Preference for high concentrations of plant pyrrolizidine alkaloids in the specialist arctiid moth Utetheisa ornatrix depends on previous experience

Secondary metabolites are one the most pervasive defensive mechanisms in plants. Many specialist herbivores have evolved adaptations to overcome these defensive compounds. Some herbivores can even take advantage of these compounds by sequestering them for protection and/or mate attraction. One of the most studied specialist insects that sequesters secondary metabolites is the arctiid moth Utetheisa ornatrix. This species sequesters pyrrolizidine alkaloids (PAs) from its host plant, the legume Crotalaria spp. The sequestered PAs are used as a predator repellent and as a mating pheromone. We used this species to test larval preference for different concentrations of PAs. We purified PAs from plant material and added them at different concentrations to an artificial diet. Larvae of U. ornatrix previously feeding on low and high PA concentration artificial diets were allowed to choose between two new artificial diets with different PA concentrations. The amount of PAs sequestered and larval preference were dependent on their previous exposure to low or high PA content in the diet. Larvae that were pretreated with a low PA diet significantly consumed more diet with the high PA concentration, while larvae that were pretreated with a high PA diet showed no discrimination between future feeding of different PA concentration diets. We discuss our results using mechanistic and evolutionary approaches. Finally, we discuss how these results have important implications on the evolution of plant herbivore interactions and how specialist herbivores may decrease the levels of chemical defenses on plant populations.     

Complex effects of parasitoids on pharmacophagy and diet choice of a polyphagous caterpillar

This study investigates complex effects of parasitoid infection on herbivore diet choice. Specifically, we examine how immunological resistance, parasitoid infection stage, and parasitoid taxonomic identity affect the pharmacophagous behavior of the polyphagous caterpillar, Grammia incorrupta (Arctiidae). Using a combination of lab and field experiments, we test the caterpillar’s pharmacophagous response to pyrrolizidine alkaloids (PAs) over the course of parasitoid infection, as well as the effect of dietary PAs on the caterpillar’s immunological response. Previous work from other systems gave the prediction that dietary PAs would be detrimental to the immune response and thus less acceptable to feeding early in the infection, when encapsulation of the parasitoid is most crucial. We found that the feeding acceptability of PAs was indeed low for caterpillars with early-stage parasitoid infections; however, this was not explained by PA interference with immune function. When allowed to choose among three host plant species, individuals harboring early-stage parasitoids increased their consumption of a nutritious plant containing antioxidants. This result was driven by wasp-parasitized caterpillars, whereas fly-parasitized caterpillars increased their consumption of plants containing iridoid glycosides. Individuals in the later time phase of infection exhibited an increase in PA intake that was consistent with previously reported self-medication behavior during late-stage parasitoid infection. This study reveals the depth of complexity and the dynamic nature of herbivore host plant choice, and underscores the importance of considering multitrophic interactions when studying insect diet choice.     

Detection of pyrrolizidine alkaloids in German licensed herbal medicinal teas

BackgroundBecause of the hepatotoxic, mutagenic, and cancerogenic effects of pyrrolizidine alkaloids (PAs) the German Federal Institute for Risk Assessment (BfR) recommends not to exceed a daily PA intake of 0.007 µg/kg body weight (0.42 µg/60 kg adult). In a recent study conducted by the BfR, up to 5647 µg PA/kg dried herbal material were detected in tea products marketed as food.PurposeThe present study aimed at elucidating whether medicinal teas licensed or registered as medicinal products contain PAs as well.Study designOne hundred sixty-nine different commercially available medicinal teas, i.e. 19 nettle (Urtica dioica L.), 12 fennel (Foeniculum vulgare Mill.), 14 chamomile (Matricaria recutita L.), 11 melissa (Melissa officinalis L.) and 4 peppermint (Mentha piperita L.) teas as well as 109 tea mixtures were analyzed for the presence of 23 commercially available PAs.MethodLC/MS was used for the determination of the PAsResultsIn general, the total PA contents ranging 0–5668 µg/kg. Thirty percent of the tested single-ingredient tea products and 56.9% of the tested medicinal tea mixtures were found to contain PA concentrations above the limit of quantification (LOQ) of 10 µg/kg. In 11 medicinal teas PA contents >300 µg/kg dry herb were determined thus exceeding the recommended limit for PA intake by BfR. In addition three products of the investigated tea mixtures revealed extremely high PA contents of 4227, 5137, and 5668 µg/kg. Generally, single-ingredient tea products contained much less or even no detectable amounts of PAs when compared to the tea mixtures. PAs in the range between 13 and 1080 µg/kg were also detected in five analyzed aqueous herbal infusions of the medicinal tea mixture products with the highest PA content. Two out of the five investigated herbal infusions exceeded the recommended BfR limit for PA intake.ConclusionThis study demonstrates clearly that also medicinal teas licensed as medicinal products may partly contain high amounts of PAs exceeding current recommendations. For that reason manufacturers are advised to carry out more rigorous quality control tests devoted to the detection of PAs. This is very important to minimize PAs in medicinal teas accounting for possible additional exposure of the consumer to PAs from other food sources (e.g. honey).     

