Petra Brandt
email: petra.brandt@awi.de

Gunnar Gerdts

Maarten Boersma

Karen H Wiltshire

Antje Wichels

Comparison of different DNA-extraction-techniques to investigate the bacterial community of marine copepods

Keywords: Helgoland roads, Bacteria, Copepod, DNA extraction, DGGE

Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods" exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp. and Temora sp. from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.

Introduction

Copepods are important components of the marine food web. Their significance as a link between primary producers and higher trophic levels has long been recognised [START_REF] Cushing | A difference in structure between ecosystems in strongly stratified waters and in those that are only weakly stratified[END_REF][START_REF] Møller | Production of bacterial substrate by marine copepods: Effect of phytoplankton biomass and cell size[END_REF][START_REF] Møller | Sloppy feeding in marine copepods: Prey-size-dependent production of dissolved organic carbon[END_REF][START_REF] Olsen | Real-time quantification of microbial degradation of copepod fecal pellets monitored by isothermal microcalorimetry[END_REF]. Copepods also contribute to the microbial loop [START_REF] Azam | The ecological role of water-column microbes in the sea[END_REF][START_REF] Møller | Production of bacterial substrate by marine copepods: Effect of phytoplankton biomass and cell size[END_REF] by releasing dissolved organic carbon (DOC) through sloppy feeding, excretion or more indirectly by leakage of DOC from their faecal pellets [START_REF] Hasegawa | Fate of food nitrogen in marine copepods[END_REF][START_REF] Møller | Production of bacterial substrate by marine copepods: Effect of phytoplankton biomass and cell size[END_REF][START_REF] Møller | Production of doc by calanus finmarchicus, c. Glacialis and c. Hyperboreus through sloppy feeding and leakage from fecal pellets[END_REF][START_REF] Steinberg | Production of chromophoric dissolved organic matter (cdom) in the open ocean by zooplankton and the colonial cyanobacterium trichodesmium spp[END_REF] or via the decomposition of copepod carcasses by bacteria (Tang et al. 2006a;Tang et al. 2006b).

Bacteria typically have a strong affinity to both biotic and abiotic surfaces (Zobell 1943;[START_REF] Costerton | How bacteria stick[END_REF][START_REF] Cooksey | Adhesion of bacteria and diatoms to surfaces in the sea: A review[END_REF][START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF] and in most cases benefit from this attachment. Hence, also animal surfaces such as the chitinous exoskeleton of crustaceans can undergo extensive colonisation by bacteria [START_REF] Sieburth | Dissolved organic matter and heterotrophic microneuston in the surface microlayers of the north atlantic[END_REF][START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF] depending on the life stage or fitness of the animal [START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF]. Using scanning electron microscopy [START_REF] Nagasawa | Scanning electron microscopy investigation of bacterial colonization of the marine copepod acartia clausi[END_REF] found that between 0 and 100 % of the members of a copepod population were colonised by bacteria, and that the epibiotic load of adult Acartia sp. can be anywhere between 10 to 10 5 per specimen [START_REF] Nagasawa | Scanning electron microscopy investigation of bacterial colonization of the marine copepod acartia clausi[END_REF][START_REF] Nagasawa | Bacterial epibionts of copepods[END_REF].

From previous studies it is known that different bacterial species show different distribution or colonisation patterns on copepods [START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF].

Especially the oral region, body appendages [START_REF] Huq | Ecological relationships between vibrio cholerae and planktonic crustacean copepods[END_REF], intersegmental regions and the region around the anus [START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF] are colonised by bacteria, reflecting the bacterial exploitation of DOC released by sloppy feeding or defecation [START_REF] Hansen | Bacteria associated with a marine planktonic copepod in culture. I. Bacterial genera in seawater, body surface, intestines and fecal pellets and succession during fecal pellet degradation[END_REF].

