Heavy metal contamination and metallothionein mRNA in blood and feathers of Black-tailed gulls (Larus crassirostris) from South Korea
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- Kim, M., Park, K., Park, J.Y. et al. Environ Monit Assess (2013) 185: 2221. doi:10.1007/s10661-012-2703-0
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The objectives of this study were to determine levels of heavy metal in the feathers and blood of Black-tailed gulls (Larus crassirostris), to evaluate metallothionein (MT) mRNA level in Black-tailed gulls on three independent islets, and to examine the correlation between heavy metal concentrations and MT mRNA expression. Eleven heavy metals (Al, Cd, Mn, Pb, Cr, Fe, Cu, Zn, Se, Hg, and As) were investigated in blood and feathers of 65 chicks from breeding colonies (Seomando, Hongdo, and Dokdo islet) of South Korea in 2010. Heavy metals were assayed by PerkinElmer NexION 300 inductively coupled plasma mass spectrometry. The mean concentrations of non-essential heavy metals were found to blood containing Cd (0.002 ~ 0.02 ppm), Pb (0.06 ~ 0.18) ppm, Hg (0.03 ~ 0.05) ppm, and As (0.26 ~ 0.48 ppm), and feather containing Cd (0.05 ~ 0.30 ppm), Pb (2.47 ~ 10.80 ppm), Hg (1.18 ~ 1.57 ppm), and As (0.15 ~ 0.44 ppm). Chicks on Seomando islet showed the highest levels of metals (Cd, Pb, Mn, Cr, Cu, and Se in blood; Al, As, Mn, Cr, Fe, Cu, and Se in feathers) among the colonies. Concentrations of Pb and Hg in feathers were the highest on Hongdo, and the levels of Cd and Zn in feathers were the highest on Dokdo islet. MT mRNA in the blood of Black-tailed gulls was relatively higher in gulls from Seomando than in gulls from Hongdo and Dokdo islet. MT mRNA level is thus positively correlated to heavy metal concentrations in Black-tailed gulls.
KeywordsBlack-tailed gullsHeavy metalsMetallothioneinGene expressionIslet habitatSouth Korea
Bio-indicators have been developed to detect the effect of heavy metals on organisms. Seabirds have been used as effective bio-indicators of heavy metals. Because seabirds have been well-studied, are long-lived, are exposed to pollutants over time, and are the omnivorous top-predators in marine ecosystems, they provide information on long-term effects and bioaccumulation in marine food webs (Furness and Greenwood 1993; Cui et al. 2011). Birds contaminated with heavy metals often have lower growth rates (Eeva and Lehikoinen 1996) and behavioral changes (Janssens et al. 2003). Pollution near smelters affects not only heavy metal concentrations but also genetic diversity, which increases the mutation rates in Great tits (Parus major) (Eeva et al. 2006).
Metals are elements naturally present in the environment. Internal tissues such as kidneys and liver, as well as blood, eggs, and feathers can be used for monitoring heavy metal contamination in marine ecosystems. The level of contamination varies among tissues. The kidneys are a primary contaminated organ after heavy metal exposure in avian species (Garcá-Fernández et al. 1995). However, blood and feathers can be collected without killing birds. Blood reflects levels of heavy metals during the short-term and levels are relatively lower than those in internal tissues, whereas feathers are contaminated for relatively longer period (3 to 4 weeks) when they are growing than that of blood (Bearhop et al. 2000a). The advantage of using feathers is that they are a non-destructive assessment of heavy metals because feathers can be collected from live birds (Malik and Zeb 2009). Heavy metal levels in feathers are also consistent with other body tissue levels. The levels of lead (Pb) in feathers have been correlated with those in internal tissues and blood (Burger 1993). Brait and Antoniosi (2011) suggested that feathers of feral pigeons (Columba livia) are useful for monitoring metal levels in urban environments. Because seabirds travel long distances between breeding and wintering areas, feathers reflect the heavy metal concentrations at the area of molting. Newly grown tissue and feathers at the breeding colony can be used to monitor changes in heavy metals near breeding grounds (Abdennadher et al. 2010; Bearhop et al. 2000b), although the concentration of some elements differ between adults and chicks (Barbieri et al. 2010).
