Songbird nests on the ground as islands of diversity of ptyctimous mites (Acari: Oribatida) in the primeval Bialowieza Forest (Poland).

Due to specific microclimatic conditions and accumulation of organic matter, bird nests are microhabitats that are often inhabited by various invertebrates, including mites (Acari). We tested whether nests of the ground-nesting passerine Phylloscopus sibilatrix (Bechstein) (Passeriformes: Phylloscopidae) [wood warbler] were associated with an increased local diversity of ptyctimous mites (Acari: Oribatida) on the forest floor in the Bialowieza National Park, East Poland. In 2019-2020, we analysed 150 warbler nests shortly after they had been vacated by the birds, and additionally we sampled mites in leaf litter at 1 and 6 m distances from 21 of the collected nests. We found on average more mite species in nests sampled in 2020 than in nests sampled in 2019. Although the species composition largely overlapped between bird nests and the litter, bird nests contained a greater average number of mite species than litter samples, including species found only - Microtritia minima (Berlese) and Phthiracarus crenophilus Willmann - or mostly - Euphthiracarus cribrarius (Berlese) and Phthiracarus globosus (C.L. Koch) - in bird nests. The results suggest that the presence of bird nests may increase the local diversity of the invertebrate species assemblage.

Implication of adipocytes from subcutaneous adipose tissue and fatty acids in skin inflammation caused by λ-carrageenin in gilthead seabream (Sparusaurata).

The role of subcutaneous adipose tissue adipocytes and the effects of fatty acids on carrageenan-induced skin inflammation in gilthead seabream (Sparus aurata) were studied. Fish were injected intramuscularly with phosphate-buffered saline (control) or λ-carrageenin (1%), and skin samples collected at the injection site at 3 and 6 h post-injection (p.i.) were processed for histological study. In addition, the presence and levels of lipid classes, fatty acid methyl esters (FAME) and eicosanoids were evaluated in the skin samples obtained from the injected areas. Histological results indicated an increase in adipocyte area in fish sampled at 3 h p.i. with λ-carrageenin compared to fish in the control group. Furthermore, the frequency of adipocytes between 4500 and 5000 µm2 was increased at 6 h in the λ-carrageenin group compared to the control group. Analysis of lipid classes found that fish injected with λ-carrageenan showed increased free fatty acid (FFA) and sphingomyelin content at 3 and 6 h, respectively, compared to the control group. An increase in saturated fatty acids (SFA), n-6 polyunsaturated fatty acids (PUFA), and a decrease in the values of monounsaturated fatty acids (MUFA), n-3 PUFA and minor fatty acids were observed in fish skin at 6 h after λ-carrageenin injection, with respect to the values obtained in the control group. Regarding the analysis of eicosanoids, an increase in hydroxyeicosatetraenoic acid (5-HETE) was detected in the skin of fish at 6 h post-carrageenin injection compared to the control group. The presented results indicate the contribution of adipocytes and fatty acids in the development and regulation of the inflammatory response triggered by λ-carrageenin in gilthead seabream skin.

Endoplasmic reticulum stress as a key mechanism in stunted growth of seawater rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + -ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. RESULTS: No differences in Gill Na+, K + -ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. CONCLUSION: While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.

Interspecific attraction between ground-nesting songbirds and ants: the role of nest-site selection.