The study of microbial epibionts on crustaceans in aquatic environments was in its infancy in 1997 [START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF], and this situation did not change until Tang et al. (Tang et al. 2006a;Tang et al. 2006b) first investigated bacterial communities on decomposing copepod carcasses using molecular techniques such as PCR-DGGE. These studies showed that the bacterial community of freshly dispatched copepods was different from that of copepods which were incubated for some days. [START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF] first investigated the bacterial community associated with Calanus sp. using PCR-DGGE and compared it with the community of the surrounding water. They observed that the copepod samples consisted of less than five discernable DGGE bands, whereas much higher numbers of bands could be found in the surrounding water samples. However, part of the free-living bacteria could also be found attached to the copepods. The authors also reported that the first extraction technique they applied, an enzyme/SDS extraction protocol without use of phenol-chloroform, led to the release of PCR-inhibiting substances which required an altered DNA extraction protocol.

Therefore, not only a substantial effort in culture-independent investigations of the bacteria associated with copepods is needed, but also a proper assessment of those DNA-extraction techniques that are suited for these consortia. Only then do we stand a chance to substantially further our knowledge on the community structure and the identity of the bacteria associated with marine copepods.

Hence, the present study focuses on the evaluation of different DNA extraction techniques of copepod-bacteria consortia. To this end we used commercial lysis reagents and extraction kits as well as a common method such as phenolchloroform-DNA extraction. Furthermore, the bacterial community-DGGE-pattern of laboratory-grown Acartia tonsa and field-caught Acartia sp. and Temora sp. is described and selected DGGE bands are further analysed by DNA sequencing.

Materials and Methods

Sample collection and preparation

Zooplankton samples from Helgoland Roads in the North Sea (54°11.3" N, 7°54.0" E) were collected in June 2007 using a 150 μm and 500 μm net aboard the research vessel "Aade". With a stereo microscope the animals were sorted by genus with sterile tweezers and washed twice with sterile sea water. A culture of Acartia tonsa was grown from egg to adult stage at 18 °C and LD 16:8 in filtered sea water with a salinity of 31. These copepods were fed with Rhodomonas sp. Adult specimens from laboratory-reared cultures of Acartia tonsa, field-caught Acartia sp. and Temora sp.

were frozen in sterile tubes with one, five or ten individuals per tube.

DNA extraction

A number of cell lysis and DNA extraction techniques were applied to the copepod samples (Fig. 1) covering a range of chemical and physical treatments. Additionally, protocols for differentiation of the attached bacterial community on copepod surfaces from the entire bacterial community were tested and are described below. Obviously, it would be of interest to differentiate between individual copepods, as no information is available on the variation of the bacterial community among specimens of copepods. On the other hand, given the very low densities of bacteria on copepods reported in previous studies (see above), it was first of all necessary to determine the quantity of copepod specimens needed to obtain an adequate amount of bacterial DNA for further analysis. Due to the fact that the numbers of bacteria associated with copepods could be very low, the extraction techniques were first tested on batches of ten and five copepods per sample

Epibiotic bacterial community

Copepod surface attached bacteria were investigated using two protocols: One used the Lyse-N-Go reagent (Pierce, Rockford, IL, USA) and the other employed lysozyme and SDS for cell lysis followed by phenol-chloroform-DNA extraction and DNA precipitation with isopropanol, leaving the copepods intact.

Lyse-N-Go: Entire copepods in batches of either ten, five or one individuals were treated with 10 µl Lyse-N-Go reagent, followed by either one or two cycles of the thermo cycle program recommended by the manufacturer. Lyse-N-Go isolates were directly subjected to PCR using either 2 µl of pure extract or 2 µl of a 1:10 dilution thereof, or were stored at -20 °C until further analysis.