Metallothioneins (MTs) are metal-binding proteins that have been studied to monitor heavy metal contamination in organisms. MTs regulate both essential and non-essential metals, and protect organisms against metal stress (Klaassen et al. 1999). Production of MTs increases with the level of heavy metals such as cadmium (Cd), zinc (Zn), and copper (Cu) in the environment. In fish, the MT mRNA levels in the liver of Sand steenbras (Lithognathus mormyrus) reflect the level of heavy metals after contamination by Cd in the laboratory and in a wild population (Tom et al. 1998). In chicken (Gallus gallus), levels of hepatic MT mRNA increase after Cd, Cu, and Zn treatment (Fernando et al. 1989). When Cd is provided to Common mallards (Anas platyrhynchos) in the diet, they accumulate Cd in the kidneys and MT gene expression appears quickly (Lucia et al. 2010b). The positive relationship between MT levels and heavy metal concentrations has been studied in wild birds. MT in liver and kidney of great tits increases with Cd level in tissues (Vanparys et al. 2008). Since MTs were isolated from the Northern fulmar (Fulmarus glacialis) (Osborn 1978), they have been estimated in other wild seabirds as well. MT production is related to Cd and Cu levels in the kidney of Cory’s shearwaters (Calonectris diomedea), (Stewart et al. 1996), Rhinoceros auklets (Cerorhinca monocerata), Cassin’s auklets (Ptychoramphus aleuticus), and Ancient murrelets (Synthliboramphus antiquus) (Elliott and Scheuhammer 1997). In gulls, MT levels increase in the liver and kidney of Lesser black-backed gulls (Larus fuscus) following exposure to Cd, Zn, and Cu (Stewart et al. 1996), and MT levels have been correlated with Cd in the liver of Herring gulls (Larus argentatus) (Elliott et al. 1992). Analyses of the avian genome have been limited to captive birds, and studies of MT mRNA expression related to heavy metals are limited in wild birds including seabirds. Hence, genetic information from captive birds may be useful to analyze the genomes of wild populations and would be helpful to monitor environmental changes.
Black-tailed gulls (Larus crassirostris) are abundant seabirds in South Korea. They usually stay inshore on rivers and estuaries during winter and breed on remote islets from March to August (Kwon et al. 2006). Gulls have high levels of heavy metal concentrations due to scavenging behavior (Burger and Gochfeld 2001) and because they are a top predator. Metal concentrations often reflect trophic levels due to bioaccumulation (Alhashemi et al. 2011). Several studies of heavy metal concentrations in Black-tailed gulls have been conducted in Japan and Korea (Lee et al. 1989; Agusa et al. 2005). In Japan, Pb, Cd, and mercury (Hg) concentration in feathers of Black-tailed gulls was measured as mean 0.754, 0.044, and 4.1 μg/g dry weight [dry wt.], respectively (Agusa et al. 2005). In Korea, the levels of the heavy metals Pb, Cd, and Zn in liver of Black-tailed gull chicks ranged from 0 ~ 5.65, 3.26 ~ 0.09, and 0.23 ppm, respectively (Lee 2003; Lee et al. 1989). A seabird species of Korea, Ancient murrelets, from Yellow sea had liver Pb and Cd levels of 4.04 and 15.3 ppm, respectively (Kim et al. 2009). The levels of heavy metals have been investigated in shorebirds on Yeonjong Island, Korea (Kim and Koo 2008). Great knots (Calidris tenuirostris) contained Pb and Cd levels of 20.8 μg/g wet weight [wet wt.] and 0.50 μg/g wet wt., respectively, while Dunlins (Calidris alpina) had respective levels of 14.8 μg/g wet wt. and 1.13 μg/g wet wt. Although the levels of heavy metals in seabirds have been studied, MT mRNA expression and its variations among breeding colonies are unknown.
The objectives of this study were to determine levels of heavy metal concentrations in the feathers and blood of Black-tailed gulls, to evaluate MT mRNA level in Black-tailed gulls at three independent islets, and to examine the correlation between heavy metal concentrations and MT mRNA expression. To these aims, the MT gene was characterized from Black-tailed gulls. A comparative phylogenetic analysis was conducted to analyze the homologies among birds including seabirds and terrestrial birds. Furthermore, the expression profiles of the MT transcripts were comparatively analyzed in each Black-tailed gull on the three selected islets in South Korea.
