Inheritance of DNA methylation differences in the mangrove Rhizophora mangle.

The capacity to respond to environmental challenges ultimately relies on phenotypic variation which manifests from complex interactions of genetic and nongenetic mechanisms through development. While we know something about genetic variation and structure of many species of conservation importance, we know very little about the nongenetic contributions to variation. Rhizophora mangle is a foundation species that occurs in coastal estuarine habitats throughout the neotropics where it provides critical ecosystem functions and is potentially threatened by anthropogenic environmental changes. Several studies have documented landscape-level patterns of genetic variation in this species, but we know virtually nothing about the inheritance of nongenetic variation. To assess one type of nongenetic variation, we examined the patterns of DNA sequence and DNA methylation in maternal plants and offspring from natural populations of R. mangle from the Gulf Coast of Florida. We used a reduced representation bisulfite sequencing approach (epi-genotyping by sequencing; epiGBS) to address the following questions: (a) What are the levels of genetic and epigenetic diversity in natural populations of R. mangle? (b) How are genetic and epigenetic variation structured within and among populations? (c) How faithfully is epigenetic variation inherited? We found low genetic diversity but high epigenetic diversity from natural populations of maternal plants in the field. In addition, a large portion (up to ~25%) of epigenetic differences among offspring grown in common garden was explained by maternal family. Therefore, epigenetic variation could be an important source of response to challenging environments in the genetically depauperate populations of this foundation species.

Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence.

Phenotypic variation determines the capacity of plants to adapt to changing environments and to colonize new habitats. Deciphering the mechanisms contributing to plant phenotypic variation and their effects on plant ecological interactions and evolutionary dynamics is thus central to all biological disciplines. In the past few decades, research on plant epigenetics is showing that (1) epigenetic variation is related to phenotypic variation and that some epigenetic marks drive major phenotypic changes in plants; (2) plant epigenomes are highly diverse, dynamic, and can respond rapidly to a variety of biotic and abiotic stimuli; (3) epigenetic variation can respond to selection and therefore play a role in adaptive evolution. Yet, current information in terms of species, geographic ranges, and ecological contexts analyzed so far is too limited to allow for generalizations about the relevance of epigenetic regulation in phenotypic innovation and plant adaptation across taxa. In this report, we contextualize the potential role of the epigenome in plant adaptation to the environment and describe the latest research in this field presented during the symposium "Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence" held within the Botany 2020 conference framework in summer 2020.

Galactose-α-1,3-galactose (alpha-gal) allergy: first pediatric case in a series of patients in Spain.

INTRODUCTION AND OBJECTIVES: Allergy to galactose-α-1,3-galactose (alpha-gal) is a peculiar form of food allergy generally manifesting as an anaphylactic reaction hours after mammalian meat consumption, due to the presence of specific IgE against this oligosaccharide. In addition, immediate anaphylaxis may develop after exposure to other sources of alpha-gal, such as monoclonal antibody cetuximab, vaccines, plasma expanders or anti-snake venoms. Sensitization to alpha-gal has also been implicated in the rapid degeneration of biological valve implants, and recognized as a cause of occupational disease in cattle raisers. The implication of tick bites in this type of sensitization has been accepted by all the research groups dedicated to this disease. PATIENTS AND METHOD: The present study describes the clinical and sensitization characteristics of 39 patients diagnosed with alpha-gal allergy in the hospitals of our province (Lugo, Monforte de Lemos and Burela, Spain). RESULTS: Most patients were middle-age males. Of note, is the fact that the series includes the first pediatric patient reported in Spain to date. The predominant clinical manifestations were urticaria or delayed anaphylaxis after consumption of mammalian meat. Seventy-four percent of the patients reported having suffered a previous tick bite, and the clinical presentation of anaphylaxis was significantly more prevalent in those with a persistent local reaction following the bite than in those with no such reaction (p = 0.032). CONCLUSIONS: A review is also made of the disorder which, due to its variable clinical expression, is referred to as alpha-gal syndrome. The study concludes that a diagnosis of alpha-gal allergy should be considered in patients with urticaria-anaphylaxis of uncertain origin or manifesting after the administration of vaccines or products of bovine/porcine origin.

Genetic structuring of the moss Pseudoscleropodium purum sampled at different distances from a pollution source.