BACKGROUND: Interspecific interactions within ecological networks can influence animal fitness and behaviour, including nest-site selection of birds and ants. Previous studies revealed that nesting birds and ants may benefit from cohabitation, with interspecific attraction through their nest-site choice, but mutual interactions have not yet been tested. We explored a previously undescribed ecological link between ground-nesting birds and ants raising their own broods (larvae and pupae) within the birds' nests in a temperate primeval forest of lowland Europe. We tested whether the occurrence of ant broods within bird nests resulted from a mutual or one-sided interspecific attraction that operated through nest-site choice and was modified by weather conditions. RESULTS: We found a non-random occupation of bird nests by ants raising their own broods within them, which indicated interspecific attraction driven solely by the ants. The birds' preference to nest near tussocks of vegetation showed little overlap with the most frequent placement of ant colonies among fallen deciduous tree-leaves, dead wood and moss. Additionally, birds did not appear to select forest localities with high densities of ant colonies. The occurrence of ant broods within bird nests was also unrelated to bird nest placement near to specific habitat features. The attractiveness of bird nests to ants appeared to increase with the thermal activity of the birds warming their nests, and also during cool and wet weather when the occurrence of ant broods within bird nests was most frequent. Ants often remained in the nests after the birds had vacated them, with only a slight reduction in the probability of ant brood occurrence over time. CONCLUSIONS: The natural patterns of bird nest colonisation by ants support the hypothesis of ants' attraction to warm nests of birds to raise their broods under advantageous thermal conditions. Similar relationships may occur between other warm-blooded, nest-building vertebrates and nest-dwelling invertebrates, which depend on ambient temperatures. The findings advance our understanding of these poorly recognised interspecific interactions, and can inform future studies of ecological networks.

Lack of specialist nidicoles as a characteristic of mite assemblages inhabiting nests of the ground-nesting wood warbler, Phylloscopus sibilatrix (Aves: Passeriformes).

Bird and mammal nests provide microhabitats that support a range of other species, including invertebrates. However, the variation between communities of nest-dwelling invertebrates in different nests is poorly understood. The major aim of this study was to analyze the assemblage structure of mites from the suborder Uropodina (Acari: Mesostigmata) and from superfamily Crotonioidea (Acari: Oribatida) inhabiting nests of the wood warbler, Phylloscopus sibilatrix (Aves: Passeriformes), located on a forest floor in Bialowieza Forest, in eastern Poland. We also assessed the correlation between the nest material used by the birds with the assemblage structure of Uropodina mites, and compared the results with published studies of the nests of other birds and a mammal (common mole, Talpa europaea), and also with communities of mites inhabiting the soil. The field research was conducted in the strict nature reserve of the Bialowieza National Park, a near-primeval European temperate forest. In 2019, immediately after the breeding period, 69 wood warbler nests and 439 soil samples were collected. Analyses revealed assemblages of Uropodina mites inhabiting the nests that consisted of 14 species, mostly common soil species. Only five species of oribatid mites from superfamily Crotonioidea were present in the nest material. Analyzed nests had a high percentage of tree leaves and grass blades, whereas moss was the least frequent component of the nest material. The Uropodina mites were more abundant in the nests that had greater amounts of grass blades, but similar relationships were insignificant for the nests with varying amounts of tree leaves or moss. The assemblages of Uropodina mites inhabiting wood warbler nests were very similar to those found in soil and nests of the common mole, but they lacked typical nest-dwelling species of Uropodina (i.e., specialized nidicoles).

Angiosperm symbioses with non-mycorrhizal fungal partners enhance N acquisition from ancient organic matter in a warming maritime Antarctic.

In contrast to the situation in plants inhabiting most of the world's ecosystems, mycorrhizal fungi are usually absent from roots of the only two native vascular plant species of maritime Antarctica, Deschampsia antarctica and Colobanthus quitensis. Instead, a range of ascomycete fungi, termed dark septate endophytes (DSEs), frequently colonise the roots of these plant species. We demonstrate that colonisation of Antarctic vascular plants by DSEs facilitates not only the acquisition of organic nitrogen as early protein breakdown products, but also as non-proteinaceous d-amino acids and their short peptides, accumulated in slowly-decomposing organic matter, such as moss peat. Our findings suggest that, in a warming maritime Antarctic, this symbiosis has a key role in accelerating the replacement of formerly dominant moss communities by vascular plants, and in increasing the rate at which ancient carbon stores laid down as moss peat over centuries or millennia are returned to the atmosphere as CO2 .

