BUSCADOR RECOLECTA

Data from the extensive literature review on each protein family and their allergenic members is fully described/summarised in the Electronic supplementary material section, as an excel file. This excel file is divided into eight pages, each dedicated to a single protein family (2S albumins, nsLTP, ATI, cereal prolamins, profilins, legumins, vicilins and PR-10 proteins). All the abbreviations used in the excel file are presented in this manuscript as an abbreviation list. Based on the information gathered in this excel file, Table 3 was constructed, thus summarising the data presented in the Electronic supplementary material section., Peer reviewed

[Methods] Methodology appears in detail in DOI: 10.1242/jeb.243342. The data set contains data on genotypes in SERT SNP290 and on three experiments on coping style on free living Great tits Parus major: The first experiment was risk-taking behavior in reaction to a flag that was attached to the top of the nest box at the time of feeding of the nestlings. The method was previously used and tested by Cole and Quinn (2014), and reflects the proactivity of the birds in front of a new object. Time to enter into the flagged nest box correlated with additional assays with the same birds in captivity, in which exploration rate in a novel environment was measured. This novel environment assay additionally stresses the trade-off between risk-taking and chick provisioning. This experiment was conducted when the chicks had a median age of 9 days (range 9 - 12) by video recording at the distance of 7-36 meters from the nests (median= 16 m), depending on the presence of vegetation around each nest. Distance from the observer to the nest was set as a covariate in the analysis. To ascertain for the effect of the flag (novel object) in our experiment, we first recorded the normal latency of the birds to enter the nest-box without any flag. Then, we recorded the behavior of the birds with the flag attached at the top of the nest-box. We recorded the behavior of the birds with a video camera, and corrected for the distance of the camera from the nest, since this could affect to the time of appearance of the bird in the video. The flag was removed after 40 minutes. This behaviour was tested in 34 individuals, using the birds that entered the nestbox during the control experiment (with no flag). However, when considering only the birds that entered the nestbox during the flag trial, sample size was reduced 24 birds. The trait has been shown previously to be repeatable (Cole and Quinn, 2014). The second test focused on distress calling rate during handling, which has also been previously recognized as a rate of proactivity (boldness): in black-capped chickadees (Poecile atricapillus), distress calling rate was positively related to exploration rate, which is one of the main characteristics of the proactive coping style (Guillette and Sturdy, 2011). In siskins (Carduelis spinus), individuals uttering more often distress calls also displayed bolder behaviors in front of a novel object (Mateos-González and Senar, 2012; Pascual and Senar, 2014). Distress calling has therefore been used as a proxy of proactivity in several studies (Andersen, 2012; Pascual and Senar, 2014; Richardson et al., 2016; Senar et al., 2017; Thorsteinsen, 2015). This was tested during the capture of the parents, normally at age 15 days of the chicks. Captured animals were kept in a ringing bag for a period of about 5 min to calm down. Just after extracting the bird from the ringing bag, distress calling rate was quantified by counting the number vocalized distress calls that were emitted by the birds during the next 15s of handling while holding their legs and moving a straight finger positioned at 1-2 cm from the beak of the focal bird (Markó et al., 2013; Senar et al., 2017). We recorded this behavior on a total of 64 birds. The trait has been shown previously to be repeatable (Senar et al., 2017). The third test focused on the propensity of incubating females to utter hissing calls towards a nest predator. This behaviour was first described in Great tits by Krams et al. (2014). We measured the hissing response of incubating females to a mouse model meanwhile we inserted the model head into the entrance of the nest box. The head of the mouse was kept in this position for 15 s. We counted the number of calls of incubating females in that period. As soon as 15 s was over, the mouse was removed and the observer silently moved away. The method has been used in several other papers although using a woodpecker as a nest predator (Krams et al., 2014; Thys et al., 2021; Timm et al., 2019). We recorded this behavior on a total of 34 females. The trait has been found previously to be highly repeatable (Koosa and Tilgar, 2016; Krams et al., 2014). DNA was extracted from blood samples using Ecogen MasterPure DNA purification kit (MCD85201). Primers were designed using the great tit genome (assembly 1.3, accession number: SRS1185780, (Laine et al., 2016) blasting with the reverse complement of the SERT promoter interval of the great tit (GenBank: accession number KP869099) (Riyahi et al., 2015). The primer sequences were as follows: Forward primer 5´-TTCAGCAATGCACAAAGTCCAG-3´, Reverse primer 5´-ACTCCAGGTCTCCCTGTCCTC-3´. Genetic polymorphisms within the SERT loci were genotyped by standard PCR amplification and direct sequencing of the resulting amplicon. 50 ng genomic DNA was amplified in 25 µl reactions containing 10x reaction buffer. All the samples were sequenced at the Genomics Core Facility of the University of Pompeu Fabra (Barcelona, Spain) using an Applied Biosystems sequencing platform. All of the sequences were aligned using Sequencher v4.6 (Gene Codes Corporation, MI, Ann Arbor, USA) and visually checked for polymorphisms (SNPs). The names of the SNPs were given by the position of each SNP in the resulting amplicon. [Usage notes] We intended to conduct the three behavioral experiments in our breeding pairs in all of the nest boxes. However, the number of individuals involved in each test varied because we were unable to perform some tests on some nests because the chicks died before conducting the experiment or because the parents abandoned the nests or were not captured. This is why sample sizes for the different experiments differ., The coping style of an individual in relation to potentially dangerous situations has been suggested to be inherited in a polygenic fashion, being SERT one of the candidate genes. In this paper, we assessed in free-living great tits Parus major the association between SNP290 in the SERT promoter and three standard fear-related behaviors, namely the response of the birds to a black and white flag fixed to the top of the nest-box, distress calling rate of the birds in the hand once captured and the hissing call of incubating females when approached by a predator. We found a strong association between SNP290 polymorphism and the three risk-taking behaviors, with birds with genotype CT entering faster to the nest box with the flag and displaying more distress calls and less hissing calls. CT birds could therefore be described as more proactive than CC individuals. These results also suggest that hissing behavior should be regarded as a fear-induced shy behavior, and confirm that SERT has an important function in relation to risk aversion behaviors and coping style., Ministerio de Economía y Competitividad, Award: CGL-2016-79568-C3-3-P; Ministerio de Ciencia e Innovación, Award: CGL-2020 PID2020-114907GB-C21., Peer reviewed

