila tellinii (Rosa, 1888).