A Palmitic Acid Elongase Affects Eicosapentaenoic Acid and Plastidial Monogalactosyldiacylglycerol Levels in Nannochloropsis.

Nannochloropsis species are oleaginous eukaryotes containing a plastid limited by four membranes, deriving from a secondary endosymbiosis. In Nannochloropsis, thylakoid lipids, including monogalactosyldiacylglycerol (MGDG), are enriched in eicosapentaenoic acid (EPA). The need for EPA in MGDG is not understood. Fatty acids are de novo synthesized in the stroma, then converted into very-long-chain polyunsaturated fatty acids (FAs) at the endoplasmic reticulum (ER). The production of MGDG relies therefore on an EPA supply from the ER to the plastid, following an unknown process. We identified seven elongases and five desaturases possibly involved in EPA production in Nannochloropsis gaditana Among the six heterokont-specific saturated FA elongases possibly acting upstream in this pathway, we characterized the highly expressed isoform Δ0-ELO1 Heterologous expression in yeast (Saccharomyces cerevisiae) showed that NgΔ0-ELO1 could elongate palmitic acid. Nannochloropsis Δ0-elo1 mutants exhibited a reduced EPA level and a specific decrease in MGDG In NgΔ0-elo1 lines, the impairment of photosynthesis is consistent with a role of EPA-rich MGDG in nonphotochemical quenching control, possibly providing an appropriate MGDG platform for the xanthophyll cycle. Concomitantly with MGDG decrease, the level of triacylglycerol (TAG) containing medium chain FAs increased. In Nannochloropsis, part of EPA used for MGDG production is therefore biosynthesized by a channeled process initiated at the elongation step of palmitic acid by Δ0-ELO1, thus acting as a committing enzyme for galactolipid production. Based on the MGDG/TAG balance controlled by Δ0-ELO1, this study also provides novel prospects for the engineering of oleaginous microalgae for biotechnological applications.

Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

The functional characterization of wax biosynthetic enzymes in transgenic plants has opened the possibility of producing tailored wax esters (WEs) in the seeds of a suitable host crop. In this study, in addition to systematically evaluating a panel of WE biosynthetic activities, we have also modulated the acyl-CoA substrate pool, through the co-expression of acyl-ACP thioesterases, to direct the accumulation of medium-chain fatty acids. Using this combinatorial approach, we determined the additive contribution of both the varied acyl-CoA pool and biosynthetic enzyme substrate specificity to the accumulation of non-native WEs in the seeds of transgenic Camelina plants. A total of fourteen constructs were prepared containing selected FAR and WS genes in combination with an acyl-ACP thioesterase. All enzyme combinations led to the successful production of wax esters, of differing compositions. The impact of acyl-CoA thioesterase expression on wax ester accumulation varied depending on the substrate specificity of the WS. Hence, co-expression of acyl-ACP thioesterases with Marinobacter hydrocarbonoclasticus WS and Marinobacter aquaeolei FAR resulted in the production of WEs with reduced chain lengths, whereas the co-expression of the same acyl-ACP thioesterases in combination with Mus musculus WS and M. aquaeolei FAR had little impact on the overall final wax composition. This was despite substantial remodelling of the acyl-CoA pool, suggesting that these substrates were not efficiently incorporated into WEs. These results indicate that modification of the substrate pool requires careful selection of the WS and FAR activities for the successful high accumulation of these novel wax ester species in Camelina seeds.

Heterogeneous natural selection on oxidative phosphorylation genes among fishes with extreme high and low aerobic performance.

