ISSN , Volume 46, Number 4

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1 ISSN , Volume 46, Number 4 This article was published in the above mentioned Springer issue. The material, including all portions thereof, is protected by copyright; all rights are held exclusively by Springer Science + Business Media. The material is for personal use only; commercial use is not permitted. Unauthorized reproduction, transfer and/or use may be a violation of criminal as well as civil law.

2 Biol Fertil Soils (2010) 46: DOI /s SHORT COMMUNICATION Manure contaminated with the antibiotic sulfadiazine impairs the abundance of nirk- and nirs-type denitrifiers in the gut of the earthworm Eisenia fetida Anja Kotzerke & Sven Klemer & Kristina Kleineidam & Marcus A. Horn & Harold L. Drake & Michael Schloter & Berndt-Michael Wilke Received: 2 October 2009 / Revised: 10 December 2009 / Accepted: 11 December 2009 / Published online: 12 January 2010 # Springer-Verlag 2010 Abstract The antibiotic sulfadiazine (SDZ) can affect denitrifying bacteria in soil. However, effects on denitrifiers in the gut of earthworms have not been described so far. Therefore, the influence of SDZ-contaminated manure applied to soil on denitrifiers in the gut of the earthworm Eisenia fetida was assessed by quantitative polymerase chain reaction targeting genes coding for nirk- and nirstype nitrite reductases of denitrifiers. Gut contents of Eisenia fetida contained and gene copies of nirk and nirs, respectively, after 2 weeks in soils amended with manure only. Copy numbers of nirk and nirs in gut contents from manure treatments with SDZ were up to ten times less. Overall, the data indicate a negative impact of SDZ on denitrifiers in the gut of earthworms. Keywords Denitrifiers. nirk. nirs. Real-time PCR. Antibiotic. Sulfadiazine. Worm gut. Eisenia fetida A. Kotzerke : S. Klemer : B.-M. Wilke Berlin University of Technology, Institute of Ecology, Franklinstraße 29, Berlin, Germany A. Kotzerke Anja.Kotzerke@tu-berlin.de S. Klemer svenklemer@gmx.de B.-M. Wilke bmwilke@tu-berlin.de K. Kleineidam : M. Schloter (*) Helmholtz Zentrum München, Institute of Soil Ecology, Ingolstädter Landstraße 1, Neuherberg, Germany schloter@helmholtz-muenchen.de K. Kleineidam kristina.kleineidam@helmholtz-muenchen.de M. A. Horn : H. L. Drake Department of Ecological Microbiology, University of Bayreuth, Dr.-Hans-Frisch-Straße 1-3, Bayreuth, Germany M. A. Horn marcus.horn@uni-bayreuth.de H. L. Drake hld@uni-bayreuth.de Introduction Earthworms inhabit diverse terrestrial ecosystems and contribute fundamentally to the cycling of organic matter in soils and litter (Edwards and Bohlen 1996). Their populations can affect the structure and selected functional properties of soil microbial communities (Pawlett et al. 2009). Besides, the earthworm rather than the material ingested by earthworms determines the chemical milieu to which the microbial biome in the worm gut is exposed to. The environment of the gut is anoxic and harbors high numbers of denitrifiers (Karsten and Drake 1997; Horn et al. 2003; Ihssen et al. 2003; Wüst et al. 2009a). Denitrifiers catalyze the sequential reduction of nitrate or nitrite to nitrous oxide (N 2 O) and/or dinitrogen (N 2 ) by the sequential action of several terminal oxidoreductases including nitrite reductases encoded by the nirk and nirs gene, respectively (Zumft 1997). Earthworms emit denitrificationderived nitrogenous gasses and thus contribute to the overall emissions of N 2 OandN 2 from soils (Matthies et al. 1999; Drake and Horn 2006; Horn et al. 2006b; Drake and Horn 2007; Wüstetal.2009a). The antibiotic sulfadiazine (SDZ), often used in husbandry and reaching soil by manuring, can impact