In this study, we used amplified fragment length polymorphism analysis to investigate the genetic structure of the terrestrial moss Pseudoscleropodium purum (Hedw.) M. Fleish. naturally exposed to different levels of atmospheric deposition of heavy metals. We also determined the heavy metal concentrations in samples of this moss to evaluate whether there was a relationship between atmospheric pollution and population genetic diversity. A low level of genetic diversity and a limited gene flow among populations were observed which is in accordance to the prevalence of asexual reproduction in this species. In addition, no significant correlation was found between metal content and gene diversity in P. purum, probably because of the common history of the sampled populations and/or to the lack of a drastic reduction of the size of the population; nonetheless, a clear genetic structure was evident in relation to the existing pollution gradient. Thus, based on the results of the principal coordinate analysis and Bayesian analysis of the genotypes, the mixed structure of the second most polluted population would suggest an ongoing differentiation of metal-tolerant genotypes in the most polluted sites of the sampling area.

Serum tryptase concentrations in beekeepers with and without Hymenoptera venom allergy.

BACKGROUND: Increased tryptase concentrations are a risk marker for the severity of reactions to Hymenoptera stings or venom immunotherapy OBJECTIVE: To investigate serum tryptase concentrations in beekeepers with and without Hymenoptera venom allergy (HVA). METHODS: Serum tryptase concentrations were measured in adult patients with HVA (n = 91, 37 of whom were beekeepers), beekeepers without HVA (n = 152), and control individuals from the general adult population (n = 246). RESULTS: Multivariate analyses revealed that serum tryptase levels were positively associated with beekeeping activities (P < .001) and HVA (P < .001). Tryptase levels were also positively associated with age (P < .001) and male'sex (P = .02), and negatively associated with alcoho consumption (P = .002). CONCLUSIONS: Beekeeping and HVA are independently associated with increased concentrations of serum tryptase.

High Hg biomagnification in North Atlantic coast ecosystems and limits to the use of δ15N to estimate trophic magnification factors.

Mercury contamination is a global environmental problem. This pollutant is highly toxic and persistent which makes it extremely susceptible to biomagnify, i.e. increase its concentrations as it moves up the food chain, reaching levels that threaten wildlife and, ultimately, ecosystems' function and structure. Mercury monitoring is thus crucial to determine its potential to damage the environment. In this study, we assessed the temporal trends of the concentrations of Hg in two coastal animal species closely connected by a predator-prey interaction, and evaluated its potential transfer between trophic levels using the δ15N signatures of the two species. For this, we performed a multi-year survey of the concentrations of total Hg and the values of δ15N in the mussel Mytilus galloprovincialis (prey) and the dogwhelk Nucella lapillus (predator) sampled along ∼1500 km of the North Atlantic coast of Spain over a 30-year period (five surveys between 1990 and 2021). Concentrations of Hg decreased significantly between the first and the last survey in the two species studied. Except for the 1990 survey, the concentrations of Hg in mussels were amongst the lowest registered in the literature for the North East Atlantic Ocean (NEAO) and the Mediterranean Sea (MS) between 1985 and 2020. Nonetheless, we detected Hg biomagnification in almost all surveys. Worryingly, trophic magnification factors obtained here for total Hg were high and comparable to the found in the literature for methylmercury, the most toxic and readily biomagnified form of this element. The δ15N values were useful to detect Hg biomagnification under normal circumstances. However, we found that nitrogen pollution of coastal waters differentially affected the δ15N signatures of mussels and dogwhelks limiting the use of this parameter for this purpose. We conclude that Hg biomagnification could constitute an important environmental hazard even when found at very low concentrations in the lower trophic levels. Also, we warn that use of δ15N in biomagnification studies when there is some underlying nitrogen pollution problem might lead to misleading conclusions.

Analysis of mite allergic patients in a diverse territory by improved diagnostic tools.