BACKGROUND: Oxidative phosphorylation (OXPHOS) is the primary source of ATP in eukaryotes and serves as a mechanistic link between variation in genotypes and energetic phenotypes. While several physiological and anatomical factors may lead to increased aerobic capacity, variation in OXPHOS proteins may influence OXPHOS efficiency and facilitate adaptation in organisms with varied energy demands. Although there is evidence that natural selection acts on OXPHOS genes, the focus has been on detection of directional (positive) selection on specific phylogenetic branches where traits that increase energetic demands appear to have evolved. We examined patterns of selection in a broader evolutionary context, i.e., on multiple lineages of fishes with extreme high and low aerobic performance. RESULTS: We found that patterns of natural selection on mitochondrial OXPHOS genes are complex among fishes with different swimming performance. Positive selection is not consistently associated with high performance taxa and appears to be strongest on lineages containing low performance taxa. In contrast, within high performance lineages, purifying (negative) selection appears to predominate. CONCLUSIONS: We provide evidence that selection on OXPHOS varies in both form and intensity within and among lineages through evolutionary time. These results provide evidence for fluctuating selection on OXPHOS associated with divergence in aerobic performance. However, in contrast to previous studies, positive selection was strongest on low performance taxa suggesting that adaptation of OXPHOS involves many factors beyond enhancing ATP production in high performance taxa. The broader pattern indicates a complex interplay between organismal adaptations, ATP demand, and OXPHOS function.

Temporal avoidance as a means of reducing competition between sympatric species.

Human activity has modified the availability of natural resources and the abundance of species that rely on them, potentially changing interspecific competition dynamics. Here, we use large-scale automated data collection to quantify spatio-temporal competition among species with contrasting population trends. We focus on the spatial and temporal foraging behaviour of subordinate marsh tits Poecile palustris among groups of socially and numerically dominant blue tits Cyanistes caeruleus and great tits Parus major. The three species exploit similar food resources in mixed groups during autumn-winter. Using 421 077 winter recordings of individually marked birds at 65 automated feeding stations in Wytham Woods (Oxfordshire, UK), we found that marsh tits were less likely to join larger groups of heterospecifics, and they accessed food less frequently in larger groups than in smaller ones. Marsh tit numbers within groups declined throughout the diurnal and winter periods, while the number of blue and great tits increased. However, sites that attracted larger groups of these heterospecifics also attracted more marsh tits. The results suggest that subordinate species exhibit temporal avoidance of socially and numerically dominant heterospecifics, but have limited ability for spatial avoidance, indicating that behavioural plasticity enables only a partial reduction of interspecific competition.

The effect of cryopreservation on genomic stability in strains of the fungus Trichoderma.

Within Biological Resource Collections, the successful long-term storage of fungal cultures is essential because of their scientific and potential commercial value. Preservation procedures such as cryopreservation have traditionally been used to ensure genomic stability due to the suspension of metabolic activity at ultra-low temperatures. Genomic integrity is important with regards to conservation, as changes in the genome may compromise production of a desired metabolite, enzyme or activity. To evaluate cryopreservation as a conservation protocol, genomic integrity was assessed in five strains of the economically important fungus Trichoderma. Two polymerase chain reaction (PCR) fingerprinting techniques commonly used for molecular studies of fungi were applied. Three genetic polymorphisms were detected amongst replicates post preservation, indicating that even robust, standardised preservation cryopreservation methodologies can sometimes induce genomic change. However, the low number of polymorphisms suggests that cryopreservation is a reliable method for organism storage over long periods of time.

Weather impacts on interactions between nesting birds, nest-dwelling ectoparasites and ants.

Weather has a dominant impact on organisms, including their life histories and interspecific interactions. Yet, for nesting birds, and the arthropods inhabiting bird nests, the direct and cascading effects of weather are poorly known. We explored the influence of ambient temperatures and rainfall on the cohabitation of dome-shaped bird nests by Wood Warblers Phylloscopus sibilatrix, their blowfly Protocalliphora azurea ectoparasites, and predatory Myrmica and Lasius ants that may provide nest sanitation. We sampled blowflies and ants in 129 nests, and measured warbler nestlings during 2018-2020 in the primeval Bialowieza Forest, eastern Poland. The probability of ectoparasites occurring in nests increased with increasing ambient temperatures and declining precipitation in the early nestling stage, when adult blowflies are ovipositing. Where present, the number of ectoparasites was greater if higher ambient temperatures had prevailed in the late nestling stage, but only when ants were absent from nests. However, the nestling growth was unrelated to ectoparasite abundance or ant presence within bird nests, although it was lower at high rainfall. The results suggest that weather can have conflicting impacts on interactions between nesting birds and nest-dwelling arthropods, but birds can mostly compensate for any related costs in old-growth forest, where food is generally abundant.

