Establishing reliable DNA barcoding primers for jumping plant lice (Psylloidea, Hemiptera).

OBJECTIVES: DNA Barcoding has proven to be a reliable method for rapid insect identification. The success of this method is based on the amplification of a specific region, the 'Folmer' barcode region at the 5´ start of the cytochrome c oxidase 1 gene (cox1), with universal primers. Previous studies showed failures of standard "universal" primers to amplify this region in psyllids. The aim of the study was the design of a new alternative more reliable primer combination for taxa of the superfamily Psylloidea and its comparison with the performance of the standard "universal" Folmer-primers. RESULTS: A newly designed degenerate forward primer LCOP-F was developed following comparison of the sequence alignment of the priming site of "universal" primer LCO1490 and the standard insect forward primer LepF1. When combined with the "universal" reverse primer, HCO2198, this new primer pairing was able to generate barcode sequence for all 36 species in 20 genera across the five families of psyllids tested in this study, and these primers were found to be more universally reliable across psyllid taxa than other primer pairs tested.

Use of marine debris as nest material by ospreys.

The use of anthropogenic debris as nest-building materials may affect nest function. We study ospreys (Pandion haliaetus) on an island with scarce vegetation and high availability of beached marine debris. We describe the anthropogenic debris in osprey nests, evaluate the factors affecting its prevalence and abundance, and test its potential effects on breeding parameters. We also quantify plastic entanglements among adults and nestlings. Of the 36 studied nests, 92 % included non-natural items, with plastic being the most frequent material (88.9 %). Nests that were bigger and closer to the coast had more anthropogenic items. The abundance of anthropogenic items in nests did not correlate with osprey breeding parameters. We recorded two live entangled adult females, which represent 3.9 % of the adult population. Monitoring the abundance of anthropogenic debris and its effects on wildlife is necessary to guarantee long-term viability of coastal wildlife.

Fungal Pathogens Associated with Aerial Symptoms of Avocado (Persea americana Mill.) in Tenerife (Canary Islands, Spain) Focused on Species of the Family Botryosphaeriaceae.

Fungi of the family Botryosphaeriaceae are considered responsible for various symptoms in avocado such as dieback, external necrosis of branches and inflorescences, cankers on branches and trunks, or stem-end rot of fruits. In recent years, these problems are becoming more frequent in avocado orchards in the Canary Islands (Spain). This work includes the characterization of fungal species involved in these diseases, which were isolated from avocado crops in Tenerife Island between 2018 and 2022. A total of 158 vegetal samples were collected, from which 297 fungal isolates were culture-isolated. Fifty-two of them were selected according to their morphological features as representative isolates of Botryosphaeriaceae, and their molecular characterization was carried out, sequencing the ITS1-2 region as well as the ß-tubulin and the elongation factor 1-alpha genes. Five species of Botryosphaeriaceae were isolated, including Neofusicoccum australe, N. cryptoaustrale/stellenboschiana, N. luteum, N. parvum, and Lasiodiplodia brasiliensis. This is the first time that L. brasiliensis has been associated with avocado dieback and that N. cryptoaustrale/stellenboschiana has been cited in avocado causing symptoms of dieback and stem-end rot. However, it was not possible to assign our isolates unequivocally to N. cryptoaustrale or N. stellenboschiana even additionally using the rpb2 marker for their molecular characterization. Botryosphaeriaceae family seem to be involved in avocado dieback, in the premature fall of fruits during their development in the field and in post-harvest damage in Tenerife, but further studies are needed to clarify the fungal pathogens associated with symptoms in relation to phenological plant growth stages or less frequently observed.

Genomic basis of insularity and ecological divergence in barn owls (Tyto alba) of the Canary Islands.