BACKGROUND: There are few studies comparing the sensitization with mite allergens from different mite species which could potentially be the cause of allergy. OBJECTIVE: To improve the diagnosis of mite allergic patients from a diverse territory in which D. pteronyssinus/D. farinae mites together with storage mites could be present in the environment. METHODS: Four hundred and seventy-seven patients (both children and adults) from different regions, covering the main mite prevalent areas of Spain, were recruited. sIgE to eight allergens was measured together with SPT to whole mite extracts, level of mite allergen exposure, and specific IgG(4) . BAT and CAST was performed in a subgroup of patients. RESULTS: D. pteronyssinus and L. destructor were more prevalent in Atlantic areas, whereas D. farinae predominate in Mediterranean areas. About 90% of patients were sensitized to group 1 and/or group 2 allergens. Group 2 was the most prevalent, and the IgE response/intensity of sensitization in BAT was higher. sIgE to Der p 2/Der f 2 was almost fully cross-reactive, but no cross-reactivity was detected with Lep d 2. Group 1 allergens were also cross-reactive, but in some patients a species-specific response was observed. sIgE to Lep d 2 was associated with SPT results to storage mites. Sensitization to Der p 1 was more frequent in children, whereas Lep d 2 sensitization was more frequent in adults. A higher ratio IgE/IgG(4) to Der p 2 was associated with the presence of allergic asthma. CONCLUSION: An improved diagnosis algorithm has been established. Group 2 allergens seem to have a leading role in mite allergy, but as group 1 sensitization could be species-specific in some patients and its prevalence is higher in children, an adequate balance on major mite species and major allergens must be consider in the design of mite allergy vaccines.

Profilin as an aeroallergen by means of conjunctival allergen challenge with purified date palm profilin.

BACKGROUND: The role of profilin as a food allergen is well established, but little research has been done about its ability to elicit respiratory disease. Profilin is considered more of a confounding allergen on skin testing with whole pollen extracts than other airborne allergens. Our aim was to find out whether or not profilin can cause symptoms in sensitized individuals, which would be compatible with its role as an airborne allergen. METHODS: We performed conjunctival allergen challenges with date palm profilin in a series of consecutive pollen-allergic patients with rhino-conjunctivitis, divided in two groups: profilin sensitized (n = 17) and not sensitized (n = 14), who served as controls. We investigated the possible association between profilin sensitization and profilin allergy in these groups of patients. RESULTS: None of the patients from the not profilin-sensitized group had a positive result in conjunctival allergen challenges. In contrast, 65% of profilin sensitized patients had a positive conjunctival allergen challenge and were considered allergic to profilin. We found a significant statistical association between being profilin allergic and being profilin sensitized (χ(2) = 10.39, p < 0.005). CONCLUSIONS: Profilin seems to work as an aeroallergen in a significant proportion of profilin-sensitized patients. This might explain the uselessness of conjunctival challenges with whole pollen extracts to disclose genuine sensitization. In the future, the possibility of quantifying this allergen in pollen immunotherapy vaccines should be considered.

Heavy metal tolerance in Scopelophila cataractae: Transcriptomic and epigenetic datasets.

Studying how different plant groups deal with heavy metal exposure is crucial to improve our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used RNA sequencing (RNA-seq) and epigenotyping by sequencing (epiGBS) to assess gene expression and DNA methylation changes respectively in plants from four populations of the metallophyte moss Scopelophila cataractae treated with Cd or Cu in the laboratory. We built RNA-seq and epiGBS sequencing libraries from control and treated samples from each population and sequenced them using Illumina HiSeq 3000 (PE-150 bp) and Illumina HiSeq X-Ten System (PE-150 bp) respectively. For the RNA-seq data, we performed a read quality filter, mapped the reads to the de novo transcriptome created with Trinity, and estimated transcript abundance for each sample. For the epiGBS data, we used a custom pipeline (https://doi.org/10.5281/zenodo.7040291) to map the reads to a de novo reference genome and performed strand-specific nucleotide (single nucleotide polymorphisms, SNPs) and methylation (single cytosine methylation polymorphisms, SMPs) variant calling. We filtered out SNPs and SMPs with low coverage within (positions with <10 sequencing reads per sample) and across samples (positions with poor representation on the full set of samples). Finally, we performed pairwise comparisons between control and treated samples from each population and identified differentially expressed genes and differentially methylated cytosines associated to heavy metal exposure. We payed particular attention to the different responses of the more and the less tolerant populations of S. cataractae. These datasets could contribute to future comparative studies of abiotic stress response across plant groups.

Analysis of intra-thallus and temporal variability of trace elements and nitrogen in Fucus vesiculosus: Sampling protocol optimization for biomonitoring.

