Retrospective serological and molecular survey of myxoma or antigenically related virus in the Iberian hare, Lepus granatensis.

The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares collected over two time periods almost two decades apart, 1994-1999 and 2017-2019 for the presence of antibodies and MYXV-DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha-MYXV. The cut-off of iELISA relative index 10 = 6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related-antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n = 10, sampled in 1994-1999, none MYXV-DNA positive, and n = 28 sampled in 2017-2019 of which four were also ha-MYXV-DNA positive) and 19 found-dead and ha-MYXV-DNA-positive sampled in 2018-2019. Interestingly, four seronegative hares sampled in 1997 were MYXV-DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), seroprevalence was significantly higher in 2017-2019 (13.0%, CI95% 9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p = .009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7 vs 1.7% considering all the sample, p = .007), and lower during the winter than the autumn (p < .001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1996, they also show an increase in seroprevalence in 2018-2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018.

Factors influencing lead, mercury and other trace element exposure in birds from metal mining areas.

Non-ferrous metal mining is considered one of the largest sources of toxic metal released to the environment and may threaten ecosystems, notably biota. We explored how birds that inhabit non-ferrous metal mining sites are exposed to mercury, lead, and other trace elements by analyzing their feathers and verifying which factors may influence element concentrations in feathers. We sampled a total of 168 birds, representing 26 species, with different feeding habits and migration patterns in a non-polluted reference site and two historical metal mining areas: Almadén, which is considered one of the most heavily mercury-contaminated sites worldwide, and the Sierra Madrona mountains where lead has been mined since ancient times. The quantification of aluminum (Al), arsenic (As), barium (Ba), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), magnesium (Mg), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), selenium (Se), thorium (Th), thallium (Tl), uranium (U), vanadium (V) and zinc (Zn) was performed by inductively coupled plasma mass spectrometry (ICP-MS). Feather analysis revealed contamination by Hg and Pb, in Almadén and Sierra Madrona, respectively. We found that granivorous birds had the lowest feather Hg levels compared to those found in omnivorous, insectivorous, and piscivorous species, whereas feather Pb was about twice as high in granivores and omnivores, than in insectivorous and piscivorous birds. We also found differences among study sites in 13 elements and confirmed the influence of feather age, migratory patterns of the birds, and external deposition of elements, on metal concentrations in the feathers. Our results highlight that despite the cessation of metal mining in the study areas, local avifauna are being exposed to Hg and Pb from abandoned mines and old tailings sites, indicating that appropriate measures are needed to protect biota from overexposure to these toxic metals.

Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares.

Myxoma virus (MYXV) is naturally found in rabbit Sylvilagus species and is known to cause lethal myxomatosis in European rabbits (Oryctolagus cuniculus). In 2019, an MYXV strain (MYXV strain Toledo [MYXV-Tol]) causing myxomatosis-like disease in Iberian hares (Lepus granatensis) was identified. MYXV-Tol acquired a recombinant region of ∼2.8 kb harboring several new genes, including a novel host range gene (M159) that we show to be an orthologous member of the vaccinia virus C7 host range family. Here, to test whether M159 alone has enabled MYXV to alter its host range to Iberian hares, several recombinant viruses were generated, including an MYXV-Tol ΔM159 (knockout) strain. While MYXV-Tol underwent fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (lacking M159) nor vMyxTol-ΔM159 (deleted for M159) was able to infect and replicate, showing that the ability of MYXV-Tol to infect these cells and replicate depends on the presence of M159. Similar to other C7L family members, M159 was shown to be expressed as an early/late gene but was translocated into the nucleus at later time points, indicating that further studies are needed to elucidate its role in the nucleus. Finally, in rabbit cells, the M159 protein did not contribute to increased replication but was able to upregulate the replication levels of MYXV in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species. Altogether, these observations demonstrate that the M159 protein plays a critical role in determining the host specificity of MYXV-Tol in hare and human cells by imparting new host range functions. IMPORTANCE The coevolution of European rabbit populations and MYXV is a textbook example of an arms race between a pathogen and a host. Recently, a recombinant MYXV (MYXV-Tol) crossed the species barrier by jumping from leporid species to another species, causing lethal myxomatosis-like disease. Given the highly pathogenic nature of this new virus in hares and the incidences of other poxvirus cross-species spillovers into other animals, including humans, it is important to understand how and why MYXV-Tol was able to become virulent in a new host species. The results presented clearly demonstrate that M159 is the key factor allowing MYXV-Tol replication in hare cells by imparting new host range functions. These results have the potential to improve current knowledge about the virulence of poxviruses and provide a platform to better understand the new MYXV-Tol, rendering the virus capable of leaping into a new host species.