Genetic variation in constitutive and inducible pyrrolizidine alkaloid levels inCynoglossum officinale L.

The constitutive pyrrolizidine alkaloid (PA) concentration of both shoots and roots differed significantly between 17 selfed families. The broad-sense heritability accounted for 33–43% of the variation in PA levels. Families also differed significantly in the amount and the direction of PA induction in both shoots and roots, 24 h after punching 15 holes in the leaves. We found a significantly negative relationship between the changes in PA content of the shoots and changes in PA content of the roots. The total PA content of the plants did not increase. We thus concluded that changes in PA distribution over the plant resulted from transport of PAs within the plant. The direction of transport differed between families: some transported PAs to the shoots, others to the roots. This makes it questionable whether PAs act as damage-induced defences. The effect of damage on the PA concentration is far less than the differences found between families in the constitutive PA concentration. This again strongly suggests that damage-induced defences inCynoglossum officinale do not play an important role. We argue that the general lack of attention that is given to genotype in induction experiments, has led to false conclusions.     

Seasonal variability in pyrrolizidine alkaloids in Senecio inaequidens from the Val Venosta (Northern Italy)

Senecio inaequidens is a neophyte originating from South Africa that has managed to spread to Europe and colonize large areas. This plant has toxic pyrrolizidine alkaloids (PAs) that represent a health risk to humans and animals. The aim of this work was to measure the content of PAs of these plants in continuous intervals over the entire growing season. The plants were separated into their organs, such as sprouts, stems, leaves and inflorescences. PAs were extracted from the dried plants using an acidic methanol solution, purified and measured by GC/MS. The average PA content of plants over the growing season was 0.33% of dry weight. The highest PA levels were about 1% in the dry weight and were found in the young sprouts and flower heads. There were nine PAs present, of which six could be identified. The main alkaloid was retrorsine followed by senecivernine, senecionine, integerrimine, usaramine and seneciphylline. Due to the high PA content of the inflorescences, the long flowering period and the rapid invasion dynamics, this species presents a high health risk for humans and animals.     

A free lunch? No cost for acquiring defensive plant pyrrolizidine alkaloids in a specialist arctiid moth (Utetheisa ornatrix)

Many herbivorous insects sequester defensive chemicals from their host plants. We tested sequestration fitness costs in the specialist moth Utetheisa ornatrix (Lepidoptera: Arctiidae). We added pyrrolizidine alkaloids (PAs) to an artificial diet at different concentrations. Of all the larval and adult fitness components measured, only development time was negatively affected by PA concentration. These results were repeated under stressful laboratory conditions. On the other hand, the amount of PAs sequestered greatly increased with the diet PA concentration. Absence of a detectable negative effect does not necessarily imply a lack of costs if all individuals express the biochemical machinery of detoxification and sequestration constitutively. Therefore, we used qPCR to show that expression of the gene used to detoxify PAs, pyrrolizidine‐alkaloid‐N‐oxygenase (pno), increased 41‐fold in our highest PA treatment. Nevertheless, fitness components were affected only slightly or not at all, suggesting that sequestration in this species does not incur a strong cost. The apparent lack of costs has important implications for our understanding of the evolution of ecological interactions; for example, it implies that selection by specialist herbivores may decrease the levels of certain chemical defences in plant populations.     