To advance the methodological standardization of the biomonitoring technique using macroalgae, we comprehensively characterized the intra-thallus and temporal patterns of variation in concentrations of a wide set of elements (Al, As, Cd, Co, Cr, Cu, Fe, Hg, Ni, Pb, Zn, N) and δ15N signal in 6 consecutive Fucus vesiculosus apical dichotomous sections collected monthly over a four-year period (2015-2019) at 3 sites on the NW coast of Spain. The concentrations of Al, Co, Fe, Ni, Pb and Zn increased significantly from the youngest to the oldest dichotomies regardless of the sampling time and collection site; As, Cd, N and δ15N showed the opposite trend. Time series analysis revealed a significant and consistent seasonal variation of As, Cd, Co, Cu, Fe, Hg, Ni, Zn, N and δ15N concentrations, with maximum values in winter and minimum values in summer. We discussed the possible mechanisms driving these two sources of variation, and proposed an efficient and effective sampling strategy to minimize their impact in the results of biomonitoring studies, in which the part of the algal thallus selected for chemical analysis and the sampling frequency were carefully considered. This protocol will improve the conclusions and comparability of biomonitoring data from coastal environments.

Epigenetic effects of parasites and pesticides on captive and wild nestling birds.

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ectoparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free-living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced-representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we conducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identified a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontarget effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost-effective way to characterize genome-wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects.

Exhaled nitric oxide predicts control in patients with difficult-to-treat asthma.

We aimed to evaluate the accuracy of baseline exhaled nitric oxide fraction (F(eNO)) to recognise individuals with difficult-to-treat asthma who have the potential to achieve control with a guideline-based stepwise strategy. 102 consecutive patients with suboptimal asthma control underwent stepwise increase in the treatment with maximal fluticasone/salmeterol combination dose for 1 month. Then, those who remained uncontrolled received oral corticosteroids for an additional month. With this approach, 53 patients (52%) gained control. Those who achieved control were more likely to have positive skin results (60.4% versus 34%; p = 0.01), positive bronchodilator test (57.1% versus 35.8%; p = 0.02) and peak expiratory flow variability > or =20% (71.1% versus 49.1%; p = 0.04). Conversely, depression was more frequent in those who remained uncontrolled (18.4 % versus 43.4 %; p = 0.01). An F(eNO) value > or =30 ppb demonstrated a sensitivity of 87.5% (95% CI 73.9-94.5%) and a specificity of 90.6% (95% CI 79.7-95.9%) for the identification of responsive asthmatics. The current results suggest that F(eNO) can identify patients with difficult-to-treat asthma and the potential to respond to high doses of inhaled corticosteroids or systemic steroids.

Double (honeybee and wasp) immunoglobulin E reactivity in patients allergic to Hymenoptera venom: the role of cross-reactive carbohydrates and alcohol consumption.

BACKGROUND: Immunoglobulin (Ig) E-mediated sensitization to N-glycans (cross-reactive carbohydrate determinants, CCDs) may induce double IgE reactivity to honeybee venom (HBV) and yellow jacket venom (YJV) in patients who are monosensitized to either of these venoms. Alcohol consumption is associated with increased IgE levels and possibly with sensitization to CCDs in the general population. OBJECTIVES: This study investigated the factors associated with double (HBV and YJV) IgE reactivity in patients who are allergic to Hymenoptera venom, and in particular, alcohol consumption. METHODS: Ninety-one patients with Hymenoptera allergy (68 to HBV, 19 to YJV, and 4 to both venoms) were studied. Determinations included a multiallergen IgE test and IgE to HBV, YJV, natural (glycosylated) HBV phospholipase-A2 (nPLA2), recombinant (nonglycosylated) HBV phospholipase-A2 (rPLA2), MUXF (the N-glycan from bromelain), natural (glycosylated) rubber latex, total IgE. Double reactivity was defined as an IgE level > 0.35 kU(A)/L to HBV and YJV. RESULTS: Double reactivity was observed in 28/87 (32%) clinically monosensitized patients. Double reactivity was associated with high levels of total IgE, MUXF-specific IgE, nPLA2-specific IgE, latex-specific IgE, and false-positive results in the multiallergen IgE test, but not with rPLA2-specific IgE. Alcohol consumption was associated with double reactivity and with high levels of IgE to glycosylated allergens after adjusting for confounders in the multivariate analysis. CONCLUSIONS: Sensitization to CCDs and clinically irrelevant double (honeybee and wasp) IgE reactivity are common among Hymenoptera venom-allergic patients who drink alcohol. A simple questionnaire about alcohol consumption could be useful when interpreting levels of specific IgE in these patients.

Follow-up of venom immunotherapy (VIT) based on conventional techniques and monitoring of immunoglobulin E to individual venom allergens.