Materials and methods
Breeding colonies of Black-tailed gulls
Chick blood and feather sampling
We visited the Black-tailed gull breeding colonies at Seomando islet on 13 June, at Hongdo islet on 18 June, and at Dokdo islet on 15 July, 2010. Because gulls breed earlier on Hongdo islet than on Seomando and Dokdo islets, breeding stages were similar among breeding colonies when we visited. Blood and feather samples were collected from chicks approximately 1 month after hatching. Ages (days after hatching) of chicks were estimated by the length of tarsus according to previous data on the relationship between age and growth rate in Black-tailed gulls (Lee 2003). The length of the tarsus was measured with vernier calipers to the nearest 0.01 mm. Blood (0.25 ~ 0.5 ml) was collected from three chicks each on Hongdo and Seomando islets, and from four chicks on Dokdo islet. Blood samples were obtained from a wing vein using a needle (25 gauge × 1 in. length) and kept in EDTA-coated tubes until analysis in the laboratory. Two feathers per chick were collected from 65 chicks at the three breeding colonies. Collected feathers were stored in a paper envelope separately to prevent pollution. Sex of the chicks was not considered in light of the similar concentrations of heavy metals such as Cd, iron (Fe), Zn, Cu, manganese (Mn), and Pb in female and male Black-tailed gulls (Lee 2003). All work on breeding colonies was conducted after permission was obtained.
Heavy metal analysis
All bloods and feathers of Black-tailed gulls were analyzed at the Center for Research Facilities of Chonnam National University. A tissue sample (0.1 g dry wt.) and blood sample (0.5 ml) were soaked in digestion buffer containing 3 ml HNO3, 3 ml HF, and 1 ml H2O2. Digestion was performed using a MARS 5 (CEM, Matthews, NC) microwave digestion system. The digested solution was filtered and used for metal analysis by inductively coupled plasma mass spectrometry (ICP-MS) using a NexION 300 apparatus; (Perkin-Elmer, Waltham, MA). Eleven metals, including aluminum (Al), arsenic (As), Cd, Mn, Pb, chromium (Cr), Fe, Cu, Zn, selenium (Se), and Hg, were analyzed using ICP-MS. Each sample was analyzed in triplicate; the mean value was calculated, and its metal concentration was expressed as parts per million. The accuracy of the analysis was checked by measuring certified reference materials (CRM 278, CRM 195). Mean recoveries ranged from 97 to 104 %. Recovered concentrations of the certified samples were within 5 % of the certified values.
Gene characterization and phylogenetic analysis
The amino acid sequences were aligned with those of other organisms using Clustal X version 1.8 and then displayed using GeneDoc ver 2.6.001 software. A phylogenetic tree was constructed by neighbor-joining analysis using TreeTop (Brodsky et al. 1993). Bootstrap values were calculated based on 1,000 replicates.
MT mRNA expression analysis
Total RNA was isolated from blood and feathers of Black-tailed gulls using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Single-strand cDNA was then synthesized from 3 μg of total RNA using the SuperScript III RT kit (Invitrogen) with random hexamer primers for reverse transcription in a 20-μl reaction mix. Next, the cDNAs were used as templates for PCR with primers specific for the MT gene. Additionally, PCR was conducted using primers specific for β-actin as an internal control (JQ267800). The primer sequences for MT gene were 5′-TGCAAGTGCAAGAACTGCCGC-3′ and 5′-TCCCCGAAGCGTGCCGGCT-3′ (GenBank ID JQ267799). The primer sequences of the β-actin were the same as those used for gene characterization. Quantitative RT-PCR amplifications and subsequent measurements were performed using an AB7300 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. To quantify cDNA, RT-PCR was performed using a master mix with a final volume of 25 μl that contained 1 μl of cDNA template, 0.2 μM of each primer, and 1× SYBR Premix Ex Taq (Takara, Kyoto, Japan). The following PCR conditions were used to amplify the genes: 94 °C for 3 min, followed by 36 cycles of 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 50 s. The quality of the amplification was then determined using an AB7300 Real Time PCR system to conduct dissociation curve analysis. The cycle threshold (CT) was determined using the AB7300 System SDS software. Fold changes in the expression levels were calculated using the comparative CT method, which was normalized against β-actin expression of the same samples. Each test consisted of at least three replicates.