Dietary fat increases energy intake across the range of typical consumption in the United States

Objective: The fat content of a diet has been shown to affect total energy intake, but controlled feeding trials have only compared very high (40% of total calories) fat diets with very low (20% of total calories) fat diets. This study was designed to measure accurately the voluntary food and energy intake over a range of typical intake for dietary fat. Methods and Procedures: Twenty‐two non‐obese subjects were studied for 4 days on each of three diets, which included core foods designed to contain 26, 34, and 40% fat, respectively of total calories and ad lib buffet foods of similar fat content. All diets were matched for determinants of energy density except dietary fat. Subjects consumed two meals/day in an inpatient unit and were provided the third meal and snack foods while on each diet. All food provided and not eaten was measured by research staff. Results: Voluntary energy intake increased significantly as dietary fat content increased (P = 0.008). On the 26% dietary fat treatment, subjects consumed 23.8% dietary fat (core and ad lib foods combined) and 2,748 ± 741 kcal/day (mean ± s.d.); at 34% dietary fat, subjects consumed 32.7% fat and 2,983 ± 886 kcal/day; and at 40% dietary fat subjects consumed 38.1% fat and 3,018 ± 963 kcal/day. Discussion: These results show that energy intake increases as dietary fat content increases across the usual range of dietary fat consumed in the United States. Even small reductions in dietary fat could help in lowering total energy intake and reducing weight gain in the population.     

论保护地分类与以国家公园为主体的中国保护地建设

中国正在开展一场以国家公园为龙头的保护地建设, 面临着一个跨领域、跨行业、全空间的保护地分类管理难题。世界各国建立了各种各样的保护地, 这些保护地存在着“同物异名、同名异物、一物多名”现象。IUCN提出了一个保护地分类系统, 以期将所有的保护地纳入一个体系。人们试图按照IUCN的保护地分类系统对现有保护地分类, 但是IUCN保护地分类系统的定义模糊, 无量化标准, 而保护地的属性常常存在非唯一属性等问题。保护地分类是一个学术问题, 可以自由探讨, 百家争鸣; 而保护地的管理是行政问题, 必须有章可依, 有法可循。中国现有10多种不同的保护地类型, 本身就是一种分类体系, 有的保护地, 如自然保护区、风景名胜区、地质公园等已经有相应的行政法规和管理经验。中国国家公园应整合优化而不是取代现有的其他类型保护地。以国家公园为龙头的中国保护地体系需要顶层设计和专门立法。保护地应当推动当地社区人与自然协调发展。     

Methods for Assessment of Dietary Lead Exposure of the Inner-City Population

Abstract

Assessment of dietary lead exposure of individuals begins with the determination of food and beverage intake by the individuals, and concludes with an evaluation of the lead content of the foods and beverages consumed. Of several techniques available for assessment of dietary intake, the 24-hour food recall is recommended as the method of choice for assessing current dietary lead intakes in inner-city populations. The three-day food record can be used among cooperative and motivated subjects, while the dietary history method is available for assessing long-term intakes in the past. The unavailability of lead content values of a large number of foods will to a large extent restrict the use of these methods in large-scale dietary lead exposure studies. Until the time that such data becomes available, the most accurate estimates of lead intake can be provided by chemical analysis of duplicate samples of foods consumed, as is currently done. However, this method is feasible only for small samples.     

Polyamines in macroalgae: advances and future perspectives

Polyamines (PA) are ubiquitous, small, aliphatic cations found in all living cells. In recent years the importance of these molecules for macroalgae has become evident and a substantial body of knowledge has been accumulated over the last three decades. This review summarizes research on the PAs found in macroalgae, their transport and metabolism, and their biological significance in processes such as cell division, chloroplast development, and reproduction. The involvement of PAs in environmental stress responses in macroalgae is also addressed. The discussion of PAs in this review not only demonstrates that PAs play an important role in physiological processes in macroalgae, but also clearly demonstrates the similarities and differences between PA metabolism in macroalgae and higher plants. Key areas for future research are also discussed.     