Lysozyme/SDS: Entire copepods in batches of either ten, five or one individuals were treated with 370 µl of STE-buffer (6.7 % saccharose, 50 mM Tris, 1 mM EDTA, pH 8). Cell lysis was accomplished by incubating the samples with 100 µl lysozyme (10 mg / ml) for 30 min at 37 °C with gentle shaking at 400 rpm. Next, 50 µl EDTA-Tris (250 mM, 50 mM) and 30 µl SDS-Tris-EDTA (20 %, 50 mM, 20 mM) were added and samples were incubated for 60 min at 50 °C with gentle shaking. The salt concentration was increased by adding a 1 / 10 vol. of NaCl (5 M), and proteins were removed using 1 vol. phenol-chloroform (1:1). DNA was then precipitated by overnight incubation with isopropanol at -20 °C. Precipitates were washed with 75 % ethanol and dried in a laminar flow cabinet for 15 min prior to resuspension with 10 µl sterile water. DNA extracts were stored at -20 °C until further analysis.

Entire bacterial community DNA of bacteria internally and externally associated with sampled copepods was extracted using one of three protocols after mechanical processing of each sample: first, treatment with Lyse-N-Go reagent; second, treatment with lysozyme/SDS followed by phenol-chloroform-DNA extraction and precipitation with isopropanol; and third, a combination of two commercial DNA extraction kits.

Lyse-N-Go: Copepods in batches of either ten, five or one individuals were suspended in 10 µl of Lyse-N-Go reagent and transferred to a sterile agate mortar using sterile cut pipet tips. An additional 10 µl of Lyse-N-Go reagent was added to the copepod suspension prior to grinding the sample with agate mortar and pestle by hand for 30 s in a laminar flow cabinet. The homogenised sample was pipetted into a sterile micro reaction tube. The agate mortar and pestle were then rinsed with an additional 10 µl of Lyse-N-Go, which was added to the homogenate in the tube.

Alternatively, another grinding procedure was conducted with a pellet pestle and pellet pestle motor (Kontes, Vineland, NJ, USA) for 30 s with 10 µl Lyse-N-Go reagent inside a sterile micro reaction tube before carrying out the recommended thermal cycle program one or two times.

Commercial kit combination: BIO101 FastDNA Kit (Qbiogene, Carlsbad, CA, USA) and Blood and Tissue Kit (Qiagen, Hilden, Germany) were combined as follows: 1000 µl of CLS-TC buffer (chaotropic lysis buffer, Qbiogene) was added to a sample of copepods (10 ind.) and pipetted with a sterile cut pipet tip into a matrix tube (Qbiogene). The sample was homogenised with FastPrep Instrument for 30 s at a speed setting of 4.0. After a 5 min centrifugation step at 16 000 x g, the entire supernatant was transferred into a new micro reaction tube. A 1 / 10 vol. of proteinase K (Roche, Mannheim, Germany) was added to one of the supernatant samples and another sample was left without proteinase to test whether a difference in the released amount of DNA or in the amount of PCR product exists. Both samples were then first incubated for 30 min at 37 °C followed by 60 min incubation at 56 °C in a thermomixer with gentle shaking. A ½ vol. of buffer AL (Qiagen) and a ½ vol. ethanol (96 %) were added and the solution was transferred to a spin column tube containing a silica matrix for DNA purification (Qiagen). DNA binding and elution steps were performed as recommended by the manufacturer, with one exception: The final elution step was conducted twice using 30 µl of elution buffer AE (Qiagen) for each step.

Lysozyme/SDS: Copepods (10, 5, 1 ind.) were ground in 10 µl STE-buffer for 30 s with pellet pestle using pellet pestle motor followed by addition of 360 µl of STE and 100 µl lysozyme (10 mg / ml), respectively. The next steps were the same as described above.

PCR conditions

PCR amplification of 16S rRNA gene fragments was performed using the bacteria specific primers P3-clamp [START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rrna[END_REF]) with a 40-bp GC-rich sequence at the 5" end (5"-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC CCC TAC GGG AGG CAG CAG-3") and 907r (5"-CCG TCA ATT CCT TTG AGT TT-

3").

PCR reaction mixtures with a volume of 50 µl contained 5 µl of 10 x Taq buffer (Eppendorf, Hamburg, Germany), 8 µl of Master Enhancer (Eppendorf) for initial PCRs and no enhancer for reamplification after DGGE, 300 µM dNTPs (Promega, Mannheim, Germany), 0.2 µM of each primer, 2 U of Taq DNA polymerase (Eppendorf) and either 2 µl of pure or 1:10 diluted DNA prior to or 1 µl after DGGE. "Touchdown" PCR was performed as described by [START_REF] Sapp | Bacterial community dynamics during the winter-spring transition in the north sea[END_REF].