Determination of glyphosate exposure in the Iberian hare: A potential focal species associated to agrosystems.

Glyphosate is the most used herbicide worldwide. It is a small and highly polar pesticide whose physicochemical properties makes its analytical determination difficult. Here, a procedure based on liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was developed for glyphosate determination in samples of gastric content from wildlife. Iberian hare (Lepus granatensis), a herbivorous mammal species, strongly associated to agrosystems was selected as model species. The procedure involves direct analysis of sample without derivatization or instead of neither further cleaning steps. The procedure was validated by inter-day accuracy and precision studies with gastric content of hare spiked with glyphosate at ecologically relevant concentrations for the species (0.1-6 µg/g), and with 1 µg/g of isotopically labelled internal standard (glyphosate-2-13C,15N). Finally, glyphosate residues in hunted animals from pesticide-treated and pesticide-free areas (n = 75 and 28, respectively), as well as from hares found dead in the field (n = 11) were analysed. The linearity of both standards in extraction solutions and procedural calibration curves with spiked samples was similar, both with determination coefficients (r2) higher than 0.99. Satisfactory recoveries in spiked samples were achieved within the range of 95% to 118% (CV ≤ 20%). The limit of detection of glyphosate in hare gastric content was 0.03 µg/g. Prevalence of glyphosate in hunted animals from pesticide-treated areas ranged between 9 and 22%, increasing to 45% in animals found dead. The glyphosate concentrations detected in the gastric content of hares ranged from 0.11 to 16 µg/g. No residues were detected in animals from pesticide-free areas. In practice, the developed methodology may be particularly useful in the context of research and other work on the exposure in wildlife of one of the most used pesticides nowadays.

Trace element bioaccumulation in hypersaline ecosystems and implications of a global invasion.

Hypersaline ecosystems are under increasing threat due to anthropogenic pressures such as environmental pollution and biological invasions. Here we address the ecotoxicological implications of the Artemia franciscana (Crustacea) invasion in saltpans of southern Spain. This North American species is causing the extinction of native Artemia populations in many parts of the globe. The bioaccumulation of trace elements (As, Cd, Cu, Co, Cr, Mn, Ni, Pb and Zn) in native populations (A. parthenogenetica) from Cabo de Gata and Odiel saltpans and invasive Artemia from Cádiz saltpan was studied at different salinities. Furthermore, in Odiel, the most polluted study site, we also analysed the bioaccumulation of trace elements by Chironomus salinarius larvae (Diptera) and Ochthebius notabilis adults (Coleoptera). High levels of trace elements were detected in the studied saltpans, many of them exceeding the recommended threshold guidelines for aquatic life. Bioaccumulation of trace elements by Artemia was lowest at the highest salinity. The invasive A. franciscana showed higher potential to bioaccumulate trace elements than its native counterpart (in particular for As, Cd, Ni and Cr). In Odiel, O. notabilis stood out as showing the highest potential to bioaccumulate As and Cu. Results showed that the shift from a native to an alien Artemia species with a higher bioaccumulation capacity may increase the transfer of trace elements in hypersaline food webs, especially for waterbirds that depend on Artemia as food. Thus, our study identifies an indirect impact of the Artemia franciscana invasion that had not previously been recognised.

A multidisciplinary approach to the evaluation of the effects of foraging on landfills on white stork nestlings.

The use of landfills as foraging areas by white storks (Ciconia ciconia) is a recent well-known behaviour. While several studies have highlighted positive effects at a populational level others suggest that the presence of pollutants, pathogens and the lower presence of antioxidants in the food could pose a health risk for individuals. The objective of this study was to evaluate potential effects of the use of landfills as a food resource on the physiology and health of white stork nestlings, by a multidisciplinary approach based on the analysis of nutritional status, body condition, blood parameters, oxidative stress balance and the presence of pathogens. Results showed better body condition in individuals associated with landfills compared to the ones feeding on natural resources, as well as better nutritional status, as indicated by higher levels of albumin, cholesterol, and triglycerides in plasma. As many pollutants have a pro-oxidant effect, we evaluated oxidative stress balance, with no differences in the indicators of damage except for methaemoglobin (metHb), significantly higher in nestlings associated with landfill-origin food. Regarding antioxidants, GSH was higher in nestlings associated with landfills, which may suggest a hormetic response induced potentially by the presence of pollutants in waste. Nestlings fed food from landfills also had a higher presence of Escherichia coli with a multiresistant phenotype to antibiotics. In conclusion, our results show that nestlings fed with a higher proportion of food from landfills present a better nutritional status and body condition than those fed with a higher proportion of natural diet, being the only indicators of negative effects of the use of this food resource the higher percentage of metHb in the peripheral blood and the presence of antibiotic-resistant E. coli.

Integrating active and passive monitoring to assess sublethal effects and mortality from lead poisoning in birds of prey.

The ingestion of lead (Pb) ammunition is the most important exposure pathway to this metal in birds and involve negative consequences to their health. We have performed a passive monitoring of Pb poisoning in birds of prey by measuring liver (n = 727) and blood (n = 32) Pb levels in individuals of 16 species found dead or sick in Spain between 2004 and 2020. We also performed an active monitoring by measuring blood Pb levels and biomarkers of haem biosynthesis, phosphorus (P) and calcium (Ca) metabolism, oxidative stress and immune function in individuals (n = 194) of 9 species trapped alive in the field between 2016 and 2017. Passive monitoring results revealed some species with liver Pb levels associated with severe clinical poisoning (>30 µg/g d.w. of Pb): Eurasian griffon vulture (27/257, 10.5%), red kite (1/132, 0.8%), golden eagle (4/38, 10.5%), and Northern goshawk (1/8, 12.5%). The active monitoring results showed that individuals of bearded vulture (1/3, 33.3%), Eurasian griffon vulture (87/118, 73.7%), Spanish imperial eagle (1/6, 16.7%) and red kite (1/18, 5.6%) had abnormal blood Pb levels (>20 µg/dL). Blood Pb levels increased with age, and both monitoring methods showed seasonality in Pb exposure associated with a delayed effect of the hunting season. In Eurasian griffon, blood Pb concentration was associated with lower δ-ALAD activity in blood and P levels in plasma, and with higher blood lipid peroxidation and plasma carotenoid levels in agreement with other experimental and field studies in Pb-exposed birds. The study reveals that Pb poisoning is a significant cause of death and sublethal effects on haem biosynthesis, P metabolism and oxidative stress in birds of prey in Spain.

Effect of mineral supplementation on lead bioavailability and toxicity biomarkers in sheep exposed to mining pollution.

The chronic exposure of livestock to lead (Pb) pollution in historical mining areas may represent significant and unnecessary costs for farmers and primary producers, in addition to important food safety risks. Here, we evaluate the effect of mineral supplements, in the form of a commercial mineral block (MB), to reduce Pb bioavailability and toxicity in sheep through an experimental approach under real farming conditions in an abandoned mining area. Blood, fecal Pb levels, and soil ingestion, along with different blood and plasma biomarkers were studied. Experiment 1 was carried out with 3-months-old female lambs, n = 54, fenced in two contiguous MB and non-MB-supplemented plots. After 20 days of treatment, blood Pb level was lower in MB-supplemented sheep than in those that were non-MB-supplemented. Experiment 2 was carried out with 2-months-old female lambs, n = 34, fenced in a single plot and MB-supplemented during the first 20 days of experiment. After MB supplementation, blood Pb level in sheep was also reduced by almost half, falling below the threshold of subclinical intoxication, and then increased again after 20 days without MB. Experiment 3 was carried out with adult rams, n = 10, fenced in a single MB-supplemented plot during the first 20 days of experiment. In this case, blood Pb level decreased by day 40. Soil ingestion was not reduced by MB supplementation in any of the experiments. MB supplementation favored antioxidant status by increasing SOD activity and reducing GPX activity and MDA levels. In conclusion, the MB supplementation seemed to reduce Pb bioavailability by increasing its fecal excretion, but renal excretion and bone deposition may also have favored the reduction of blood Pb concentration. Mineral supplements may be a new easy-to-apply and cost-effective way to reduce livestock exposure in Pb polluted sites.

Concentration and origin of lead (Pb) in liver and bone of Eurasian buzzards (Buteo buteo) in the United Kingdom.

Ingestion of lead (Pb) derived from ammunition used in the hunting of game animals is recognised to be a significant potential source of Pb exposure of wild birds, including birds of prey. However, there are only limited data for birds of prey in Europe regarding tissue concentrations and origins of Pb. Eurasian buzzards (Buteo buteo) found dead in the United Kingdom during an 11-year period were collected and the concentrations of Pb in the liver and femur were measured. Concentrations in the liver consistent with acute exposure to Pb were found in 2.7% of birds and concentration in the femur consistent with exposure to lethal levels were found in 4.0% of individuals. Pb concentration in the femur showed no evidence of consistent variation among or within years, but was greater for old than for young birds. The Pb concentration in the liver showed no effect of the birds' age, but varied markedly among years and showed a consistent tendency to increase substantially within years throughout the UK hunting season for gamebirds. The resemblance of the stable isotope composition of Pb from buzzard livers to that of Pb from the types of shotgun ammunition most widely-used in the UK increased markedly with increasing Pb concentration in the liver. Stable isotope results were consistent with 57% of the mass of Pb in livers of all of the buzzards sampled being derived from shotgun pellets, with this proportion being 89% for the birds with concentrations indicating acute exposure to Pb. Hence, most of the Pb acquired by Eurasian buzzards which have liver concentrations likely to be associated with lethal and sublethal effects is probably obtained when they prey upon or scavenge gamebirds and mammals shot using Pb shotgun pellets.

In vitro assessment of mineral blocks as a cost-effective measure to reduce oral bioavailability of lead (Pb) in livestock.

Soil contamination in former mining districts is a persistent problem resulting from the historic lack of legal requirements as regards land restoration after mine closures. Much of this polluted land is currently being used worldwide for livestock and big game production, with the consequent health risks for the animals exposed and the subsequent threats to food safety. Soil remediation and restoration may be unfeasible or difficult to accomplish in the short term when pollution affects large territories and other alternatives must, therefore, be explored in order to reduce the probability of grazing animals being exposed to this contamination. In this paper, we study the use of mineral blocks (MBs) as a potential alternative by which to reduce the oral bioavailability of lead (Pb) in polluted soils by means of a simplified in vitro assay simulating gastrointestinal pH conditions. Experiments were carried out with twelve commercial MBs of different compositions in order to identify the most useful to be tested in further in vivo bioavailability studies. The results showed that one of them reduced the bioaccessibility of Pb from polluted soil by 88.2% and 75.9% under gastric and intestinal conditions, respectively, when compared with assays containing only polluted soil without MBs. The MB in question had the highest phosphorus content (7%) and one of the highest calcium contents (10%) of all those tested. Furthermore, negative correlations were detected between the content of calcium and phosphorus in the MBs and the percentage of bioaccessible Pb under gastric conditions, and between phosphorus and bioaccessible Pb under intestinal conditions. The use of MBs with a high phosphorus and calcium content should consequently be tested in vivo as a cost-effective (€ 0.6-1.5/sheep/month) tool by which to reduce the bioavailability of Pb for extensive grazing livestock reared in contaminated areas.

Tools for non-invasive sampling of metal accumulation and its effects in Mediterranean pond turtle populations inhabiting mining areas.

Among reptiles, freshwater turtle species have high potential for metal accumulation because of their long lifespan or their aquatic and terrestrial habits. In order to monitor metal bioaccumulation, determine potential toxic effects, and investigate tools for non-invasive metal sampling in reptiles, we studied lead (Pb) and mercury (Hg) accumulation in Mediterranean pond turtles (Mauremys leprosa) inhabiting two former mining areas, one of them with high environmental concentrations of Pb (Sierra Madrona-Alcudia Valley district) and the other one with high environmental concentrations of Hg (Almadén district). Individuals from the Pb mining area showed mean blood concentrations (i.e. 5.59 µg Pb/g dry weight, d.w.) that were higher than those measured in other populations. Blood Hg concentrations were highest (8.83 µg Hg/g d.w.) in the site close to the former Hg mines, whereas blood Hg concentrations in terrapins from another site of Almadén district, located ∼28 km downstream, were not different from locations at the non-mining area. Animals from the Pb-contaminated site showed evidence of oxidative stress, whereas those from the Hg-contaminated site showed increased activity of the antioxidant enzyme glutathione peroxidase, as well as reduced circulating levels of the main endogenous antioxidant peptide, glutathione. Concentrations measured in feces and carapace scutes were useful indicators to monitor blood concentrations of Pb, but not of Hg. Our results provide evidence of the usefulness of freshwater turtles as sentinels of chronic metal pollution, and validate non-invasive tools to advance Pb monitoring in reptiles.

Life history and physiological responses of native and invasive brine shrimps exposed to zinc.

Although a substantial amount of research exists on pollution and biological invasions, there is a paucity of understanding of how both factors interact. Most studies show that pollution favours the establishment of invasive species, but pollution may also promote local adaptation of native species and prevent the establishment of new incomers. However, evidence for this is extremely limited because most studies focus on successful invasions and very few on cases where an invasion has been resisted. Here we provide evidence of local adaptation of native species to pollution combining life history and physiological data. We focused on the invasion of the North American brine shrimp Artemia franciscana, which is causing a dramatic biodiversity loss in hypersaline ecosystems worldwide, and one of the last native Artemia populations in SW Europe (A. parthenogenetica from the historically polluted Odiel estuary, SW Spain). Life table response experiments were carried out in the laboratory to compare the demographic responses of A. parthenogenetica and a nearby A. franciscana population to long-term Zn exposure (0.2 mg L-1). We also evaluated oxidative stress by measuring antioxidant defences (catalase, glutathione reductase and superoxide dismutase) and lipid peroxidation (thiobarbituric acid reactive substances). A high concentration of Zn induced strong mortality in A. franciscana, which also showed high levels of lipid peroxidation, suggesting relatively poor physiological resistance to pollution compared with A. parthenogenetica. The age at maturity was shorter in A. parthenogenetica, which may be an adaptation to the naturally high mortality rate observed in the Odiel population. Exposure to Zn accelerated age at first reproduction in A. franciscana but not in A. parthenogenetica. In contrast, Zn had a stimulatory effect on offspring production in A. parthenogenetica,which also showed higher reproductive parameters (number of broods, total offspring and offspring per brood) than A. franciscana. Overall, the results of this study strongly suggest that native Artemia from Odiel estuary is locally adapted (at both, reproductive and physiological levels) to Zn contamination and that A. franciscana is highly sensitive. This is a good example of how pollution may play a role in the persistence of the last native Artemia populations in the Mediterranean.

Exposure of black-necked grebes (Podiceps nigricollis) to metal pollution during the moulting period in the Odiel Marshes, Southwest Spain.

European populations of black-necked grebes (Podiceps nigricollis) congregate every year to moult at the salt ponds of the Odiel Marshes (SW Spain). However, the Odiel Marshes are part of one of the most metal-polluted coastal estuaries in the world, which may pose risks to wildlife. We assessed the exposure of grebes to metal pollution during the critical moulting period in the Odiel Marshes and its potential to cause adverse health effects. Levels of metals in red blood pellet (as a biomarker of exposure), plasma carotenoids, eye redness, and body condition (as biomarkers of effects) were studied. Metal content was also analyzed in the brine shrimp Artemia parthenogenetica, the most important food for grebes in this hypersaline ecosystem during the moulting period. Results showed that, in comparison to toxicity thresholds, grebes had relatively high blood levels of arsenic (As), mercury (Hg) and zinc (Zn). The high loads found in Artemia and the way blood levels vary during the moulting period indicate that shrimp consumption may be the main route of metal exposure for grebes. Plasma carotenoids and body condition showed a positive association with exposure to As, while the relationship of lutein-like carotenoids with Hg accumulation was negative at the beginning of the moulting period to become positive afterwards. Moreover, eye redness was negatively affected by As accumulation. Factors including food resource availability, seasonal fluctuations in physiological status, and interannual variations in the degree of environmental contamination should be considered in monitoring efforts when using moult migrant waterbirds as sentinel species.

Age-dependent changes in essential elements and oxidative stress biomarkers in blood of red deer and vulnerability to nutritional deficiencies.

Changes in the concentration of circulating essential elements in animals over life may be indicative of periods of vulnerability to deficiencies and associated diseases. Here we studied age-related variations in essential elements (Se, Cu, Zn and Mn) and some selected oxidative stress biomarkers (GPx, SOD, vitamin A and vitamin E) in blood of an Iberian red deer (Cervus elaphus hispanicus) population living in semicaptive conditions. Animals during their first year of life showed to be especially vulnerable to suffer Se- and Cu-related diseases and disorders. Older female deer had lower blood levels of Zn and Mn, which was accompanied by a lower blood SOD activity. On the contrary, GPx blood activity was elevated in older deer, which may help to compensate the reduction of other antioxidants with during aging. Age-related changes in GPx and SOD and their positive relationships with the essential elements suggest that the observed nutritional deficiencies at certain age stages may have a detrimental effect on the antioxidant system, increasing the risk of oxidative stress. Thus, the biomarkers used in the present study may be important tools for the subclinical diagnosis of nutritional disorders and diseases related to the generation of oxidative stress in both domestic and wild ungulates.

May arsenic pollution contribute to limiting Artemia franciscana invasion in southern Spain?

Limited information exists regarding the complex interactions between biological invasions, pollution, and climate change. Most studies indicate that pollution tends to favor invasive species. Here, we provide evidence that arsenic (As) pollution may have a role in limiting the invasion of the exotic brine shrimp Artemia franciscana. We tested As toxicity in natural populations of Artemia parthenogenetica (a native taxon) and A. franciscana from localities in southern Spain with differing degrees of As contamination. Tests were conducted both under current mean temperature conditions (25 °C), and as per a future climate scenario (i.e., an increase in mean temperature of 4 °C). Acute toxicity was estimated on the basis of the median lethal concentration (at 24 h), and chronic toxicity was evaluated by measuring Artemia survival and growth under sublethal exposures (after 26 days). At 25 °C, native A. parthenogenetica from the highly polluted Odiel and Tinto estuary was much more resistant to acute As stress (LC50-24 h, 24.67 mg L(-1)) than A. franciscana (15.78 mg L(-1)) and A. parthenogenetica from unpolluted sites (12.04 mg L(-1))-suggesting that local adaptation to polluted conditions may occur. At 29 °C, resistance of A. parthenogenetica from Odiel decreased significantly, and there were no statistical differences in sensitivity between the three species/populations, suggesting that climate change may enhance the probability of invasion. Resistance increased with developmental stage from nauplii to adults, and was extremely high in cysts which still hatched at As concentrations of up to 6400 mg L(-1). Under sublethal chronic exposure A. franciscana performed better (survival and growth) than A. parthenogenetica, and both species experienced a faster growth when exposed to As, compared with unexposed (control) individuals, probably due to the hormesis. We discuss the ecological implications of our results.

When Parasites Are Good for Health: Cestode Parasitism Increases Resistance to Arsenic in Brine Shrimps.

Parasites and pollutants can both affect any living organism, and their interactions can be very important. To date, repeated studies have found that parasites and heavy metals or metalloids both have important negative effects on the health of animals, often in a synergistic manner. Here, we show for the first time that parasites can increase host resistance to metalloid arsenic, focusing on a clonal population of brine shrimp from the contaminated Odiel and Tinto estuary in SW Spain. We studied the effect of cestodes on the response of Artemia to arsenic (acute toxicity tests, 24h LC50) and found that infection consistently reduced mortality across a range of arsenic concentrations. An increase from 25°C to 29°C, simulating the change in mean temperature expected under climate change, increased arsenic toxicity, but the benefits of infection persisted. Infected individuals showed higher levels of catalase and glutathione reductase activity, antioxidant enzymes with a very important role in the protection against oxidative stress. Levels of TBARS were unaffected by parasites, suggesting that infection is not associated with oxidative damage. Moreover, infected Artemia had a higher number of carotenoid-rich lipid droplets which may also protect the host through the "survival of the fattest" principle and the antioxidant potential of carotenoids. This study illustrates the need to consider the multi-stress context (contaminants and temperature increase) in which host-parasite interactions occur.

Assessing estuarine quality: A cost-effective in situ assay with amphipods.

In situ assays based on feeding depression can be powerful ecotoxicological tools that can link physiological organism-level responses to population and/or community-level effects. Amphipods are traditional target species for toxicity tests due to their high sensitivity to contaminants, availability in the field and ease of handling. However, cost-effective in situ assays based on feeding depression are not yet available for amphipods that inhabit estuarine ecosystems. The aim of this work was to assess a short-term in situ assay based on postexposure feeding rates on easily quantifiable food items with an estuarine amphipod. Experiments were carried out under laboratory conditions using juvenile Echinogammarus marinus as the target individual. When 60 Artemia franciscana nauplii (as prey) were provided per individual for a period of 30 min in dark conditions, feeding rates could be easily quantified. As an endpoint, postexposure feeding inhibition in E. marinus was more sensitive to cadmium contamination than mortality. Assay calibration under field conditions demonstrated the relevance of sediment particle size in explaining individual feeding rates in uncontaminated water bodies. An evaluation of the 48-h in situ bioassay based on postexposure feeding rates indicated that it is able to discriminate between unpolluted and polluted estuarine sites. Using the harmonized protocol described here, the in situ postexposure feeding assay with E. marinus was found to be a potentially useful, cost-effective tool for assessing estuarine sediment and water quality.

Patchy sediment contamination scenario and the habitat selection by an estuarine mudsnail.

Since mudsnails are able to avoid contaminated sediment and that the contaminants in sediment are not uniformly distributed, the mudsnail Peringia ulvae was exposed to cadmium (Cd) spiked sediment and assessed for avoidance response in a heterogeneous contamination scenario. Four Cd concentrations were prepared and disposed in patches on dishes, which were divided in 25 fields (six fields for each sediment concentration); 24 organisms were deployed in the central field, with no sediment. Observations were made at 2, 4 and 6 h (corresponding to immediate response), 8, 10 and 12 h (very short term), and 24 h (short term). A trend to avoid contaminated patches was observed in the immediate and very short term. After 24 h exposure, the organisms exposed to the highest level of contamination seemed to have lost the ability to move and avoid contaminated patches. In a contamination scenario in which non- and contaminated sediment patches are heterogeneously distributed, local mudsnail populations can simply rearrange their locality without needing to move to a different habitat. Such less contaminated patches can become donor areas in a future recolonization scenario.

Altered immune response in mallard ducklings exposed to lead through maternal transfer in the wild.

Lead (Pb) poisoning has caused significant mortality in waterfowl populations worldwide. In spite of having been banned since 2003, prevalence of Pb shot ingestion in mallards (Anas platyrhynchos) from the Ebro delta was still 15.5% in 2011-12. We collected mallard eggs from this area to study the effects of maternally transferred Pb on eggshell properties and on immune response and oxidative balance of ducklings. Eggshell Pb levels were positively correlated with Pb levels in the blood of ducklings. Ducklings with blood Pb levels above 180 ng mL(-1) showed reduced body mass and died during the first week post hatching. Blood Pb levels positively correlated with humoral immune response, endogenous antioxidants and oxidative stress biomarkers, and negatively correlated with cellular immune response. Pb shot ingestion in birds can result in maternal transfer to the offspring that can affect their developing immune system and reduce their survival in early life stages.

Effects of Agricultural Management Policies on the Exposure of Black-Winged Stilts (Himantopus himantopus) Chicks to Cholinesterase-Inhibiting Pesticides in Rice Fields.

Levels of exposure to pesticides in rice fields can be significant depending on the environmental policies practiced. The aim of European Union integrated management policy is to reduce pesticide use and impact on environment. Rice fields provide an alternative breeding habitat for many waterbirds that are exposed to the pesticides used and therefore can be valuable indicators of their risk for wildlife. To evaluate integrated management success we examined exposure of Black-winged Stilts (Himantopus himantopus) to cholinesterase-inhibiting pesticides in rice fields under different types of management by measuring plasma cholinesterase activity. Cholinesterase activity was lower in birds sampled in (a) 2008 after a period of intense pesticide application, than in (b) 2005-2007 and 2011 in rice fields subject to integrated management in Doñana (SW Spain) and (c) in control natural wetlands in Spain and Morocco. During 2009 and 2010, cholinesterase activity was lower in rice fields in Doñana than in rice fields in Larache and Sidi Allal Tazi (NW Morocco). Our results suggest that integrated management successfully reduced the exposure of Black-winged Stilts to pesticides in most of the years. Care should be taken to implement mosquito and pest crop controls on time and with environmentally friendly products in order to reduce its impact on wildlife.