Why do we lose protected areas? Factors influencing protected area downgrading,downsizing and degazettement in the tropics and subtropics

Protected areas (PAs) are an essential tool for the conservation of biodiversity globally. Previous studies have focussed on the effectiveness of PAs and the design of optimal PA networks. However, not all PAs remain intact permanently; many PAs undergo downgrading, downsizing and/or degazettement (PADDD), a fact largely ignored until recently. The drivers of enacted PADDD events and the factors influencing its spatial occurrence are poorly understood, potentially undermining the efficacy of PAs and PA networks. Here we examine the spatial relationship between PADDD and economic, demographic and structural variables, using a 110‐year data set of 342 enacted PADDD events across 44 countries in the tropics and subtropics. We find that the probability of an enacted PADDD event increases with the size of the PA and through a synergistic interaction between PA size and local population densities. Our results are robust to the under‐reporting of enacted PADDD events that occur among smaller PAs and in regions with lower population density. We find an economic motive for PADDD events, given that the opportunity costs associated with larger PAs are higher, on average, than smaller PAs. Our findings suggest a need for conservation practitioners to better consider PA characteristics, as well as the social, economic and political context in which PAs are situated, to aid the creation of more efficient and sustainable PA networks. In particular, the dynamics of enacted PADDD events highlight the need to explicitly consider PA robustness as a core component of systematic conservation planning for PA networks.     

A Preliminary Study of a Peruvian Diet Using Dietary Analysis and Hair Mineral Content as Indicators

Observations among former American residents living long-term in Peru suggested that hair health improved while in Peru. To determine if a Peruvian diet correlates with hair composition, dietary intake of nutrients and mineral content of hair were measured among Peruvian and matched US residents. Selected foods from Peru were also analyzed for mineral and antioxidant content and compared with equivalent foods available in the USA. Statistically significant differences between Peruvian and US residents’ hair were found for sodium (decreased in Peru, p?=?0.007) and vanadium (decreased in Peru, p?=?0.03). Differences in hair composition between residencies may be explained by lower dietary sodium and vanadium intake among Peruvian residents or by lower concentrations of these minerals in Peruvian drinking water. Many significant mineral differences were also identified between Peruvian foods and their US equivalents. Although no statistically significant correlations between dietary intake and hair mineral content were found, results indicate that a Peruvian diet contributes differently to hair composition than a US diet. More research is needed to elucidate the link between a Peruvian diet and specific aspects of hair health.     

Phytoestrogen consumption and association with breast, prostate and colorectal cancer in EPIC Norfolk

Phytoestrogens are polyphenolic secondary plant metabolites that have structural and functional similarities to 17β-oestradiol and have been associated with a protective effect against hormone-related cancers. Most foods in the UK only contain small amounts of phytoestrogens (median content 21 μg/100 g) and the highest content is found in soya and soya-containing foods. The highest phytoestrogen content in commonly consumed foods is found in breads (average content 450 μg/100 g), the main source of isoflavones in the UK diet. The phytoestrogen consumption in cases and controls was considerably lower than in Asian countries. No significant associations between phytoestrogen intake and breast cancer risk in a nested case-control study in EPIC Norfolk were found. Conversely, colorectal cancer risk was inversely associated with enterolignan intake in women but not in men. Prostate cancer risk was positively associated with enterolignan intake, however this association became non-significant when adjusting for dairy intake, suggesting that enterolignans can act as a surrogate marker for dairy or calcium intake.     

Involvement of polyamines in the inhibiting effect of injury caused by cutting on K+ uptake through the plasma membrane

The inhibition of K⁺ uptake through the plasma membrane resulting from injury caused by cutting, or from application of polyamines (PAs), has been investigated in root segments of maize (Zea mays L.) and pea (Pisum sativum L.). It was found, for both treatments, that K⁺ uptake recovered if the segments were washed for 2 h. The K⁺ uptake inhibited by cutting and that inhibited by spermidine treatment were stimulated to the same extent by fusicoccin. In addition, there was a correlation between the extent of the recovery of K⁺ uptake caused by washing and the distribution, along the root axis, of both PAs and the activities of enzymes responsible for PA degradation. In apical segments of maize, where the PA content and the activity of the degradative enzyme polyamine oxidase (EC 1.5.3.3) were higher than in the more distal segments, the recovery of K⁺ uptake caused by washing was also higher. On the other hand, the opposite trend was observed in root segments of pea, where the PA content and the activity of the degradative enzyme diamine oxidase (EC 1.4.3.6) were higher in distal segments in which K⁺ uptake was greatly stimulated by washing. The effect of the amine-oxidase inhibitor, aminoguanidine, indicates that the degradation products of PAs are involved in the mechanism of inhibition of K⁺ uptake by PAs. The data also seem to indicate that PAs and their degradation products are responsible for the inhibition of K⁺ uptake occurring as a result of injury sustained by cutting roots into segments.Abbreviations DAO diamine oxidase - FC fusicoccin - PA polyamine - PAO polyamine oxidase - PUT putrescine - SPD spermidine     