All analyses were conducted using SPSS 18.0 for Windows (SPSS, Chicago, IL). Normality of data was tested using a Kolmogorov–Smirnov test. Data were normally distributed, and a parametric test was used for analysis. Differences in heavy metal concentrations among three islets were evaluated using one-way ANOVA. Differences of heavy metal distribution between blood and feathers were tested using paired t test. A P value <0.05 was considered as statistically significant.
Results and discussion
Trace element distributions of breeding colonies
Mean concentrations of 11 heavy metals (Al, As, Cd, Mn, Pb, Cr, Fe, Cu, Zn, Se, and Hg) in blood and feathers of Black-tailed gull chicks on Seomando islet, Hongdo islet, and Dokdo islet, South Korea
Seomando islet is located near the large city of Incheon, which is industrialized and located near Incheon International Airport. These higher concentrations on Seomando islet might be related to pollution near the breeding colony. The chicks concentrated higher levels of Al in feathers (3,547 ppm) than seabirds in the other colonies. Feathers of adult Flesh-footed shearwater (Puffinus carneipes) in Australia concentrated 90 ~ 222 ppm of Al (Bond and Lavers 2011). Elevated Al has been linked with increased mortality and reduced reproductive success in wild birds and reduced growth of juveniles (Lucia et al. 2010a; Capdevielle et al. 1998), although Al is an essential element. It may be that Black-tailed gull chicks on Seomando islet experience depressed growth due to high levels of Al. Mn and Fe levels in feathers were also highest in chicks on Seomando islet. The Mn level (122 ppm) in feathers of Black-tailed gull chicks on Seomando islet was much higher than that in the feathers of adult kelp gulls (3.11 ppm) during the breeding season in Namibia (Burger and Gochfeld 2001) and in feathers of adult Black-tailed gulls (0.32 ppm) on Rishiri island, Japan (Agusa et al. 2005). High levels of Se also induce higher mortality, lower reproductive rates, and result in behavioral changes (Ohlendorf et al. 1990; Burger and Gochfeld 2001). Gulls from Seomando islet displayed higher Se concentrations in the blood and feathers than gulls from Hongdo and Dokdo islets.
The non-essential element Pb is a neurotoxic element that affects physiology as well as behavioral and intellectual development in vertebrates (Burger and Gochfeld 2005). The Pb level (10.80 ppm) in feathers of birds on Hongdo islet was above 4 ppm, which can negatively affect birds (Burger and Gochfeld 2000). Pb in feathers on Seomando islet was much higher than in the feathers from Siberian gulls (Larus heuglini) sampled in Iran (Mansouri et al. 2011) and from Black-tailed gulls in Japan (Agusa et al. 2005). A previous study on Hongdo islet reported that Black-tailed gull chicks with higher Pb in their tissues died because of attacks by neighbors (Lee 2003). In our study, Pb was also detected at significantly high concentrations in blood and feathers of chicks on Hongdo islet. The reason for the higher Pb concentration at this locale is unclear. We often observed many fishing boats around Hongdo islet, and Black-tailed gulls typically feed around fishing boats. Fishing weights have caused lead poisoning in other studies (Scheuhammer and Norris 1996; Zabka et al. 2006). Chicks on Dokdo islet had significantly higher levels of Zn than chicks on other colonies, and the Zn level was three times higher than that in adult feathers of an Japanese breeding population (Agusa et al. 2005).
Differences of heavy metal distribution between blood and feathers
Heavy metal concentrations varied among breeding colonies and between blood and feathers (Table 1). The concentrations of ten heavy metals except to As were higher in feathers than in blood. Cd (p < 0.05), Pb (p < 0.01), Cr (p < 0.001), Cu (p < 0.001), Zn (p < 0.001), Se (p < 0.001), and Hg (p < 0.001) significantly differed between blood and feathers. As was higher in blood than that in feathers of birds on Hongdo islet and Dokdo islet, whereas the level of As in blood was lower than in feathers of gulls recovered from Seomando islet. Metal concentrations decreased in the order Fe > Al > Zn > Cu > Se > Cr > As > Mn > Pb > Hg > Cd in blood, and Fe > Al > Zn > Mn > Cu > Pb > Se > Cr > Hg > As > Cd in feathers.
Blood reflects a short-term exposure such as immediate dietary intake, whereas feathers represent long-term exposure during feather growth (Furness 1993). In our study, the concentration of Fe, Al, and Zn was high in both feathers and blood of chicks. Because the metal burden in blood and feathers is obtained from food that the parents provided during the breeding season, chick blood and feathers can be used to monitor metal concentration near breeding colonies. This pattern was consistent with previous observations in another field study (Lucia et al. 2010a).