PCR fragments on 1.2 % agarose gels were visualised by ethidium bromide (0.5 mg l -1 ) and images were captured with a ChemiDoc XRS System (BioRad, München, Germany). The thickness and intensity of each band visualised was used to gauge the relative volume of the corresponding product used for DGGE (see below).

DGGE

All 16S rRNA gene amplicons were resolved on 6 % (w/v) polyacrylamide gels in 0.5 x TAE buffer (20 mM TrisHCl, 10 mM acetic acid, 0.5 mM EDTA) with denaturing gradients of 15-55 % urea/formamide (100 % denaturant contains 7 M urea and 40 % formamide). Electrophoresis was performed at 60 °C and 150 V for 10 h [START_REF] Sigler | Electrophoresis time impacts the denaturing gradient gel electrophoresis-based assessment of bacterial community structure[END_REF]) using a DCode mutation detection system (BioRad). DGGE gels were stained with SYBRGold (Invitrogen, Karlsruhe, Germany). Imaging was performed with a ChemiDoc XRS System (BioRad). Prominent DGGE bands were excised, eluted [START_REF] Sambrook | Molecular cloning: A laboratory manual[END_REF]), reamplified and confirmed by an additional DGGE.

Sequencing and phylogenetic analysis

Selected DGGE bands were excised, the DNA was eluted and reamplified using the primers 341f (without GC-clamp) [START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rrna[END_REF]) and 907r. PCR-products were checked on 1.2 % (w/v) agarose gels prior to sequencing. PCR products with the right size (~566 bp) were excised from the agarose gels and used for sequencing.

DNA sequencing of PCR products was performed by Qiagen GmbH using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing primers were 341f and 907r. Nearest relatives were searched using BLAST (http://www.ncbi.nlm.nih.gov; [START_REF] Altschul | Gapped blast and psi-blast: A new generation of protein database search programs[END_REF].

Sequence data were checked for the presence of PCR amplified chimeric sequences by the CHECK_CHIMERA program [START_REF] Cole | The ribosomal database project (rdp-ii): Previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy[END_REF]. The ARB software package (http://www.arb-home.de) was used for phylogenetic analysis [START_REF] Ludwig | Arb: A software environment for sequence data[END_REF]).

After addition of sequences to the ARB 16S rDNA sequences database (release May 2005) alignment was carried out with the Fast Aligner integrated in the program and refined by comparison of closest relatives retrieved by BLAST. Sequences with more than 1300 nucleotides were used to calculate phylogenetic trees. The ARB "parsimony interactive" tool was used to add partial sequences to respective trees.

Phylogenetic relationships were deduced by the neighbour joining method including the correction algorithm of Felsenstein [START_REF] Felsenstein | Phylip (phylogeny inference package), version 3.5c[END_REF].

Nucleotide sequence accession numbers

The sequences obtained in this study are available from GenBank under accession numbers FJ226496 -FJ226515.

Results

DNA extraction and PCR

DNA extracts yielded from the different extraction techniques were not quantified before the use as PCR template, because it was very plausible that especially with the ground copepods also copepod DNA was extracted. Nevertheless, PCR amplification of the targeted 16S rRNA gene region was successful. The differences observed between the extraction methods tested here are described in the following paragraphs.

Epibiotic bacterial community

Lyse-N-Go: Applying the Lyse-N-Go treatment using one thermo cycle on ten isolated copepods yielded sufficient bacterial DNA for successful bacterial 16S rRNA gene amplification by PCR, regardless of the copepod group (laboratory-grown Acartia tonsa, field-caught Acartia sp. or field-caught Temora sp.) selected. Employing an additional thermo cycle during DNA extraction yielded a higher quantity of PCR product. Replicate extractions using Lyse-N-Go reagent with one or two thermo cycles on one or five copepods per sample yielded DNA which, when used as a PCR template, led to only inconsistent results regarding the amplification of PCR product.