Sequence microheterogeneity of parvalbumin,the major fish allergen

The microheterogeneity of amino acid sequence observed in various allergens may affect immune response, but incidence of sequence microheterogeneity in allergens and its relation to their allergenicity are unclear. The occurrence of sequence microheterogeneity in major fish allergen, parvalbumin (PA), has been explored using bioinformatics approaches. 44% of 111 species with known PA sequence have PA isoforms. 41% of these species exhibit from 1 to 4 cases of PA sequence microheterogeneity, i.e. unique pairs of PA isoforms with sequence identity above 90%. 29% of 210 PA sequences studied are characterized by microheterogeneity. The occurrence of allergens among them is 2.5-fold higher than among other PAs. The incidence of sequence microheterogeneity within more allergenic β isoform of PA is 2.0-fold lower than that for its less allergenic α isoform. 39 residues affected by PA microheterogeneity are concentrated in the region of helices A, B, F, while helices D and E are the most conservative region. 44% and 11% of the microheterogeneous substitutions are located in the species-specific and cross-reactive IgE-binding epitopes of PAs, respectively. 45% and 48% of the substitution cases are predicted to cause notable changes in protein disorder propensity and protein stability, respectively. Hence, the increased allergenicity rate among PAs having microheterogeneous isoforms can be related to differences in their IgE-binding caused directly or in an allosteric manner. Overall, sequence microheterogeneity is shown to be inherent to many of PAs and likely contributes to PA allergenicity.     

Evaluating whether protected areas reduce tropical deforestation in Sumatra

Aim  This study determines whether the establishment of tropical protected areas (PAs) has led to a reduction in deforestation within their boundaries or whether deforestation has been displaced to adjacent unprotected areas: a process termed neighbourhood leakage.
Location  Sumatra, Indonesia.
Methods  We processed and analysed 98 corresponding LANDSAT satellite images with a c.  800 m² resolution to map deforestation from 1990 to 2000 across 440,000 km² on the main island of Sumatra and the smaller island of Siberut. We compared deforestation rates across three categories of land: (1) within PAs; (2) in adjacent unprotected land lying with 10 km of PA boundaries; and (3) within the wider unprotected landscape. We used the statistical method of propensity score matching to predict the deforestation that would have been observed had there been no PAs and to control for the generally remote locations in which Sumatran PAs were established.
Results  During the period 1990–2000 deforestation rates were found to be lower inside PAs than in adjacent unprotected areas or in the wider landscape. Deforestation rates were also found to be lower in adjacent unprotected areas than in the wider landscape.
Main conclusions  Sumatran PAs have lower deforestation rates than unprotected areas. Furthermore, a reduction in deforestation rates inside Sumatran PAs has promoted protection, rather than deforestation, in adjacent unprotected land lying within 10 km of PA boundaries. Despite this positive evaluation, deforestation and logging have not halted within the boundaries of Sumatran PAs. Therefore the long-term viability of Sumatran forests remains open to question.     

Changes in Large Pulmonary Arterial Viscoelasticity in Chronic Pulmonary Hypertension

Conduit pulmonary artery (PA) stiffening is characteristic of pulmonary arterial hypertension (PAH) and is an excellent predictor of mortality due to right ventricular (RV) overload. To better understand the impact of conduit PA stiffening on RV afterload, it is critical to examine the arterial viscoelastic properties, which require measurements of elasticity (energy storage behavior) and viscosity (energy dissipation behavior). Here we hypothesize that PAH leads to frequency-dependent changes in arterial stiffness (related to elasticity) and damping ratio (related to viscosity) in large PAs. To test our hypothesis, PAH was induced by the combination of chronic hypoxia and an antiangiogenic compound (SU5416) treatment in mice. Static and sinusoidal pressure-inflation tests were performed on isolated conduit PAs at various frequencies (0.01–20 Hz) to obtain the mechanical properties in the absence of smooth muscle contraction. Static mechanical tests showed significant stiffening of large PAs with PAH, as expected. In dynamic mechanical tests, structural stiffness (κ) increased and damping ratio (D) decreased at a physiologically relevant frequency (10 Hz) in hypertensive PAs. The dynamic elastic modulus (E), a material stiffness, did not increase significantly with PAH. All dynamic mechanical properties were strong functions of frequency. In particular, κ, E and D increased with increasing frequency in control PAs. While this behavior remained for D in hypertensive PAs, it reversed for κ and E. Since these novel dynamic mechanical property changes were found in the absence of changes in smooth muscle cell content or contraction, changes in collagen and proteoglycans and their interactions are likely critical to arterial viscoelasticity in a way that has not been previously described. The impact of these changes in PA viscoelasticity on RV afterload in PAH awaits further investigation.     