Fridericia chica (Bonpl.) L.G. Lohmann (synonym Arrabidaea chica Verlot) (Bignoniaceae) is widely used in folk medicine in Brazil for health benefits. Considering overcoming pitfalls of scaling up production of plant extracts, herein the effects of N2 atomizing gas in spray-drying process of standardized plant extract production is reported. Samples were monitored by in vitro antioxidant activity and microbiological evaluation. The type of gas used did not interfere in the product’s color. However, the drying atmosphere influenced 3-deoxyanthocyanines content when using air as atomizing gas, decreasing carajurin (37.5%) content with concomitant increase in luteolin yield (24.1%). Both drying processes preserved the samples’ pharmacological profile. The antioxidant activity was also similar IC50 (445.60 ± 12.73 and 466.99 ± 46.11). In the cell migration test with HaCaT cells, the extract dried under air flow (5 μg/mL) promoted wound closure by 78% (12 hours) whereas the extract dried using N2 flow promoted 49% (12 hours), with 98% closure (12 hours) for the positive control. The antimicrobial evaluation for Staphylococcus aureus did not differ with the different drying atmospheres, with MIC (minimum inhibitory concentration) at 0.39 mg/mL. Therefore, the drying process reported herein did not interfere with the outcome of the biological activity., Peer reviewed

[Methods] Distribution data was collected manually from Reptile Database (https://reptile-database.reptarium.cz/), and the biogeographic analyses were entirely performed in the R framework. [Usage notes] Information about directory structure can be found in the README.md file., The geographic distribution of biodiversity is central to understanding evolutionary biology. Paleogeographic and paleoclimatic histories often help to explain how biogeographic patterns unfold through time. However, such patterns are also influenced by a variety of other factors, such as lineage diversification, that may affect the probability of certain types of biogeographic events. The complex and well-known geologic and climatic history of Afro-Arabia, together with the extensive research on reptile systematics in the region, makes Afro-Arabian squamate communities an ideal system to investigate biogeographic patterns and their drivers. Here we reconstruct the phylogenetic relationships and the ancestral geographic distributions of several Afro-Arabian reptile clades (totaling 430 species) to estimate the number of dispersal, vicariance and range contraction events. We then compare the observed biogeographic history to a distribution of simulated biogeographic events based on the empirical phylogeny and the best-fit model. This allows us to identify periods in the past where the observed biogeographic history was likely shaped by forces beyond the ones included in the model. We find an increase in vicariance following the Oligocene, most likely caused by the fragmentation of the Afro-Arabian plate. In contrast, we did not find differences between observed and expected dispersal and range contraction levels. This is consistent with diversification enhanced by environmental processes and with the establishment of a dispersal corridor connecting Africa, Arabia and Eurasia since the middle Miocene. Finally, here we show that our novel approach is useful to pinpoint events in the evolutionary history of lineages that might reflect external forces not predicted by the underlying biogeographic model., Peer reviewed