Lysozyme/SDS: Enzymatic/detergent cell lysis followed by phenol-chloroform-DNA extraction performed on ten, five, or one copepod(s) in suspension yielded PCR amplifiable bacterial DNA in all cases. Quality checks of the amplification products revealed consistently higher band intensity than products derived from Lyse-N-Go DNA extracts. No variation among the three investigated copepod groups and replicates were observed.

Entire bacterial community

Lyse-N-Go: Grinding ten, five, or one copepod individual(s) in Lyse-N-Go reagent with an agate mortar resulted in inconsistent success with PCR amplification. PCR products generated using DNA extracts from pellet-pestle-homogenised copepod suspensions as template yielded PCR products with a higher concentration than those generated from agate mortar DNA extracts. Amplification was inconsistent when the pellet pestle technique was applied to five or one copepod(s). The procedure with two thermo cycles yielded higher amounts of detectable PCR product than that with one thermo cycle, irrespective of which extraction steps were taken before. Up to 20 distinct DGGE bands were generated using PCR products from Temora sp. and approximately 10 for both groups of Acartia sp. irrespective of the number of copepods used for extraction. Clear differences are notable between the laboratorygrown and field-caught Acartia sp. groups (Fig. 3). The highest number of DGGEbands and a very stable and reproducible pattern could be observed using PCR products of phenol-chloroform extracted DNA regardless of whether the copepods were left intact or ground by pellet pestle prior to analysis. DGGE fingerprint patterns showed higher numbers of bands when using the ground copepods than the intact ones (Fig. 4). Unexpectedly, some bands are only visible in the intact copepods (Fig. 4, lower white box).

Sequencing and phylogenetic analysis

Prominent DGGE bands were sequenced from excised agarose gel bands. Sequence data of 20 excised bands from ground copepods could be retrieved representing nine different bacterial phylotypes (Tab. 1). Closest relatives of the sequenced bands derived from BLAST analyses are also listed in Table 1. The results revealed many close matches with 98 -100 % similarity to bacterial 16S rRNA gene sequences in the GenBank. 17 Aca_104/21, Aca_104/22 and Aca_104/48 are strongly related to an uncultured marine bacterium (DQ372849) [START_REF] Morris | Pirellula and om43 are among the dominant lineages identified in an oregon coast diatom bloom[END_REF], while the sequences Tem_104/44, Tem_104/52 and Tem_104/45 (Tem_104/39 and Tem_104/56 are not shown in the tree) are grouped with the sequences of copepod associated bacteria (DQ839253 and DQ839261) found by [START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF]. Phylotype Aca_104/1 clusters with a bacterium detected in a Taiwanese mangrove ecosystem (DQ234217) [START_REF] Liao | Microbial community composition of the danshui river estuary of northern taiwan and the practicality of the phylogenetic method in microbial barcoding[END_REF], while sequence Tem_104/37 is related to a surface water bacterium from the German Bight (DQ911789). Sequence Tem_104/50 is grouped with an uncultured surface-attached bacterium (EF215784) [START_REF] Dang | Cross-ocean distribution of rhodobacterales bacteria as primary surface colonizers in temperate coastal marine waters[END_REF], whereas Aca_104/36 is related to a deep sea octacoral bacterium (DQ395758).