Islands, and the particular organisms that populate them, have long fascinated biologists. Due to their isolation, islands offer unique opportunities to study the effect of neutral and adaptive mechanisms in determining genomic and phenotypical divergence. In the Canary Islands, an archipelago rich in endemics, the barn owl (Tyto alba), present in all the islands, is thought to have diverged into a subspecies (T. a. gracilirostris) on the eastern ones, Fuerteventura and Lanzarote. Taking advantage of 40 whole-genomes and modern population genomics tools, we provide the first look at the origin and genetic makeup of barn owls of this archipelago. We show that the Canaries hold diverse, long-standing and monophyletic populations with a neat distinction of gene pools from the different islands. Using a new method, less sensitive to structure than classical FST, to detect regions involved in local adaptation to insular environments, we identified a haplotype-like region likely under selection in all Canaries individuals and genes in this region suggest morphological adaptations to insularity. In the eastern islands, where the subspecies is present, genomic traces of selection pinpoint signs of adapted body proportions and blood pressure, consistent with the smaller size of this population living in a hot arid climate. In turn, genomic regions under selection in the western barn owls from Tenerife showed an enrichment in genes linked to hypoxia, a potential response to inhabiting a small island with a marked altitudinal gradient. Our results illustrate the interplay of neutral and adaptive forces in shaping divergence and early onset speciation.

Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of 'Candidatus Liberibacter Solanacearum' by qPCR.

'Candidatus Liberibacter solanacearum' (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsureTM, HotSHOT, and DNeasy®). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study.

Landscape and Climatic Variations Shaped Secondary Contacts amid Barn Owls of the Western Palearctic.

The combined actions of climatic variations and landscape barriers shape the history of natural populations. When organisms follow their shifting niches, obstacles in the landscape can lead to the splitting of populations, on which evolution will then act independently. When two such populations are reunited, secondary contact occurs in a broad range of admixture patterns, from narrow hybrid zones to the complete dissolution of lineages. A previous study suggested that barn owls colonized the Western Palearctic after the last glaciation in a ring-like fashion around the Mediterranean Sea, and conjectured an admixture zone in the Balkans. Here, we take advantage of whole-genome sequences of 94 individuals across the Western Palearctic to reveal the complex history of the species in the region using observational and modeling approaches. Even though our results confirm that two distinct lineages colonized the region, one in Europe and one in the Levant, they suggest that it predates the last glaciation and identify a secondary contact zone between the two in Anatolia. We also show that barn owls recolonized Europe after the glaciation from two distinct glacial refugia: a previously identified western one in Iberia and a new eastern one in Italy. Both glacial lineages now communicate via eastern Europe, in a wide and permeable contact zone. This complex history of populations enlightens the taxonomy of Tyto alba in the region, highlights the key role played by mountain ranges and large water bodies as barriers and illustrates the power of population genomics in uncovering intricate demographic patterns.

Assessment of Multilocus Sequence Analysis (MLSA) for Identification of Candidatus Liberibacter Solanacearum from Different Host Plants in Spain.

Liberibacter is a bacterial group causing different diseases and disorders in plants. Among liberibacters, Candidatus Liberibacter solanaceraum (CLso) produces disorders in several species mainly within Apiaceae and Solanaceae families. CLso isolates are usually grouped in defined haplotypes according to single nucleotide polymorphisms in genes associated with ribosomal elements. In order to characterize more precisely isolates of CLso identified in potato in Spain, a Multilocus Sequence Analysis (MLSA) was applied. This methodology was validated by a complete analysis of ten housekeeping genes that showed an absence of positive selection and a nearly neutral mechanism for their evolution. Most of the analysis performed with single housekeeping genes, as well as MLSA, grouped together isolates of CLso detected in potato crops in Spain within the haplotype E, undistinguishable from those infecting carrots, parsnips or celery. Moreover, the information from these housekeeping genes was used to estimate the evolutionary divergence among the different CLso by using the concatenated sequences of the genes assayed. Data obtained on the divergence among CLso haplotypes support the hypothesis of evolutionary events connected with different hosts, in different geographic areas, and possibly associated with different vectors. Our results demonstrate the absence in Spain of CLso isolates molecularly classified as haplotypes A and B, traditionally considered causal agents of zebra chip in potato, as well as the uncertain possibility of the present haplotype to produce major disease outbreaks in potato that may depend on many factors that should be further evaluated in future works.

'Candidatus Liberibacter Solanacearum' Is Unlikely to Be Transmitted Spontaneously from Infected Carrot Plants to Citrus Plants by Trioza Erytreae.