Profiling Phospholipids within Atlantic Salmon Salmo salar with Regards to a Novel Terrestrial Omega-3 Oil Source.

The development and inclusion of novel oils derived from genetically modified (GM) oilseeds into aquafeeds, to supplement and supplant current terrestrial oilseeds, as well as fish oils, warrants a more thorough investigation into lipid biochemical alterations within finfish species, such as Atlantic salmon. Five tissues were examined across two harvesting timepoints to establish whether lipid isomeric alterations could be detected between a standard commercial diet versus a diet that incorporated the long-chain polyunsaturated fatty acids (LC-PUFA), EPA (eicosapentaenoic acid), and DHA (docosahexaenoic acid), derived from the GM oilseed Camelina sativa. Tissue-dependent trends were detected, indicating that certain organs, such as the brain, have a basal limit to LC-PUFA incorporation, though enrichment of these fatty acids is possible. Lipid acyl alterations, as well as putative stereospecific numbering (sn) isomer alterations, were also detected, providing evidence that GM oils may modify lipid structure, with lipids of interest providing a set of targeted markers by which lipid alterations can be monitored across various novel diets.

Slow development of woodland vegetation and bird communities during 33 years of passive rewilding in open farmland.

Passive rewilding is a potential tool for expanding woodland cover and restoring biodiversity by abandoning land management and allowing natural vegetation succession to occur. Land can be abandoned to passive rewilding deliberately or due to socio-economic change. Despite abandonment being a major driver of land use change, few have studied the long-term outcomes for vegetation and biodiversity in Western Europe. Studies are also biased towards sites that are close to seed sources and favourable to woodland colonisation. In this case-study, we reconstruct a time series of passive rewilding over 33 years on 25 ha of former farmland that had been subject to soil tipping, far from woodland seed sources. Natural colonisation by shrubs and trees was surveyed at three points during the time series, using field mapping and lidar. Breeding birds were surveyed at three time points, and compared with surveys from nearby farmland. Results showed that natural colonisation of woody vegetation was slow, with open grassland dominating the old fields for two decades, and small wetlands developing spontaneously. After 33 years, thorny shrub thickets covered 53% of the site and former hedgerows became subsumed or degraded, but trees remained scarce. However, the resulting habitat mosaic of shrubland, grassland and wetland supported a locally distinctive bird community. Farmland bird species declined as passive rewilding progressed, but this was countered by relatively more wetland birds and an increase in woodland birds, particularly songbirds, compared to nearby farmland. Alongside biodiversity benefits, shrubland establishment by passive rewilding could potentially provide ecosystem services via abundant blossom resources for pollinators, and recreation and berry-gathering opportunities for people. Although closed-canopy woodland remained a distant prospect even after 33 years, the habitat mosaic arising from passive rewilding could be considered a valuable outcome, which could contribute to nature recovery and provision of ecosystem services.

Long-term trends of second generation anticoagulant rodenticides (SGARs) show widespread contamination of a bird-eating predator, the Eurasian Sparrowhawk (Accipiter nisus) in Britain.