MT gene characterization in Black-tailed gulls
To compare MT mRNA levels in chicks of three islets, partial sequences of the Black-tailed gull MT gene were amplified by PCR using primers designed from higher avian consensus sequences. We obtained a 234-bp nucleotide sequence of the Black-tailed gull MT (GenBank accession no. JQ267799; Fig. 2a). The sequence shared 96 % identity with Great cormorants. In genome analysis of Black-tailed gull MT, the MT nucleotide sequence region was 95 and 94 % similar to that of Dunlins (Calidris alpina) and Black-tailed godwits (Limosa limosa), respectively (Fig. 2a). The MT amino acid sequence was 98 and 95 % similar to that of Great cormorants and Dunlins, respectively (Fig. 2a). The amino acid sequence of Black-tailed gull MT exhibited a 73 % identity with human MT2 (NP005944). Additionally, the phylogenetic analysis shows the evolutional relationships between the Black-tailed gull MT gene and other avian species (Fig. 2). The phylogenetic analysis revealed MT from chickens formed clusters with MTs from other avian species, whereas the Black-tailed gull MT formed clusters with MT genes from Great cormorants, Dunlins, Red knots, and Black-tailed godwits (Fig. 2b).
Analysis of the MT sequence from Black-tailed gulls suggests that avian MT2 genes are divergent between seabirds and passerine birds. Considering the slow evolutionary rate of nucleotide sequence changes, high conservation of avian MT2 nucleotides is persuasive (Shartzer et al. 1993). The critical cellular function of MT2 might have resulted in a constraint, leading to the slow evolution between Galliformes and Anseriformes (Lee et al. 1996). This report was also confirmed by a sequence analysis of MT isoforms in Great cormorants and Common mallards (Nam et al. 2007).
MT expression and relationship with heavy metal levels in Black-tailed gulls
We also investigated the relationship between heavy metal concentrations and MT mRNA expression. Relative MT mRNA levels and metal concentrations in blood and feather were higher in birds on Seomando islet than in those on Hongdo islet and Dokdo islet. In our study, MT expression was higher in birds on Seomando islet accompanied by significantly higher levels of Al, As, Mn, Fe, Zn, and Se in feathers among colonies.
Previous studies correlated tissue MT expression with heavy metals in the environment (Elliott et al. 1992; Vanparys et al. 2008; Stewart et al. 1996). In seabirds, MT has been positively correlated with Cd concentration in the kidneys of Herring gulls and to Hg and Se concentration in the liver of Leach's storm-petrels (Oceanodroma leucorhoa) (Elliott et al. 1992). In gulls, MT expression was positively correlated with Cd, Zn, and Cu in the liver and kidney (Stewart et al. 1996). Hence, it is still unclear whether certain elements directly correlate with MT expression in feathers and blood or whether any metal influences MT expression in black tailed gulls. Despite this lack of clarity, Black-tailed gulls can still be considered as a potential bioindicator for monitoring the pollution of the marine environment using analysis of heavy metal and MT mRNA levels.
Black-tailed gulls on Seomando islet displayed relatively higher levels of Cd and Pb of non-essential elements in the blood and As in feathers and most essential heavy metals compared to chicks on Hongdo and Dokdo islets. The concentrations of 11 heavy metals were higher in feathers than in blood of Black-tailed gulls on Seomando islet. Additionally, chicks on Seomando islet showed relatively higher expression of MT mRNA than those on Hongdo and Dokdo islets. These results suggest that MT gene expression in seabirds can be used to monitor heavy metal-exposed environments and to investigate biological effects of heavy metals in the marine environment.
We thank Dr. Young-soo Kwon of the National Park Research Institute for his help on Hongdo and Dokdo islet and Prof. Wonchoel Lee of Hanyang University. Dr. Ki-sup Lee of the Waterbirds Network Korea also supported for traveling to Seomando islet and help with the fieldwork. This study has been partially funded by “Ecosystem changes of Dokdo and Ulleungdo under climate change in Korea” of the National Institute of Environmental Research. This study was supported by the National Research Foundation of Korea Grant funded by the Korean Government [NRF-2012-0004186] and [NRF-2012-0004352].