Supplementary data 1: Table 1S. Analytical characteristics (volatile and non-volatile composition) of the wines employed in this study. Supplementary data 2: Table S2. Average oral aroma release values and results from one-way ANOVA, performed with each wine type (red or white), to check for significant differences (p < 0.05) among the four age-gender groups: young-female (YF), young-male (YM), senior-female (SF), senior-male (SM)., Peer reviewed

Supplemental Table S1. Complete overview of the inclusion and exclusion criteria for this study. Supplemental Table S2. Composition of the EuroFlow B-cell panel and technical information on the reagents for the IMI-2 PERISCOPE BERT study. Supplemental Table S3. Phenotypic descriptions used to define B-cell subsets stained with the EuroFlow B-cell panel by manual analysis. Supplemental Table S4. Baseline distribution of leukocytes, lymphocytes, T cells, and NK cells in donor groups. Supplemental Table S5. Spearman Ranking Correlation between IgG1+ plasma cell and memory B-cell kinetics and vaccine-component-specific serum IgG. Supplemental Table S6. Spearman Ranking Correlation between IgA1+ plasma cell and IgA memory B-cell kinetics and vaccine-component-specific serum IgA. Supplemental Figure S1. No clear over-time postvaccination changes in major populations in any of the donor groups. Supplemental Figure S2. Over-time maturation of total plasma cells. Supplemental Figure S3. No significant changes in IgG1+ memory B-cell subsets upon vaccination. Supplemental Figure S4. Correlation between cellular changes as measured by flow cytometry and ELISpot. Supplemental Figure S5. Correlation between cellular changes and the vaccine-specific serum IgG level postvaccination as determined by Spearman’s Ranking Correlation per age cohort. Supplemental Figure S6. Impact of sex on cellular responses after vaccination in the young adult cohort (all wP-primed). Supplemental Figure S7. IgG1+ and total plasma cell expansion is more prominent in non-age-matched donors after wP priming., Peer reviewed