Bacteria belonging to 'Candidatus Liberibacter spp.' are associated with various severe diseases in the five continents. The African citrus psyllid Trioza erytreae (Hemiptera: Triozidae) is an efficient vector of citrus huanglongbing-HLB disease, absent in the Mediterranean basin. This psyllid is currently present in the islands and mainland Portugal and Spain, where the prevalence of 'Ca. Liberibacter solanacearum' (CaLsol) associated to a carrot disease is high. Trioza erytreae normally feeds on citrus plants but has also been observed on other crops. It would be a great concern to the Mediterranean citrus industry if T. erytreae could transmit this bacterium from carrots to citrus and cause disease; therefore, the transmission of CaLsol from carrot plants to citrus plants was experimentally assessed. Although CaLsol was initially detected on receptor citrus plants in transmission assays by dodder and budding, the infection was not established. The feeding behavior by electrical penetration graphs and oviposition of T. erytreae on carrot plants versus citrus plants was evaluated. Trioza erytreae only reached the phloem in citrus plants. However, it was able to acquire CaLsol from infected carrots but unable to transmit it to citrus plants. CaLsol was detected in some carrot plants immediately after 7 and 14 days (inoculation access period), but it was not detected after one month. Trioza erytreae was unable to complete its life cycle on carrot plants. In conclusion, the efficient vector of bacteria associated to huanglongbing was unable to transmit CaLsol from carrot to citrus plants, but it acquired and transmitted the bacterium from carrot to carrot plants with low efficiency.

Survey in Nurseries and Irrigation Water Reservoirs as Sources of Oomycetes Found in Avocado Orchards in the Canary Islands.

Nursery stocks and irrigation water are important sources of Phytophthora spp. In this study, 20 irrigation water reservoirs and 10 avocado nurseries were surveyed in the Canary Islands between 2013 and 2015 to investigate their potential role in disseminating pathogenic species in avocado orchards. Phytophthora multivora was isolated from one of the irrigation reservoirs, whereas, in two surveys conducted in nurseries, Phytophthora cinnamomi, the primary pathogen in avocado, was detected in addition to other Phytophthora species (P. lacustris, P. multivora, P. nicotianae, P. niederhauserii, and P. palmivora) and Phytopythium vexans. The species recovered from nurseries were isolated mostly from propagated plants but also from nursery irrigation water, soil used for substrate preparation, and soil samples collected in orchards that supply seeds for seedling propagation. Species recovered from nurseries correlated with those found in avocado orchards in a previous study, except for P. lacustris, suggesting that nurseries could be involved in their dissemination in avocado orchards. The improved sanitary status of nurseries resulted in reduced incidence in the second survey, indicating the importance of nursery monitoring to reduce infestations.

Factors affecting the spatial distribution and breeding habitat of an insular cliff-nesting raptor community.

The specific spatial distribution and habitat association-strongly influenced by environmental factors or competitive interactions-are major issues in ecology and conservation. We located and georeferenced nesting sites of five cliff-nesting raptors (Egyptian vulture Neophron percnopterus [a locally extinct species], common buzzard Buteo buteo, osprey Pandion haliaetus, common kestrel Falco tinnunculus, Barbary falcon Falco peregrinus pelegrinoides), and common raven Corvus corax on one of the most biodiverse hotspot within the Canary Islands (Teno, Tenerife). We used generalized linear models to evaluate the factors affecting abundance, richness, and intra- and interspecific interactions. Raptor abundance increased with slope, shrub-covered area, and habitat diversity, and decreased with altitude, and forested and grassed areas. Richness increased with slope and decreased with altitude. Threatened species (osprey, Barbary falcon, and raven) occupied cliffs farther away from houses and roads, and more rugged areas than the non-threatened species. The models suggested that the probability of cliff occupation by buzzards, falcons, and ravens depended only on inter-specific interactions. Buzzard occupation increased with the distance to the nearest raven and kestrel nests, whereas falcons and ravens seek proximity to each other. Teno holds between 75% and 100% of the insular breeding populations of the most endangered species (osprey and raven), indicating the high conservation value of this area. Our study suggests that the preservation of rugged terrains and areas of low human pressure are key factors for raptor conservation and provide basic knowledge on the community structure and habitat associations to develop appropriated management actions for these fragile island populations.