OBJECTIVES: To assess the efficacy of venom immunotherapy (VIT) and monitor changes in in vivo and in vitro test results after 5 years of treatment and subsequent follow-up. To study the profile of immunoglobulin (Ig) E to individual allergens prior to treatment and 1 year afterwards. METHODS: We studied 562 patients with hymenoptera venom allergy (438 to bee, 124 to wasp), all of whom underwent immunotherapy with Apis or Vespula extract. The patients were followed up using conventional in vivo and in vitro tests, and in 51 cases, specific IgE against the main hymenoptera allergens was measured before starting and after 1 year of treatment. RESULTS: Of the 387 patients who completed VLT, 130 sensitized to Apis and 68 to Vespula suffered spontaneous re-stings during treatment. Of these, 123 (94.6%) did not suffer any reaction and 64 (94.1%) suffered only a local reaction. Sixty-two patients sensitized to Apis and 14 sensitized to Vespula suffered spontaneous re-stings after stopping treatment. Only 3 patients suffered a systemic reaction (grade I Müller). At the end of treatment, the results of skin tests and specific IgE to whole extract improved significantly. Reductions in IgE to the main allergens were observed after 1 year of treatment (median differences in Ves v 5, -238.0, P = .0425; and in Api m 1, -183.0, P = .0024). CONCLUSION: The high rate of spontaneous re-stings shows that efficacy is maintained for years after completing treatment in a real-world setting. Determination of IgE to individual venom allergens may offer new perspectives in the diagnosis and follow-up of these patients.

Matching times: Trying to improve the correlation between heavy metal levels in mosses and bulk deposition.

The relationship between the concentrations of metals in moss tissues and atmospheric deposition is highly complex, resulting in a general lack of correlations between these two matrices. Here, we tried to improve the significance of the moss-bulk deposition (BD) relationship by eliminating the mismatch between the time that the moss tissue selected for analysis is exposed to atmospheric deposition, and the time during which BD is collected. For this, we analysed the concentrations of Cd, Hg and Pb in new grown tissue of Pseudoscleropodium purum and BD collected monthly, for one year, in 21 sampling sites (SS) under different degrees of pollution. Additionally, we assessed how different moss tissues, including native moss (green parts and new grown tissues of P. purum) and moss transplants of Sphagnum denticulatum, reflect BD to find out which moss tissues provide a better estimate of the atmospheric deposition of heavy metals. First of all, our results showed that eliminating the mismatch between native moss exposure time and BD collection period is not enough to improve their correlation. Environmental variation emerged as the main driver of tissue content variation altering the moss-BD relationship unpredictably. Secondly, native P. purum represents BD values better than devitalized transplants by displaying a greater number of significant correlations with BD. Specifically, green parts of P. purum generally represent better BD than new grown tissues. Overall, we conclude that neither native mosses nor transplants are good estimators of atmospheric heavy metal deposition rates. However, they are good qualitative indicators of the atmospheric deposition, by allowing us to differentiate SS subject to a wide range of pollution levels. Additionally, green parts of P. purum, and likely of other mosses with similar growth forms, should be used in passive biomonitoring studies to make results from different studies comparable.

Epigenetic Approaches in Non-Model Plants.

Reduced representation bisulfite sequencing is an emerging methodology for evolutionary and ecological genomics and epigenomics research because it provides a cost-effective, high-resolution tool for exploration and comparative analysis of DNA methylation and genetic variation. Here we describe how digestion of genomic plant DNA with restriction enzymes, subsequent bisulfite conversion of unmethylated cytosines, and final DNA sequencing allow for the examination of genome-wide genetic and epigenetic variation in plants without the need for a reference genome. We explain how the use of several combinations of barcoded adapters for the creation of highly multiplexed libraries allows the inclusion of up to 144 different samples/individuals in only one sequencing lane.

Application of macroalgae analysis to assess the natural variability in selected pollution concentrations (N and Hg), and to detect sources of it in coastal environments.

Here we present a new method in which algae are used to detect sources of heavy metal and organic pollution in coastal areas. The procedure involves characterization of the natural range of concentrations of the elements in areas not affected by local sources of pollution and subsequent comparison of concentrations in the study site to these natural range levels. To develop the method, the concentrations of various elements were determined in specimens of the macroalgae Fucus vesiculosus collected at >150 sampling sites along the shoreline. The natural variability in the element concentrations in these zones was established by determining the differences in the tissue concentrations of the elements between pairs of samples separated by different distances. The method was then tested in the surroundings of possible sources of nitrogen and was found to be a tool for detecting sources of small scale nitrogen contamination and for monitoring and evaluating water quality.