3 tables. -- Stefanescu_et_al_JAE_data_butterfly_counts.csv: overwintering adult, fresh adult and larval nest counts of Aglais urticae for the three study regions. Counts were made fortnightly (15 visits, conditions permitting). -- Stefanescu_et_al_JAE_data_nettle_phenology.csv: nettle height (cm) and quality (1, 2, 3, 4) for the three study regions. Data were recorded fortnightly (16 visits per site, conditions permitting). -- Stefanescu_et_al_JAE_data_larval_parasitism.csv: larval nests collected for estimating parasitism rates for the three study regions. -- All models were run in R (R Core Team, 2018)., Parasitism is a key factor in the population dynamics of many herbivorous insects, although its impact on host populations varies widely, for instance, along latitudinal and altitudinal gradients. Understanding the sources of geographical variation in host-parasitoid interactions is crucial for reliably predicting the future success of the interacting species under a context of global change. Here, we examine larval parasitism in the butterfly Aglais urticae in south-west Europe, where it is a mountain specialist. Larval nests were sampled over two years along altitudinal gradients in three Iberian mountain ranges, including the Sierra Nevada, home to its southernmost European population. Additional data on nettle condition and adult butterflies were obtained in the study areas. These data sources were used to investigate whether or not differences in parasitism rates are related to the geographical position and phenology of the host, and to the availability of the host plants. Phenological differences in the host populations between regions were related to the severity of summer drought and the corresponding differences in host plant availability. At the trailing-edge of its distribution, the butterfly’s breeding season was restricted to the end of winter and spring, while in its northern Iberian range the season was prolonged until mid-summer. Although parasitism was an important source of mortality in all regions, parasitism rates and parasitoid richness were highest in the north and lowest in the south. Moreover, within a region, there was a notable increase in parasitism rates over time, which probably led to selection against an additional late-summer host generation in northern regions. Conversely, the shorter breeding season in Sierra Nevada resulted in a loss of synchrony between the host and one important late-season parasitoid, Sturmia bella, which may partly explain the high density of this butterfly species at the trailing-edge of its range. Our results support the key role of host phenology in accounting for differences in parasitism rates between populations. They also provide insights into how climate through host plant availability affects host phenology and, ultimately, the impact of parasitism on host populations., [Study system] We studied the complex of larval parasitoids of Aglais urticae in three regions encompassing the whole of its latitudinal range in the Iberian Peninsula: the Pyrenees in the north of the Iberian Peninsula, the Sierra de Guadarrama in central Spain, and Sierra Nevada in southern Spain. Sampling sites were established along an altitudinal gradient in each region that covered most of the altitudinal range in which this species breeds. Nine sites were sampled for parasitoids in the Pyrenees at 1,127–2,560 m a.s.l., seven sites in the Sierra de Guadarrama at 1,150–2,004 m a.s.l., and six sites in Sierra Nevada at 975–2,532 m a.s.l. As part of a larger project aimed at investigating various aspects of the ecology of A. urticae, additional sites were surveyed in each region. The information gathered at these additional sites was used in this work to improve knowledge of the phenology of this butterfly., [Field sampling] To study the phenology of A. urticae adults, we used 500-m transects on which butterflies were counted every two weeks from March to September (a total of 15 sampling visits), following the standard methodology of the Pollard walks (Pollard & Yates, 1993). Butterflies were classified either as overwintered or freshly emerged based on wing colouration (i.e. dull or brightly coloured, respectively). Transects were walked at 15, 24 and 14 sites in 2016, and at 16, 24 and 20 sites in 2017, in the Pyrenees, Sierra de Guadarrama and Sierra Nevada, respectively. To study the phenology of A. urticae larvae, we counted all larval nests found in four (exceptionally, just two and three at two sites) focal U. dioica patches in a subsample of the sites used for adult counts in each region. The focal patches were randomly selected along the butterfly transects and, if not available, at other accessible sites that were as close as possible to the transect route. The focal patches were visited every two weeks from March to September, whenever possible during the same visits as for the adult transect counts. To study larval parasitism, larval nests detected at focal U. dioica patches (see above) were marked and, if larvae were in the third or later instars, 20 individuals were collected to assess parasitism. Because the total number of larvae per nest was sometimes less than 20, the overall average number of larvae per sample (± SD) was 16.2 ± 6.9. Moreover, given that the opportunistic parasitoid, Cotesia vestalis, is known to parasitize first instar larvae of the small tortoiseshell and to emerge mainly from the second instar (Audusseau et al., 2021), in 2017 we also collected 8 samples of five second instar larvae in all three regions (3 samples in the Pyrenees, 2 in the Sierra de Guadarrama and 3 in Sierra Nevada). We did not assess pupal parasitism, even if it may be important (Pyörnilä,1977; Shaw et al., 2009) because pupae are difficult to locate in the field, thereby precluding any reliable estimates of mortality. Larvae were reared indoors in transparent plastic containers (155 x 105 x 45 mm) in groups of up to five individuals, all from the same sample. To avoid possible contamination, larvae were always reared with nettle leaves collected from their original nettle patch; if not available, nettles were harvested from sites where A. urticae and its closest congener, A. io, were absent, since some common parasitoids (e.g. the tachinids Sturmia bella and Pales pavida) lay microtype eggs on nettle leaves that can infect caterpillars if they eat these leaves. When a larva or pupa (in the case of larva-pupal parasitoids) produced a parasitoid, we recorded the stage at which the host was killed and kept the parasitoid individually in a vial until the adult emerged. Adults were preserved in pure ethanol for identification (Ichneumonoidea by MRS, Tachinidae by DH). Although hatching success was generally poor for most tachinids, careful inspection of puparia allowed for correct identification in almost all cases. To examine host plant availability, we recorded the growing condition (i.e. quality level and height) of nettles over the season at a subsample of sites used for larval nest counts in each region. At each visit, two stems were randomly selected from each nettle patch. Their height was measured (in cm) and they were given a categorical value from 1 (worst quality) to 4 (best quality) in which (1) corresponds to already dry or withered plants, with senescent leaves; (2) to flowering plants and plants with green but not fresh leaves; (3) to old plants in which regrowth leaves were beginning to become visible (a common situation at the end of summer after rain or after herbivory); and (4) to vigorous plants, with fresh leaves. Category ranking was based on previous work showing how nettles in these various phenological stages differently affect larval growth rates, pupal and adult weights in the small tortoiseshell (Pullin, 1987) and the map butterfly, Araschnia levana (Mevi-Schütz & Erhardt, 2005)., [Host phenology] A combination of the standardized adult and larval count data was used to define the phenology of the species. GAM models were fitted to the adult (overwintering and fresh butterflies separately) and larval nest counts, which allowed us to extract the Julian day corresponding to each peak of abundance in a given region and season. GAM models were built using the package mgcv in R (Wood, 2011). In these models we used pooled data from 2016 and 2017 to increase the sample size and to improve the overall phenological picture in each of the study regions. To investigate the potential altitudinal delay in larval phenology, we regressed separately the timing of larval nest appearance against site altitude for each year and region. The timing of larval nest appearance was summarised as the weighted mean appearance date., [Host plant phenology] We tested for differences in the phenology of nettles between regions using GAMM models, in which either nettle condition or height were the response variables and altitude, region, year, visit number (i.e. the timing of the season, used as the smoothing term) and the interactions of region with both visit number and altitude were the predictors. In these models, each individual stem was used as a data point and 'nettle patch' was entered as a random factor., [Impact of parasitism on host populations] To test whether or not the number of parasitoid species was comparable between regions (because sample sizes differed greatly between regions, see below), we computed the most common nonparametric estimators of species richness for each region and year separately (based on all recorded parasitoid species and genera) using the SpadeR package in R (Chao & Chiu, 2016). To assess the impact of parasitoids on host populations, we calculated the parasitism rate for each larval nest as the number of larvae killed as a result of parasitism, after discounting those that died for unknown reasons (i.e. our calculations were always based on effective larval samples). To avoid biases resulting from low sample sizes, the parasitism rate was calculated for effective samples of ≥ 5 larvae. We obtained very similar results (not shown) when models of parasitism rate were built following a more restrictive criterion of effective samples of ≥ 10 larva. The parasitism rate was modelled with generalized linear models (GLMs) using a binomial distribution and logit link function, with region, Julian day (date of nest collection) and altitude as predictors. However, because altitude and Julian day were highly correlated (r=0.76), only models with just one of these two variables were retained in the end. Models were built separately for 2016 and 2017 because the sampling sites in Andorra differed slightly between the two years. All possible models were built using the package lme4 in R (Bates et al., 2015); the best models were selected using the MuMIn package in R (Barton, 2015), with model selection being based on the Akaike Information Criterion (AIC). Models that differed by < 2 points from the lowest AIC (∆AIC < 2) were considered the top-ranked models (statistically equivalent to the best model of the set)., [Phenological overlap between the host and its main parasitoids] The overlap (i.e. temporal co-occurrence) between the host and its two main parasitoids, Pelatachina tibialis and Sturmia bella, was estimated using the Overlap Parasitoid-Host index (OPH), as described by Audusseau et al. (2020), which is bouinbded from zero to one. This index was calculated for all possible combinations of site and year in each region. The maximum value of 1 is obtained in the hypothetical case when all individuals recorded in a given season, both of the parasitoid and the host, are concentrated in the same sampling event k. The opposite situation occurs (index value equaling to zero) when in all available samples one of the interacting species is always missing. To understand which factors explain the degree of overlap between the parasitoid and the host, we used GLM models with a binomial distribution and a logit-link function. For each parasitoid species and year we applied a model in which OPH was the dependent variable and site altitude and region were the variable predictors., Ministerio de Ciencia e Innovación, R+D Programa Nacional, Proyecto I+D+I , Award: CGL2014-57784-P, Stefanescu_et_al_JAE_data_butterfly_counts.csv; Stefanescu_et_al_JAE_data_nettle_phenology.csv; Stefanescu_et_al_JAE_data_larval_parasitism.csv, Peer reviewed