Second generation anticoagulant rodenticides (SGARs) are widely used to control rodents around the world. However, contamination by SGARs is detectable in many non-target species, particularly carnivorous mammals or birds-of-prey that hunt or scavenge on poisoned rodents. The SGAR trophic transfer pathway via rodents and their predators/scavengers appears widespread, but little is known of other pathways of SGAR contamination in non-target wildlife. This is despite the detection of SGARs in predators that do not eat rodents, such as specialist bird-eating hawks. We used a Bayesian modelling framework to examine the extent and spatio-temporal trends of SGAR contamination in the livers of 259 Eurasian Sparrowhawks, a specialist bird-eating raptor, in regions of Britain during 1995-2015. SGARs, predominantly difenacoum, were detected in 81% of birds, with highest concentrations in males and adults. SGAR concentrations in birds were lowest in Scotland and higher or increasing in other regions of Britain, which had a greater arable or urban land cover where SGARs may be widely deployed for rodent control. However, there was no overall trend for Britain, and 97% of SGAR residues in Eurasian Sparrowhawks were below 100 ng/g (wet weight), which is a potential threshold for lethal effects. The results have potential implications for the population decline of Eurasian Sparrowhawks in Britain. Fundamentally, the results indicate an extensive and persistent contamination of the avian trophic transfer pathway on a national scale, where bird-eating raptors and, by extension, their prey appear to be widely exposed to SGARs. Consequently, these findings have implications for wildlife contamination worldwide, wherever these common rodenticides are deployed, as widespread exposure of non-target species can apparently occur via multiple trophic transfer pathways involving birds as well as rodents.

Current and historical hybridization with differential introgression among three species of cyprinid fishes (genus Cyprinella).

Hybridization is common among freshwater fishes, particular among the Cyprinidae. We used two mitochondrial genes and one nuclear gene to characterize hybridization among two species pairs of Cyprinella in southwestern North America. Genalogical patterns revealed that C. lutrensis and C. venusta are currently hybridizing in several localities producing apparent F(1), F(2) and backcross generations, yet there was no evidence for introgression outside of local hybrid zones. Alternatively, mitochondrial haplotypes from C. lutrensis appear to have introgressed into a C. lepida population in the Nueces River completely replacing the native C. lepida haplotype. There was no evidence of introgression of nuclear DNA and there does not appear to be ongoing hybridization. The population of C. lepida from the nearby Frio River exhibits no evidence of hybridization with C. lutrensis. Thus, contact between C. lutrensis and C. venusta results in the formation of localized hybrid swarms, while contact between C. lutrensis and C. lepida has resulted in complete mitochondrial introgression in the Nueces River or no apparent hybridization in the Frio River. The three different outcomes of contact between these species illustrate the variable nature of interspecific reproductive interactions and provide an excellent system in which to better understand the factors influencing hybridization among freshwater fishes.

Analysis of Free and Esterified Sterol Content and Composition in Seeds Using GC and ESI-MS/MS.

Total sterol content and composition in plant tissues can be easily determined by gas chromatography (GC) after saponification of the total lipid extract. However, in oleogenic tissues a significant proportion of the sterol is esterified to fatty acids, with GC methodologies unable to provide information about the proportion and the molecular species composition of intact steryl esters (SEs). Here we describe an electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and Multiple Reaction Monitoring (MRM) method which, in parallel with GC analysis, allows for the accurate determination of both free and esterified sterol content and composition in seeds. After extraction of seed oil with hexane, free sterols are derivatized with undecanoyl chloride, total steryl esters are then purified from triacylglycerol (TAG) by liquid chromatography, infused and ionized as ammonium adducts, with molecular species identified and quantified by fragmentation in the presence of internal standards.

Long-term woodland restoration on lowland farmland through passive rewilding.

Natural succession of vegetation on abandoned farmland provides opportunities for passive rewilding to re-establish native woodlands, but in Western Europe the patterns and outcomes of vegetation colonisation are poorly known. We combine time series of field surveys and remote sensing (lidar and photogrammetry) to study woodland development on two farmland fields in England over 24 and 59 years respectively: the New Wilderness (2.1 ha) abandoned in 1996, and the Old Wilderness (3.9 ha) abandoned in 1961, both adjacent to ancient woodland. Woody vegetation colonisation of the New Wilderness was rapid, with 86% vegetation cover averaging 2.9 m tall after 23 years post-abandonment. The Old Wilderness had 100% woody cover averaging 13.1 m tall after 53 years, with an overstorey tree-canopy (≥ 8 m tall) covering 91%. By this stage, the structural characteristics of the Old Wilderness were approaching those of neighbouring ancient woodlands. The woody species composition of both Wildernesses differed from ancient woodland, being dominated by animal-dispersed pedunculate oak Quercus robur and berry-bearing shrubs. Tree colonisation was spatially clustered, with wind-dispersed common ash Fraxinus excelsior mostly occurring near seed sources in adjacent woodland and hedgerows, and clusters of oaks probably resulting from acorn hoarding by birds and rodents. After 24 years the density of live trees in the New Wilderness was 132/ha (57% oak), with 390/ha (52% oak) in the Old Wilderness after 59 years; deadwood accounted for 8% of tree stems in the former and 14% in the latter. Passive rewilding of these 'Wilderness' sites shows that closed-canopy woodland readily re-established on abandoned farmland close to existing woodland, it was resilient to the presence of herbivores and variable weather, and approached the height structure of older woods within approximately 50 years. This study provides valuable long-term reference data in temperate Europe, helping to inform predictions of the potential outcomes of widespread abandonment of agricultural land in this region.

First microsatellite markers for the European Robin (Erithacus rubecula) and their application in analysis of parentage and genetic diversity.

The European Robin is a small passerine bird associated with woodlands of Eurasia and North Africa. Despite being relatively widespread and common, little is known of the species' breeding biology and genetic diversity. We used Next Generation Sequencing (NGS) to develop and characterize microsatellite markers for the European Robin, designing three multiplex panels to amplify 14 microsatellite loci. The level of polymorphism and its value for assessing parentage and genetic structure was estimated based on 119 individuals, including seven full families and 69 unrelated individuals form Poland's Bialowieza Primaeval Forest and an additional location in Portugal. All markers appeared to be highly variable. Analysis at the family level confirmed a Mendelian manner of inheritance in the investigated loci. Genetic data also revealed evidence for extra-pair paternity in one family. The set of markers that we developed are proven to be valuable for analysis of the breeding biology and population genetics of the European Robin.

Development of a C18 Supercritical Fluid Chromatography-Tandem Mass Spectrometry Methodology for the Analysis of Very-Long-Chain Polyunsaturated Fatty Acid Lipid Matrices and Its Application to Fish Oil Substitutes Derived from Genetically Modified Oilseeds in the Aquaculture Sector.

Lipidomics methodologies traditionally utilize either reverse phase- or hydrophilic interaction liquid chromatography-type separations; however, supercritical fluid chromatography can offer a rapid normal phase type separation while reducing the dependence on organic solvents. However, normal phase type lipid separations typically lack pronounced intraclass separation, which is problematic for complex lipidomes containing very-long-chain polyunsaturated fatty acids, especially those from genetically modified organisms. A high-strength silica C18 method was developed, which benefitted from discrete class separation, as well as displaying intraclass selectivity sufficient for profiling flesh of salmon fed with a diet supplemented with oil from the genetically engineered oilseed Camelina sativa, a terrestrial oilseed with a fish oil-type profile. Salmon fed a diet containing this Camelina oil were found to have flesh enriched in triacylglycerols and phospholipids containing 18:3, 20:5, and 22:6, whereas salmon fed the control diet were differentiated by shorter chain plant-type fatty acids integrated within complex lipids. Coupled with active scanning quadrupole technology, data acquisition was enhanced, allowing for fragmentation data to be acquired in a data independent fashion, permitting acyl chain identification of resolved isomers. Therefore, we have developed a method, which is amenable for lipidomics studies of complex lipidomes, specifically those altered by synthetic biology approaches.