3 416 Biol Fertil Soils (2010) 46: denitrifying microbial communities in soil (Kotzerke et al. 2008; Schauss et al. 2009a). However, it is not clear if also denitrifier populations in the gut of earthworms are affected by antibiotics. Only one study so far (Matthies et al. 1999) observed a decreased in vivo emission of N 2 O by 80% when earthworms were preincubated in soil supplemented with the antibiotics streptomycin and tetracycline. Therefore, the aim of this study was to investigate the effects of SDZ-contaminated manure on denitrifying bacteria in the gut of earthworms using quantitative polymerase chain reaction (PCR) targeting nirk and nirs genes. Material and methods The experiments were performed with soil samples from the A p horizon of an arable soil, a silt loam (Orthic Luvisol) from Merzenhausen near Cologne (Germany). The soil had the following characteristics: 15.4% clay, 78.2% silt, and 6.4% sand; organic C content 2.1%; the water-holding capacity is 45.8% and the is ph 7.2 (CaCl 2 ). Four treatments, each containg 200 g of soil, were prepared: unamended (treatment C) and pig manureamended soils (treatments M) served as controls. Two concentrations of SDZ-contaminated manure (40 ml manure kg 1 dry soil) samples were used, 10 mg SDZ kg 1 dry soil (treatment S10) and 100 mg SDZ kg 1 dry soil (treatment S100), to measure the antibiotic effects on the denitrifiers in the gut of the earthworms. Four earthworms with an age of 3 weeks (Eisenia fetida) were incubated for 1 week in 2 kg of the unamended soil and subsequently transferred to each tray. The trays were incubated aerobically at 20 C, maintained under a fixed illumination cycle of 12 h:12 h (light/dark, about 400 lx light intensity) and a water content of 50% of the maximum water holding capacity. Two time points after application of SDZ were A nirs gene copies g -1 worm gut 2.0E E E+06 nirs week 1 nirs gene copies g -1 worm gut 2.0E E E+06 nirs week 2 5.0E E+05 B nirk gene copies g -1 worm gut 1.2E E E+06 nirk week 1 nirk gene copies g -1 worm gut 1.2E E E+06 nirk week 2 3.0E E+06 Fig. 1 Abundances of nirs (a) andnirk (b) genes in worm gut material after maintaining Eisenia fetida in unamended soil (c), soil amended with 40 g manure kg 1 soil (M), 40 g manure+10 mg SDZ kg 1 soil (S10), and 40 g manure+100 mg SDZ kg 1 soil (S100) for 1 and 2 weeks of incubation; data presented as box plots (n=4)

