Molecular genetic characterization of plant somatic hybrids

An efficient and easy method for genetic characterization of plant somatic hybrids is proposed. In a first qualitative approach, four somatic hybrids and their parental species (Nicotiana tabacum andN. plumbaginifolia) were characterized by DNA fingerprinting and Random Amplification of Polymorphic DNA (RAPD). After this, a quantitative estimation of the degree of parental contribution to the hybrids was carried out by means of a slot-blot analysis. Both qualitative methods, showed one hybrid identical toN. tabacum, two almost identical toN. plumbaginifolia, and a fourth similar to this parental species, but with someN. tabacum admixture. The quantitative method, for the same hybrids, gave 83%, 7%, 7%, and 37%N. tabacum DNA contribution, respectively.     

Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.     

Response of ‘Clausellina’ Satsuma mandarin to 3,5,6-trichloro-2-pirydiloxyacetic acid and fruitlet abscission

The application of the synthetic auxin 3,5,6-trichloro-2-piridyloxyacetic acid (3,5,6-TPA) isopropyl ester at the onset of cell enlargement stage, significantly thinned fruitlets in ‘Clausellina’ Satsuma mandarin. The magnitude of the response was related to the concentration applied, increasing the percentage of abscised fruit with higher concentrations, which was up to 70% at 25 mg l⁻¹. The magnitude of the response also depended on the organ subject to treatment, abscission being greater when applied to the leaves rather than to fruit. Results suggest that a photosynthetic disorder was responsible for a reduction in fruit growth rate, triggering abscission mechanisms producing ethylene and abscission.     

Evidence of non‐stationary relationships between climate and forest responses: Increased sensitivity to climate change in Iberian forests

Climate and forest structure are considered major drivers of forest demography and productivity. However, recent evidence suggests that the relationships between climate and tree growth are generally non‐stationary (i.e. non‐time stable), and it remains uncertain whether the relationships between climate, forest structure, demography and productivity are stationary or are being altered by recent climatic and structural changes. Here we analysed three surveys from the Spanish Forest Inventory covering c. 30 years of information and we applied mixed and structural equation models to assess temporal trends in forest structure (stand density, basal area, tree size and tree size inequality), forest demography (ingrowth, growth and mortality) and above‐ground forest productivity. We also quantified whether the interactive effects of climate and forest structure on forest demography and above‐ground forest productivity were stationary over two consecutive time periods. Since the 1980s, density, basal area and tree size increased in Iberian forests, and tree size inequality decreased. In addition, we observed reductions in ingrowth and growth, and increases in mortality. Initial forest structure and water availability mainly modulated the temporal trends in forest structure and demography. The magnitude and direction of the interactive effects of climate and forest structure on forest demography changed over the two time periods analysed indicating non‐stationary relationships between climate, forest structure and demography. Above‐ground forest productivity increased due to a positive balance between ingrowth, growth and mortality. Despite increasing productivity over time, we observed an aggravation of the negative effects of climate change and increased competition on forest demography, reducing ingrowth and growth, and increasing mortality. Interestingly, our results suggest that the negative effects of climate change on forest demography could be ameliorated through forest management, which has profound implications for forest adaptation to climate change.     

Experimentally increased testosterone affects social rank and primary sex ratio in the spotless starling

It has been suggested that the amount of maternal testosterone allocated into the eggs might be implicated in the process of sex determination. However, recent findings on the effect that female social rank has on the level of egg testosterone suggest that reported associations between male-biased sex ratios and yolk testosterone may represent an indirect hormonal effect mediated by the interdependence among maternal hormones, female social rank, and sex ratio. Here, we report the results of a field experiment in which we manipulated the circulating levels of testosterone in female spotless starlings (Sturnus unicolor) before egg formation. Focal females were controlled in subsequent years to explore possible delayed effects of hormone manipulation on primary sex ratio and social status that could persist because of permanent hormonal change or through hormone-dominance interactions. The results indicate that testosterone-implanted females (T-females) produced significantly more sons than control females (C-females) in the year in which they were manipulated. These differences in offspring sex ratio between T- and C-females persisted in the next 3 years, although no additional hormone treatments were given. These results were not mediated by an eventual effect of testosterone treatment on the quality of the females' mates. A similar proportion of T- and C-females acquired a nest box and bred either in the manipulation year or in Year 1 after manipulation, but T-females tended to be more successful in acquiring a nest box than C-females in Years 2 and 3 after manipulation. These results suggest that added testosterone had a direct role on the acquisition and maintenance of high social rank. Delayed effects of testosterone on primary sex ratio might have been caused by altered endogenous production of T-females. Alternatively, the maintenance of sex ratio differences between T- and C-females long after having being implanted might be attributed to the positive effect that enhanced social rank of T-females has on their circulating testosterone levels.     

The impact of management practices and past demographic history on the genetic diversity of red deer (Cervus elaphus): an assessment of population and individual fitness

The influences of management practices and past demographic history on genetic diversity are of critical relevance to sustainable practices and the conservation of wildlife populations. The red deer (Cervus elaphus) is an interesting model species to address these questions because it has a wide geographical distribution and it has been intensively managed for humans in the last decades. In the present study, we have analyzed the impact of recent management practices on the genetic diversity of Iberian red deer populations and assessed the genetic variation effects on population and individual fitness‐related traits. Four populations subjected to distinct management systems were selected: Cabañeros (CB) and Doñana (DN), not hunted populations; Fraga/Caspe (FG/CP), open hunting area with very low or absent management; and PE, fenced private hunting estate founded 31 years ago through the introduction of deer of different origins. Ten microsatellites were amplified in a total of 172 individuals. Additionally, several fitness‐related traits such as the presence of tuberculosis compatible lesions (TBCL), spleen weight (SW), and body length (BL) were estimated. We found a marked genetic variation and differentiation among populations, suggesting a strong population structure. In the fenced population, the introduction of genetically distinct animals has led to high genetic variability (no evidence of inbreeding) despite intensive management. Lower levels of genetic diversity were observed in two historically isolated natural populations (DN and FG/CP). The past demographic history of Iberian populations appears to be more relevant than the current management policy in shaping the genetic variability of natural populations. Population genetic diversity may correlate with life‐history traits and disease susceptibility, which could compromise the conservation and management of these wildlife populations. Although no significant effects of individual genetic diversity (general and local effect hypotheses) were observed on TBCL, SW and BL, some single‐locus effects had almost significant trends for the TBCL and SW traits. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 209–223.     

Nuclear but not cytosolic phosphoinositide 3-kinase beta has an essential function in cell survival

Class I(A) phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival.