Chronic lead intoxication decreases intestinal

Science of the Total Environment 644 (2018) 151–160

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Chronic lead intoxication decreases intestinal helminth species richness and infection intensity in mallards (Anas platyrhynchos) Prüter Hanna a,⁎, Franz Mathias a, Auls Susanne a, Czirják Gábor Á. a, Greben Oksana b, Greenwood Alex D. a,c, Lisitsyna Olga b, Syrota Yaroslav b,e, Sitko Jilji d, Krone Oliver a a

Leibniz Institute for Zoo and Wildlife Research, Department of Wildlife Diseases, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany National Academy of Sciences of Ukraine, I. I. Schmalhausen Institute of Zoology, Vul. B. Khmelnytskogo, 15, 01030 Kiev, Ukraine Freie Universität Berlin, Department of Veterinary Medicine, Berlin, Germany d Komenský Museum, Horní nám. 7, 750 11 Přerov 2, Czech Republic e Kyiv Zoological Park of National Importance, prosp. Peremohy, 32, Kyiv 04116, Ukraine b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• We demonstrate the impact of lead intoxication on Mallard intestinal helminth species richness and infection intensity. • Lead levels were compared between coracoid bones and livers of Mallards. • Chronic lead intoxication in Mallards from Germany is higher than expected. • Lead intoxication can negatively impact intestinal helminth species richness. • Cestode and acanthocephalan infection intensity was low at high lead concentrations.

a r t i c l e

i n f o

Article history: Received 11 April 2018 Received in revised form 23 June 2018 Accepted 24 June 2018 Available online xxxx Editor: Henner Hollert Keywords: Sublethal lead intoxication Parasite diversity Duck German waters

⁎ Corresponding author. E-mail address: [email protected] (H. Prüter).

https://doi.org/10.1016/j.scitotenv.2018.06.297 0048-9697/© 2018 Elsevier B.V. All rights reserved.

a b s t r a c t Lead (Pb) pollution of aquatic habitats is a known threat to vertebrate health. Depending on Pb dosage, resulting symptoms can be chronic (sublethal) or acute (lethal). While acute exposure results in death of the animal, chronic sublethal exposure can also have consequences, reproduction, antioxidant defense and immunity being the most affected traits. While a great deal is known about Pb intoxication on avian health, relatively little is known about how intoxication impacts parasites dependent on their avian hosts. The effect of Pb on intestinal helminth species richness and infection intensity was investigated in mallards (Anas platyrhynchos, n = 100) from German waters. Coracoid bones were used to measure chronic Pb exposure. Intestinal helminths were characterized morphologically. Molecular approaches were also applied to identify poorly morphologically preserved parasites to obtain sequence data (cox1 gene) for species identification and future parasitological studies. Parasite species richness and infection intensity was found to be significantly lower in birds with higher chronic Pb levels suggesting both host and parasites respond to Pb exposure. Altered immune modulation in the avian host may be the underlying mechanisms of Pb triggered decrease of parasites. However, it also likely reflects differences in the susceptibility of different helminths to Pb. Cestode and acanthocephala species richness were particularly impacted by Pb exposure. We conclude that, Pb intoxication may both negatively impact avian host and parasite diversity in aquatic habitats. © 2018 Elsevier B.V. All rights reserved.

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H. Prüter et al. / Science of the Total Environment 644 (2018) 151–160

1. Introduction Environmental lead (Pb) pollution is a global health issue for wildlife (Arnemo et al., 2016). For example, spent rifle bullets constitute a health threat to raptors and scavengers which directly consume them (Krone et al., 2009). However, the main source of environmental Pb contamination originates from Pb based shot gun pellets used for waterfowl hunting (Scheuhammer and Norris, 1995; Mateo et al., 1997, 1998; Newth et al., 2013; Ferreyra et al., 2014). Additionally, Pb from fishing weights (Birkhead and Perrins, 1985; Sears, 1988; Scheuhammer and Norris, 1995; Kelly and Kelly, 2004) and mining (Meharg et al., 2002; GómezRamírez et al., 2011) build up in water sources. As a consequence some of the most Pb affected species are found among the water birds (Scheuhammer et al., 2003; Arnemo et al., 2016). Ducks accumulate high levels of Pb because they often ingest hunting pellets from contaminated sediments as grit (Mateo et al., 1997; Ferreyra et al., 2014). Depending on the Pb dosage, acute (lethal) or chronic (sublethal) symptoms can manifest. Acute intoxication may result in severe clinical symptoms leading to death (Sears, 1988; Degernes et al., 2006; Newth et al., 2013). Lower, sublethal dosages or chronic intake may result in accumulation of Pb in tissues (mainly bones), leading to different sublethal effects of Pb (Martinez-Haro et al., 2011; Vallverdú-Coll et al., 2016) with important health consequences. Such chronic effects of Pb on the host have been examined with respect to negative effects on reproduction, oxidative balance and the immune response of birds (Eeva et al., 2005; Vallverdú-Coll et al., 2015a, 2016). It is well known that the immune systems of birds and other vertebrates are sensitive to Pb exposure (Franson, 1986; Singh et al., 2003), which can result in immunosuppression (Trust et al., 1990; Rocke and Samuel, 1991; Grasman and Scanlon, 1995; Youssef et al., 1996; Pikula et al., 2010). Because parasites can benefit from weakened immune function it might be expected that Pb intoxication would increase parasite load (Scheuhammer and Norris, 1995; Pain, 2009). However, immune suppression could be counteracted by two additional effects. First, the parasite related immune defenses (in particular helper T-cell 2 (Th2) related immunity) might be enhanced by Pb intoxication. Recent studies indicate an increase of antibody production as the main immune response against helminths (Degen et al., 2005; Bertellotti et al., 2016) in some avian species in polluted environments (Eeva et al., 2005; Vallverdú-Coll et al., 2015b). Whether the increase of Th2 response, which has also been shown for mammals (Gao et al., 2007; Cizauskas et al., 2014), is a general effect of Pb exposure on the avian immune system remains unclear. Second, Pb might also directly poison intestinal parasites in the intestine of the hosts. Helminth biodiversity was shown to be reduced in polluted compared to less polluted environments (Kennedy, 2006). Intestinal parasites are particularly exposed to Pb, as oral intake of hunting pellets is the main route of Pb intoxication in mallards (Mateo et al., 1997; Ferreyra et al., 2014). After swallowing Pb shot pellets, parasites in the intestines of mallards are directly confronted with concentrated pollutants. Both altered immunity and direct poisoning could harm parasites after Pb intoxication and decrease the parasite load. Additionally, acanthocephalan and cestode parasites have been found to accumulate Pb in higher dosages than their hosts. Thus, intestinal parasites may function as a pollution sink and reduce Pb levels affecting vertebrate hosts (Sures et al., 2017). We investigated intestinal helminth species richness and infection intensity in mallards (Anas platyrhynchos Linneaus) exposed to environmental Pb. The mallard is the most common duck species and the most heavily hunted game duck in Germany (Deutscher Jagdverband e.V., 2018). Although, hunting with Pb ammunition in aquatic habitats is forbidden in most German states (JWMG; BayJG, 1978; LJG-NRW, 1994; NJagdG, 2001; BbgJagdDV, 2004; LJG, 2010), we expect a general intake of Pb from the environment (contamination of the sediments with old ammunition and other anthropogenic Pb pollution of aquatic habitats) in mallards leading to chronic Pb intoxication. Furthermore, the mallard is a particularly suitable model species to study Pb effects as the species

can be immunosuppressed by chronic Pb intake (Vallverdú-Coll et al., 2016). They also harbor a variety of intestinal helminth species belonging to all major parasitic helminth classes (Boch and Schneidawind, 1988). We additionally investigated potential differences in the strength of Pb intoxication among the helminthic classes, which may differ in their susceptibility to Pb and/or in their ability to resist a potential shift in host immune defense. To investigate Pb burden originating from the environment, we measured Pb levels in coracoids (chronic or long-term) and liver (acute) of free-ranging mallards. Intestinal helminth species were determined by applying morphological and molecular methods. As measures of intestinal helminth diversity, we used intestinal helminth species richness and infection intensity. Species richness was defined as number of helminthic species per individual. Intestinal helminth infection intensity was measured as the number of intestinal helminth parasite individuals per duck. General linear mixed models were applied to the data to test for the effects of Pb on both, helminth species richness and intestinal helminth infection intensities. 2. Material and methods 2.1. Sampling A total of 106 mallards shot in winter hunting seasons (November till mid-January) at 11 different water bodies in Germany (50.5°N, 10.5°E) between 2014 and 2016 were examined for Pb burden and parasitic infections (Fig. 1). The sampling locations are waters where regular hunting of waterfowl takes place and Pb contamination of the sediment from old ammunition sources is likely. All ducks were kept frozen at −20 °C after hunting until further analysis. Out of 106 birds, 58 were adult males, 18 adult females, 3 one-year-old males and 27 one-year-old females. Age of the birds was determined according to Baker (1993). Necropsies and parasite isolation were performed for all 106 mallards according to Doster and Goater (1997). Parasite species determination is reported for all 106 mallards examined (Table 1). Coracoid bone was used as a long time and liver tissue as short time accumulation matrix for Pb intoxication (Scheuhammer, 1987; Guitart et al., 1994). Coracoid Pb was measured in the coracoids of 100 mallards and liver of 98 mallards (6 animals were excluded, whose coracoids were damaged and two additional individuals of whose livers were damaged and therefore potentially contaminated by shot pellets). A subset of humerus bones (n = 62) was measured to compare coracoid and humerus Pb accumulation to ensure comparability to other data sets (Ferreyra et al., 2014). 2.2. Lead (Pb) analysis Samples were carefully processed during necropsies to avoid external contamination (e.g. from used ammunition) and stored at −20 °C until chemical analysis. Approximately 1–2 g of the liver samples was weighed with a reproducibility of 0.02 g weighing balance into a Teflon digestion vessel and microwave digested in an acid solution (34.5% nitric acid and 3.75% hydrogen peroxide) using a microPREP A 1500 microwave system (MLS, Germany). The digested solutions were adjusted with deionised water to 25 ml and stored in polypropylene vials at room temperature. The concentrations of Pb in the digests were measured by GFAAS technique using ZEEnit 700 (Analytik Jena, Germany). Two percent ammonium dihydrogen phosphate was used as a matrix modifier. A standard solution of Pb TraceCERT for AAS (Sigma Aldrich) was used to generate a calibration curve. The calibration curve was prepared by serial dilution of the stock solution with 11% nitric acid. The bones were crushed and subsequently dried for 24 h at 60 °C. Subsequently they were finely powdered using a swing mill MM301 (Retsch, Germany). About 0.5 g of the powdered bone was weighed and microwave digested as described above. The concentration of Pb was determined by measuring a 1:10 dilution of the digest. Nitric acid


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Fig. 1. Sampling locations (labeled in alphabetic order from north to south) of free-ranging mallards in Germany.

(11%) was used as diluent. The measurement was performed as described for the liver samples. Data quality was assessed by simultaneous analysis of certified reference material BCR-185R-Bovine Liver (10% of all liver samples) and NIST SRM-1486-Bone Meal (10% of all bone samples). Data obtained for Pb in both reference materials was found in the confidence interval given by the producers of the certified reference material for this element. The background was monitored by analysis of 10% blanks prepared under the same conditions, but without sample added. The concentrations of Pb in all blanks were b0.8 μg/l. Pb concentrations were calculated as mg/kg (1 ppm) as wet weight (ww) in the liver and dry weight (dw) in the bones. While the limit of detection for the liver samples was 0.05 ppm ww, it was 0.10 ppm dw for the bone samples. The limit of quantification was 0.125 ppm ww for the liver samples and 0.25 ppm dw for the bone samples.

2.3. Parasite determination During necropsy, intestinal helminths were put in either formaldehyde (4%) or glycerin ethanol (5%) for morphological examination. Cestodes were stained with iron acetocarmine or Mayer's haematoxylin, dehydrated in an ascending alcohol series, cleared up in clove oil and mounted in Canada balsam. Some fragments and scoleces were mounted in Berlese's medium to facilitate the examination of the copulatory organs and hooks. Trematodes were cleared in xylene and mounted in Canada balsam. Nematodes were cleared in lactophenol. Acanthocephalans were mounted in Berlese's medium. Helminth morphology was studied under a Zeiss Axio Imager M1 microscope with digital camera and AmScope T690B microscope. Parasite identities were determined morphologically after staining. The species were identified according to Czaplinski (1956), Dubinina (1966), Spasskaya (1966) and Tolkacheva

(1991) for cestodes; McDonald (1974) for nematodes; Petrochenko (1958) and Khohlova (1986) for acanthocephalans. Where possible, characterization of intestinal helminths was performed morphologically to the species level. In addition, molecular sequences (cox1 gene) were used to identify parasites that were unidentifiable morphology (damaged or of low abundance) by aligning to sequences generated from the parasites that were morphologically determined (GenBank accession numbers MH142821, MH482552, MH523360-MH523402). For the molecular approach, DNA-extraction was performed applying the DNeasyTM Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Polymerase chain reaction (PCR) using the primer set JB3 (5′-TTTTTTGGGCATCCTGAGG TTTAT-3′) and JB4.5 (5′-TAAAGAAAGAACATAATGAAAATG-3′) ((Bowles et al., 1992; Liu et al., 2011)), targeting a 450 bp fragment of the cox1 gene, was carried out. The PCR products were sequenced on a 3130xl Genetic Analyzer (Applied Biosystems) using the PCR primers. Raw sequences were manually inspected using MEGA6 (Tamura et al., 2013) and subsequently aligned to each other and searched against the NCBI nucleotide database (Altschul et al., 1990).

2.4. Statistics To investigate potential determinants of Pb levels we used general linear mixed models (GLMM) with Pb levels in the coracoids or Pb levels in the liver as responses. As predictors, we included sex, age (one-yearolds and adults) and infection with acanthocephalans, cestodes and trematodes (either ‘yes’ or ‘no’) as fixed effects and origin of the ducks as a random effect. To investigate whether Pb accumulates equally in coracoids and humerus we estimated the Pearson's correlation coefficient. Additionally, we performed a Wilcoxon signed rank test to test for potential differences in the Pb levels between the two types of bones.

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Table 1 Intestinal helminths in free-ranging mallards from Germany. Gastrointestinal helminths

n

Prevalence (%)

Infection intensity

Organs

Origin

Acanthocephala Filicolis anatis (Schrank, 1788) Polymorphus diploinflatus (Lundström, 1941) Polymorphus contortus (Bremser, 1821) Acanthocephala undetermineda

7 22 (3) 1 2

6.60 20.75 0.94 1.89

1–25 1–20 10 1

Small and large intestine Small and large intestine Large intestine Caecum

C, I, L A, B, C, D, F, G, H, K, L C D, L

Cestoda Aploparaksis furcigera (Rudolphi, 1819) Aploparaksis sp. Cloacotaenia megalops (Nitzsch in Creplin, 1829) Diorchis stefanskii (Czaplinskii, 1956) Diorchis parvogenitalis (Skrjabin and Matevossian, 1945) Diorchis sp. Dicranotaenia coronula (Dujardin, 1845) Hymenolepididae sp. Sobolevicanthus gracilis (Zeder, 1803) Sobolevicanthus sp. Microsomacanthus paramicrosoma (Gasowska, 1932) Microsomacanthus paracompressa (Czaplinsi, 1956) Microsomacanthus sp. 1 sensu (Spassky et Yurpalova, 1966) Microsomacanthus sp. Schistocephalus pungiti (Dubinia, 1959) cestoda_sp_1° cestoda_sp_2° cestoda_sp_3° Cestoda undetermineda

6 (1) 3 12 11 2 4 1 4 7 5 1 1 1 2 4 1 1 1 3

5.66 2.83 11.32 10.38 1.89 3.77 0.94 3.77 6.60 4.72 0.94 0.94 0.94 1.89 3.77 0.94 0.94 0.94 2.83

1–17 1–3 1–20 1–30 2 1–18 3 1–4 1–22 1–12 2 4 1 1–15 11–50 5 2 10 1–10

Large intestine Large intestine Claoca Large intestine (1 small intestine) Large intestine Large intestine Large intestine Large intestine Large intestine Large intestine Large intestine Large intestine Large intestine Small and large intestine Large intestine, caecum, body cavity Small intestine Small intestine Large intestine Small and large intestine

B, D H, I, K A, B, G, I, L B, C, D, G, K, L A, K G, K B A, D, G A, B, C, E, G A, C, G, K A D C E F C G E C, G, L

Trematoda Psilochasmus oxyurus (Creplin, 1825) Notocotylus attenuatus (Rudolphi, 1809) Echinostoma revolum (Fröhlich, 1802) Bilhaziella polonica (Kowalewski, 1895) Hypoderaeum conoideum (Block, 1872) Trematoda_sp_1° Trematoda_sp_2°

1 3 7 2 1 1 1

0.94 2.83 6.60 1.89 0.94 0.94 0.94

30 2–6 1–11 1–3 5 1 1

Small intestine Caecum Cloaca, caecum Large intestine, caecum Caecum Small intestine Caecum

C A, F, L C, D, E, F, G, H, L A, K C G B

Nematoda Porrocaecum crassum (Deslongchamps, 1824) Nematoda undetermineda

(1) 1

0.94 0.94

1 1

Small intestine Caecum

H H

a Parasites that could not be identified further; °Sequences do not match with any of the other sequences. Morphological determination not performed due to bad quality of the material; n = number of infected mallards, numbers in brackets () indicate mallards where no coracoid lead was measured and that were subsequently excluded from the dataset that was studied in the frame of chronic lead burden and parasite species richness; Prevalence = percentage of mallards infected; Infection intensity = number of parasites per mallard; Organs = infested by the parasite; Origin = Location where the mallard was shot (see Fig. 1).

Four GLMMs with Poisson distributed errors were used to investigate the effects of Pb on species richnesses. In the first model we included the total species richness as response. In the other three models we included the species richness of trematodes, cestodes and acanthocephalans, respectively as the response variable. Another four GLMMs with errors following a negative binomial distribution were used to investigate the effects of Pb on total intestinal helminth infection intensities and infection intensities of the different helminth groups, respectively. In all models we included as predictor variables Pb levels in coracoid, Pb levels in liver, sex and age as fixed effects and origin of the ducks as a random effect. All GLMMs were fitted using the R package glmmTMB version 0.2.0 (Brooks et al., 2017). For all models, potential collinearity of predictors was tested using the R package car version 2.1-6 (Fox and Weisberg, 2011). To avoid a highly skewed distribution of Pb values, we log transformed coracoid and liver Pb levels. Visual inspections of residuals of the first GLMM did not indicate any violations of assumptions of normality and homogeneity of error variances. All statistical analyses were performed using R version 3.3.2 (R Core Team, 2016). 3. Results Coracoid Pb levels ranged from 0.0–53.7 ppm, humerus Pb levels from 0.0–42.5 ppm and liver Pb levels from 0.0–8.081 ppm (Table A.1 and Fig. A.1). No evidence for a statistically significant difference between Pb levels in coracoid and humerus bones was observed (Wilcoxon signed rank test, V = 1041, p = 0.5). Additionally, humerus

and coracoid Pb levels were highly positively correlated (Pearson's product-moment correlation, t = 12.1, df = 60, p b 0.001, cor = 0.84) (Fig. A.2), which confirms that Pb absorption by the two bones is very similar. This suggests that comparative studies that use different bones to investigate chronic Pb accumulation will yield meaningful and comparable results. Ducks infected with acanthocephalans or cestodes showed significantly lower Pb levels in coracoids (acanthocephalans: z = −2.67, p = 0.008; cestodes: z = −2.62, p = 0.009). No statistically significant effect of age, sex and infections with trematodes on levels of Pb in the coracoids was observed (age: z = −0.16, p = 0.87; sex: z = 1.42, p = 0.15; trematodes: z = −0.1, p = 0.92) (Fig. 2). None of the predictors was found to have a statistically significant effect on levels of Pb in liver (age: z = −1.1, p = 0.92; sex: z = 0.28, p = 0.78; acanthocephalans: z = −0.6, p = 0.55; cestodes: z = −1.14, p = 0.25; trematodes: z = −075, p = 0.45). A total of nineteen species from 4 classes of intestinal helminths could be identified to the species level (morphologically). The number of species and genera identified is summarized in Table 1. Overall prevalence of intestinal helminths was 66% in all investigated mallards (70 of 106). The prevalence of intestinal helminths in the subset of the mallards where Pb was measured in coracoids (n = 100) was 65% (65 out of 100). Species richness was calculated for the described subset of mallards. Species richness ranged from zero to 7 helminth species in mallards. Mean species richness of one-year-old birds was 1.5 (SD = 1.26, min = 0, max = 4, n = 28) and of adult individuals 1.03 (SD = 1.28, min = 0,


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Fig. 2. Lead (Pb) concentration (ppm) measured in coracoids of free-ranging mallards from Germany being either infected (yes) or not infected (no) with acanthocephalans (a), cestodes (b) or trematodes (c). Boxplots showing the distribution of Pb concentrations.

max = 7, n = 72). A total of 37 (37%) individuals were infected by a single helminth species. Fifteen (15%) individuals showed coinfection with two species, 7 with three species (7%) and 4 mallards (4%) were coinfected with 4 intestinal helminth species. One individual had 5 helminth species and another seven. Cestodes were the most diverse, abundant and prevalent class, followed by trematodes, acanthocephalans and nematodes with only two species represented (Porrocaecum crassum and undetermined) (Table 1). Polymorphus diploinflatus was the most prevalent parasite (20.75%), while Schistocephalus pungiti was the most abundant one with 11 to 50 parasites per individual mallard. Both, total parasite species richness and infection intensity was significantly lower in birds with higher chronic Pb levels in coracoids (species richness: z = −2.74, p = 0.006; infection intensity: z = −2.04, p = 0.041) (Fig. 3). No statistically significant effect on total species richness and infection intensity was associated with age, sex or Pb levels in liver (species richness: age: z = 0.93, p = 0.353; sex: z = 0.55, p = 0.584, Pb levels in livers: z = −0.88, p = 0.379; infection intensity: age: z = 0.62, p = 0.533; sex: z = −0.17, p = 0.863, Pb levels in liver: z = −0.26, p = 0.794). When analyzing coracoid Pb effects on species richness separately for the helminthic classes, there was no significant decrease of species richness for the three groups, although the effect for acanthocephalans and cestodes showed a trend towards statistical significance (acanthocephalans: z = −1.89, p = 0.059; cestodes z = −1.94, p = 0.052, trematodes: z = −0.73, p = 0.464). Infection intensities of acanthocephalans and cestodes were significantly lower at higher Pb levels in coracoids (acanthocephalans: z = −2.26, p = 0.024; cestodes z = −2.42, p = 0.015, trematodes: z = −0.56, p = 0.575) (Fig. 4). However, confidence intervals for these effects overlapped, which means that we cannot rule out the possibility that there are in fact no systematic differences among these helminthic classes (Fig. 5). 4. Discussion Intestinal helminth species richness and infection intensity is lower at higher Pb levels in coracoids in mallards. This indicates that intestinal

helminths do not benefit from a decrease in the immune function in their avian host but rather are negatively affected by long-term Pb exposure. This effect may be related to a shift in the avian immune system in response to Pb or driven by direct Pb intoxication of intestinal helminths. The avian immune system is sensitive to toxicological substances, including heavy metals such as Pb (see review Grasman, 2002). Immune suppression by Pb, particularly of the constitutive innate immune system was shown in mallards. Both total white blood cell counts and the number of specific immune cells (e.g. lymphocytes, monocytes, and heterophils) were reported to decrease after Pb exposure (Rocke and Samuel, 1991; Grasman and Scanlon, 1995). Although, immunosuppressive effects on the humoral immune response have been suggested by different authors (Trust et al., 1990; Rocke and Samuel, 1991; Grasman and Scanlon, 1995), comprehensive data to draw general conclusions about the effect of Pb on humoral immunity in birds are lacking. Some studies indicate up regulation of the avian immune system triggered by Pb. Fair and Ricklefs (2002) reported increasing numbers of granulocytes in Japanese quail chicks after treatment with Pb. Moreover, recent studies indicate a species specific effect of Pb on antibodies (increased antibody levels in red-legged partridges (Vallverdú-Coll et al., 2015b) but not mallards (Vallverdú-Coll et al., 2016)). Eeva et al. (2005) reported an increase in adaptive humoral response in passerines from moderately polluted areas. Thus, Pb might intensify the antihelminthic immune competence of birds by elevating the Th2 dependent immune mechanisms (increased eosinophil activity and antibody response), which might explain the results of this study. Robustness of parasite species to secondary extinction is reduced in parasites with more complex lifecycles (Lafferty, 2012). Also the resilience to threats such as habitat loss differs among helminthic groups (Carlson et al., 2017). Moreover, susceptibility to Pb exposure might differ between parasite species, being higher in heteroxenous (lifecycle including intermediate and final host) parasites than in monoxenous (single host) (Sures et al., 2017). Acanthocephalans and cestodes in particular were found to accumulate Pb in high dosages (Sures et al., 2002; Sures, 2004). Based on the differences in robustness, accumulation of Pb and potential defense mechanisms against Pb, tolerance of Pb burden

156


Fig. 3. Relationship between lead (Pb) concentration (ppm) in coracoids and total intestinal helminth species richness (a) and infection intensity (b) in free-ranging mallards from Germany. Circles: observed data points. Solid line: effect that was estimated by the applied GLMM (species richness: p = 0.006; infection intensity: p = 0.041).

Fig. 4. Relationship between lead (Pb) concentration (ppm) in coracoids and intestinal helminth species richness of different classes: acanthocephalans (a), cestodes (b), and trematodes (c) in free-ranging mallards from Germany. Relationship between Pb concentration in coracoids and intestinal helminth infection intensity of different classes: acanthocephalans (d), cestodes (e), and trematodes (f) in free-ranging mallards from Germany. Circles: observed data points. Solid lines: effects that were estimated by the applied GLMM (intestinal helminth species richness: acanthocephalans: p = 0.059, cestodes: p = 0.052, and trematodes: p = 0.464; intestinal helminth infection intensity: acanthocephalans: p = 0.024, cestodes: p = 0.015, and trematodes: p = 0.575).


157

Fig. 5. Estimated effects of Pb on intestinal helminth species richnesses (a) and intestinal helminth infection intensities (b) and corresponding 95% confidence intervals for different helminth classes (acanthocephalans, cestodes, trematodes).

most likely differs among helminths. Moreover, parasite susceptibility to Pb might be strongly influenced by the parasites digestive physiology, such as route of ingestion and type of digestion. Acanthocephalans and cestodes ingest food through the body surface and lack internal digestive organs. In contrast, trematodes rely on internal digestive organs for ingestion and digestion (Eckert, 2008). Thus, acanthocephalans and cestodes might adsorb higher levels of Pb than trematodes. These differences might explain why we found that Pb levels in coracoids were lower in ducks infected with acanthocephala or cestode species respectively, whereas we found no significant effect for trematodes (Fig. 2). Helminth parasites may function as Pb sinks. Thus, intestinal helminths might protect the host from higher Pb dosages. In fish, infections with acanthocephala or cestode species were shown to decrease the Pb levels of the host (Filipović Marijić et al., 2013, 2014). In red foxes (Vulpes vulpes), Pb levels were found to be decreased in individuals infected with a cestode species compared to non-infected individuals (Jankovská et al., 2010). Our results show a similar Pb correlation in ducks infected with acanthocepahla or cestode species. Although acanthocephalan and cestode species might protect infected ducks from Pb, lower infection intensities in ducks with higher Pb levels in bone indicate susceptibility to Pb for these parasites (Fig. 4). The total number of intestinal helminth species in our study is lower than reported by (Nowak et al., 2012). Especially the near absence (except from two mallards) and extremely low abundance (only one individual parasite per infected mallard) of nematodes in our study is remarkable. Comparing two studies from Poland, a decrease in nematode species richness over time between the studies can be observed (Kavetska et al., 2008; Nowak et al., 2012). This might potentially be related to a seasonality of nematode infections in mallards or indicate a higher susceptibility to external influences (such as toxins) of nematodes in particular. Consequently, nematodes are hypothetically more vulnerable and threatened than other helminth groups. Unfortunately, our data cannot resolve phylogenetically which species are most affected by Pb. Parasite biodiversity was shown to be reduced in polluted areas (Kennedy, 2006). Although we cannot distinguish, whether helminths are mainly affected by direct Pb toxicity or an elevated Th2 defense in their hosts, the decrease in parasite species richness and infection intensity shows that intestinal helminth biodiversity is reduced by chronic Pb pollution in mallards. Some parasite species may benefit from higher Pb levels, whereas others suffer. Thus, Pb may have a larger influence on parasite community composition and population dynamics within water ecosystems than suspected.

Although Pb shot has officially been phased out in most German states, we show that Pb is still affecting mallards in water ecosystems in Germany. Low Pb levels in liver indicate that the investigated mallards were not affected by acute Pb intoxication. While mean Pb levels in bones were lower than reported from mallards in the Ebro delta in Spain by (Guitart et al., 1994), Pb levels in bones were still higher than reported by Ferreyra et al. (2014, 2015), who studied water birds in Argentina, an area where hunting with Pb ammunition is still legal. This is somewhat surprising because the use of Pb ammunition is prohibited in German aquatic habitats and the intake of Pb ammunition should have decreased. Water body sediments in Germany might still be highly contaminated by residual Pb ammunition or Pb is entering the system from other unknown sources.

5. Conclusion Our findings indicate that long-term Pb intoxication can negatively impact intestinal helminth biodiversity. Given the threats to many parasite host species, parasite biodiversity is not only threatened by the loss of hosts, habitats and climate change (Carlson et al., 2017) but also the impact of toxic substances from the environment. Lafferty (2012) suggested that parasites are useful as bioindicators for changes in ecosystems, as total parasite species richness decreases in systems with overall decreasing biodiversity. The current study indicates that biodiversity losses due to Pb pollution in German water ecosystems may be underestimated. Comprehensive knowledge of long time effects of Pb is of great importance for conservation of both, hosts and parasites. Future studies on parasite and host diversity in aquatic systems, chronic effects of heavy metals and the detailed immunosuppressive effect of Pb in birds should help clarify the mechanisms that lead to reduced parasite diversity. Also, additional factors such as other pollutants or a potential negative effect of pollutants on invertebrate intermediate hosts (Spehar et al., 1978; Grosell et al., 2006; Offem and Ayotunde, 2008) should be included into future studies on host and parasite diversity in aquatic habitats.

Conflict of interest The authors declare that they have no conflict of interest.

158


Acknowledgments This study was part of the Graduate School IMPact-Vector funded by the Senate Competition Committee grant (SAW-2014-SGN-3) of the Leibniz Association. Hanna Prüter is also an associated doctoral student of the GRK2046 from the German Research Foundation (DFG). We are

thankful to all the collaborating hunters, assistants during necropsies and laboratory work by Nina Hartmann, Sophie Ewert, Lisa Giese, Lorena Derezanin and Manuela Merling de Chapa. Additionally, we thank Arne Ludwig and Anke Schmidt for help with the sequencing of helminths. We are thankful to Tatjana Kuzmina for helping with the shipment of samples.

Appendix A. Appendix

Fig. A.1. Lead (Pb) concentration (ppm) measured in coracoid (a), humerus (b) and liver (c) of free-ranging mallards from Germany. Boxplots showing the distribution of Pb concentrations. Table A.1 Lead (Pb) levels in coracoid and humerus bones and liver of free-ranging mallards from Germany. Material

Mean Pb level (ppm)

Min

Max

SD

n

Coracoid Humerus Liver

6.67 6.09 0.39

0 0.001 0

53.66 42.5 8.081

9.56 8.05 0.13

100 62 98

Fig. A.2. Relationship between lead (Pb) concentrations (ppm) measured in coracoid and humerus bones of 62 free-ranging mallards from Germany (p b 0.001). Circles: observed data points. Solid line: line of best fit.


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