4 Biol Fertil Soils (2010) 46: analyzed: 7 and 14 days. Each sampling and treatment was replicated four times. Worms were washed with distilled water, wiped, and sacrificed in ethanol (70%) before the extraction of gut contents by abscission of the anus and pressing worms from anterior to posterior with sterilized tweezers (Horn et al. 2006a). From each replicate sample, gut contents of three worms were pooled to form a sample of 1 g (fresh weight). DNA from the gut was obtained by phenol-chloroform extraction according to published protocols (Griffiths et al. 2000). Quantity and purity of DNA were checked spectrophotometrically (NanoDrop ND-1000, Peqlab Biotechnologie GmbH, Erlangen, Germany) by recording absorbance at 230, 260, and 280 nm. Quantitative real-time PCR was conducted on a 7300 Real-time PCR System (Applied Biosystems, Germany) using SybrGreen as fluorescent dye. The reaction mixture (25 µl volume) was composed of 12.5 µl Power SybrGreen PCR Master Mix (Applera, Germany), 2 µl BSA (3%, Sigma, Germany), µl DMSO (Sigma, Germany), 0.5 µl of each primer (10 pmol µl 1, Metabion, Germany), and 2 µl template (standard, sample, or water). The primers nirk1f and nirk5r (Braker et al. 1998) and cd3af (Michotey et al. 2000) and R3cd (Throbäck et al. 2004) were used for the nirk and nirs assay, respectively. Dilution series of the different DNA extracts were tested in advance to avoid inhibition of PCR. Dilutions of 1:16 turned out to be best suited for this purpose (data not shown). Amplifications were conducted in 96-well plates in triplicates for all standards, non-template controls, and samples. The assays comprised an initial activation at 95 C for 10 min, five touchdown cycles at 95 C for 15 s, 63 C for 30 s ( 1 C cycle 1 ), 72 C at 30 s, and 40 cycles at 95 C for 15 s, 58 C for 30 s, and 72 C for 30 s. After each PCR run, a dissociation curve was recorded consisting of 95 C for 15 s, 60 C for 30 s, and a subsequent temperature increase until 95 C with a ramp rate of 0.03 C s 1. Purity of amplified products was checked by the observation of a single melting peak and the presence of a unique band of the expected size in a 1.5% agarose gel stained with ethidium bromide. Efficiency of qpcr for nirs and nirk gene amplification was around 85%. Results and discussion Quantification of nirs revealed to copies g 1 per gram gut after 1 week of incubation and higher gene copy numbers ranging from to copies g 1 gut after 2 weeks of incubation in all treatments (Fig. 1a). Treatment S10 had no detectable effect on the abundance of nirs genes in the gut of the earthworms investigated, whereas treatment S100 showed a trend of reduced nirs gene copy numbers compared to treatment M at both sampling time points (Fig. 1a). The lowest gene copy numbers of nirk genes were also detected in treatment S100 (Fig. 1b). In all other treatments, copy numbers of nirk genes were significantly higher compared to nirs at both time points (Fig. 1). The number of nirk genes ranged from to g 1 gut (Fig. 1b). In contrast to nirs genes, treatments S10 and S100 showed both a tendency of decreased gene abundances of nirk compared to treatment M, but differences were not significant. Consequently, the nirs to nirk ratio increased in all SDZ treatments after application of the SDZcontaminated manure, indicating that nirs-type denitrifiers are more persistent to SDZ than nirk-type denitrifiers. Interestingly, the nirk-type denitrifiers in the gut of the worms seemed to be stimulated by the manure in the second week, comparable to denitrifiers in soil (Paul and Beauchamp 1989; Dambreville et al. 2006). This observation is in line with earlier studies indicating that nirk-type rather than nirs-type denitrifiers dominate nutrient-rich habitats (Sharma et al. 2005). However, a study concerning SDZ effects in the same soil showed higher nirs than nirk gene copy numbers in the soil and different response pattern of denitrifiers (Schauss et al. 2009b). In the present study, nirk-type denitrifiers were also more affected by SDZ than nirs-type denitrifiers. They possibly have a higher level of susceptibility to SDZ compared to nirs-type denitrifiers. This might be related to a higher activity of nirk-type denitrifiers in the gut due to the manure application. Moreover, alphaproteobacterial denitrifiers are more frequently detected in gut than in soil (Furlong et al. 2002; Ihssen et al. 2003; Singleton et al. 2003; Horn et al. 2006a; Wüst et al. 2009b), and nirk rather than nirs is prevalent among alphaproteobacterial denitrifiers (Heylen et al. 2006), which is in line with the present data. Overall, the data indicate that not only denitrifiers in soil are affected by the application of antibiotics but also those ingested into the gut of earthworms. The consequences of these findings should be investigated in the future and should also be integrated in an ecotoxicological assessment. Acknowledgments We acknowledge the financial support provided by German Research Foundation DFG as a part of the research group: Veterinary Medicines in Soils Basic Research for Risk Analysis. References Braker G, Fesefeldt A, Witzel KP (1998) Development of PCR primer systems for amplification of nitrite reductase genes (nirk and nirs) to detect denitrifying bacteria in environmental samples. Appl Environ Microbiol 64: Dambreville C, Hallet S, Nguyen C, Morvan T, Germon JC, Philippot L (2006) Structure and activity of the denitrifying community in a maize-cropped field fertilized with composted pig manure or ammonium nitrate. FEMS Microbiol Ecol 56:

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