Supplementary material: Supplementary captions: Figure S1 – Kinetic behavior of the extraction yield (g 100g-1) of supercritical fluid extracts from Pereskia aculeate leaves. Conditions: 40 °C, 25 MPa and 5 SLPM CO2 flow rate. Figure S2 – Kinetic behavior of the extraction yield (g 100g-1) of gas-expanded liquid extraction (GXL EtOH 25%, GXL EtOH 45%, GXL EtOH 75%) and pressurized liquid extraction (PLE EtOH 100%) from Pereskia aculeate leaves. Conditions: 40 °C, 7 MPa and 4 mL min-1. Figure S3 – Global yield (%) from subcritical water extractions (SW) at 5, 10, 15, 20 and 30 min and conventional alkaline extraction (CA) at 45 min of Pereskia aculeate leaves. Conditions: 80°C and 10.5 MPa., Peer reviewed

Multimedia component 1: Appendix. Panel A- ATR/FTIR spectra Panel B- Solid-state 13C CP MAS NMR spectra of the different feedstocks used., Peer reviewed

Supplementary data 1: Supplementary Table 1. Retention index, bioaccessibility, and potential bioavailability (%) of phenolic families from cocoa shell flour (CSF) and extract (CSE) after in vitro digestion. Supplementary Table 2. Physicochemical properties and intestinal absorption of the phenolic metabolites from the cocoa shell in silico colonic metabolism. Supplementary Figure 1. Association between the molecular weight of the cocoa shell phenolic metabolites and their Caco-2 (A) and human intestinal (B) absorption., Peer reviewed