Metabolic engineering and potential functions of proanthocyanidins in poplar

Poplar (Populus spp.) is a widely distributed tree genus of significant economic and ecological importance. Poplar trees accumulate proanthocyanidins (PAs) in leaves, roots, and a variety of other tissues. Damage to leaves by insects causes a rapid accumulation of PAs, both at the site of damage and distally in undamaged leaves. This rapid PA accumulation is mediated by the activation of genes encoding enzymes involved in PA synthesis. PAs have been hypothesized to deter insect feeding and reduce the nutritive value of poplar leaf tissue, but experimental evidence supporting a role for PAs as an effective inducible defense against herbivores is lacking. Our recent paper described the identification of a MYB gene that regulates the PA pathway under multiple stress conditions, and we used this gene to constitutively activate the PA pathway in poplar. Here we describe observations that suggest that poplar PAs may have roles besides insect defense, for example, responses to UV light. The PA-modified trees will be a useful tool for analyzing the biological roles of PAs in this important model tree.Key words: tannins, herbivory, flavonoid, UV light, light stress     

Toxicity of pyrrolizidine alkaloids to humans and ruminants

1,2-dehydro pyrrolizidine ester alkaloids (PA) are toxic for human and livestock. The PAs undergo a metabolic toxication process in the liver which is the first target organ for PA poisoning. World-wide many episodes of PA intoxications have been reported involving humans as well as ruminants. This intoxication is not only related to the amount and duration of the exposure to PAs but also to species, age and gender. Besides the metabolic toxification, detoxication processes are also important. The paper discusses the toxification and detoxication processes and gives an overview about PA poisoning cases in humans and ruminants.     

Protected areas in the world’s ecoregions: How well connected are they?

Protected areas (PAs) are the main instrument for biodiversity conservation, which has triggered the development of numerous indicators and assessments on their coverage, performance and efficiency. The connectivity of the PA networks at a global scale has however been much less explored; previous studies have either focused on particular regions of the world or have only considered some types of PAs.Here we present, and globally assess, ProtConn, an indicator of PA connectivity that (i) quantifies the percentage of a study region covered by protected connected lands, (ii) can be partitioned in several components depicting different categories of land (unprotected, protected or transboundary) through which movement between protected locations may occur, (iii) is easy to communicate, to compare with PA coverage and to use in the assessment of global targets for PA systems.We apply ProtConn to evaluate the connectivity of the PA networks in all terrestrial ecoregions of the world as of June 2016, considering a range of median dispersal distances (1–100 km) encompassing the dispersal abilities of the large majority of terrestrial vertebrates.We found that 9.3% of the world is covered by protected connected lands (average for all the world’s ecoregions) for a reference dispersal distance of 10 km, increasing up to 11.7% for the largest dispersal distance considered of 100 km. These percentages are considerably smaller than the global PA coverage of 14.7%, indicating that the spatial arrangement of PAs is only partially successful in ensuring connectivity of protected lands. The connectivity of PAs largely differed across ecoregions. Only about a third of the world’s ecoregions currently meet the Aichi Target of having 17% of the terrestrial realm covered by well-connected systems of PAs. Finally, our findings suggest that PAs with less strict management objectives (allowing the sustainable use of resources) may play a fundamental role in upholding the connectivity of the PA systems.Our analyses and indicator make it possible to identify where on the globe additional efforts are most needed in expanding or reinforcing the connectivity of PA systems, and can be also used to assess whether newly designated sites provide effective connectivity gains in the PA system by acting as corridors or stepping stones between other PAs. The results of the ProtConn indicator are available, together with a suite of other global PA indicators, in the Digital Observatory for Protected Areas of the Joint Research Centre of the European Commission.