Discussion

Marine bacteria have a strong affinity to surfaces (Zobell 1943;[START_REF] Costerton | How bacteria stick[END_REF][START_REF] Cooksey | Adhesion of bacteria and diatoms to surfaces in the sea: A review[END_REF][START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF]. In many cases, such as with marine snow, this can be explained by the greater availability of resources in these aggregates, or in case of surface layers and biofilms, by some sort of protective features of these structures [START_REF] Matz | Biofilm formation and phenotypic variation enhance predation-driven persistence of vibrio cholerae[END_REF]). In the case of the bacterial communities associated in-and outside of the marine copepods, little is known about their function and even less about their identity. Obviously, bacteria located around the mouth and anus could well be consuming waste and excretory products of the animals, but to our knowledge no information as to their functional role exists. In fact, it is not even known precisely whether these bacteria are simply epibionts, or possibly harmful or pathogenic for the copepods themselves, for other aquatic animals or even for humans. In earlier studies on the identification of the copepod associated bacteria [START_REF] Sochard | Bacteria associated with the surface and gut of marine copepods[END_REF] used culture techniques to characterise and quantify the bacteria with the well known disadvantages of cultivation [START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rrna[END_REF]. So the use of culture-independent techniques, or a combination of culture-dependent and -independent techniques [START_REF] Peter | An evaluation of methods to study the gut bacterial community composition of freshwater zooplankton[END_REF], could possibly shed more light on the identity of the copepod associated bacteria. For example, Tang and co-workers [START_REF] Tang | Copepods as microbial hotspots in the ocean: Effects of host feeding activities on attached bacteria[END_REF]Tang et al. 2006a;Tang et al. 2006b) investigated the bacteria on copepod carcasses and faecal pellet colonisation using molecular techniques. Their DGGE results showed that the bacterial community of copepod carcasses alter over the time of incubation. But from the results of this study, however, it remains unclear whether the bacteria were already associated with the living copepods or whether they colonised the animals after their death. In the case of the faecal pellets it is unclear whether they were colonised by the bacteria in the gut or after defecation. Additionally, the bacteria found in these studies were not identified.

To our knowledge [START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF] were the first to investigate the bacterial community of the marine copepod Calanus sp. with PCR-DGGE followed by sequencing of DGGE bands. They reported difficulties with bacterial DNA-extraction from these copepods. These authors initially used a modified enzyme/SDS-extraction technique without the use of phenol-chloroform, but it seemed that the complete lysis of the copepods with this method resulted in inhibition of the following PCR reaction.

The extraction method was changed accordingly and they continued with a commercial DNA extraction kit where the copepods were not visibly lysed. In the present study a modified lysozyme/SDS phenol-chloroform-DNA extraction protocol was compared with a combination of two commercially available DNAisolation kits and a commercial cell lysis reagent. These techniques differ in sample processing time and also in their efficiency. In addition, three different grinding procedures were tested to have the possibility to compare the surface-attached as well as the entire internal and external bacterial communities of the copepods. Due to the fact that, especially when using grinding techniques, also copepod DNA was extracted, the extracted DNA amounts were not quantified in this study. When the DNA extracts were used as PCR template, however, bacterial PCR product could be obtained in most cases.

Generally, the Lyse-Go-procedure was the fastest of the investigated techniques. It took only a few minutes to prepare the samples without grinding the copepods. With this procedure there was no visible lysis of the copepods. Grinding with the pellet pestle took another 30 seconds, while grinding with the agate mortar required more time for transferring the copepods into the mortar and back to the test tube.

Additionally, during this transfer a lot of material was lost due to the large surface area of pestle and mortar, resulting in very faint PCR bands. For PCR reaction of the Lyse-N-Go processed samples the mixture had to be diluted 10 fold, to get an adequate PCR product. This might be due to the presence of some inhibitory substances, as already described by [START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF], when using two or more microliters of the original mixture.

The combination of two commercial DNA extraction kits was even more timeconsuming. A combination of two kits was required because of the grinding procedure of the copepods. The lysing-matrix tube and the CLS-TC buffer (Bio1O1) were used with the corresponding FastPrep Instrument (Bio1O1) to homogenise the copepods, while the spin column of the Qiagen Blood and Tissue Kit was used to purify the DNA.

The lysozyme/SDS phenol-chloroform-DNA extraction was the most time consuming procedure, because the precipitation step took place over night. However, the PCR product amount yield was best of all investigated procedures, regardless of whether the copepods were intact or ground. In contrast, [START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF] reported that their modified enzyme/SDS extraction protocol led to a release of PCR-inhibiting substances which was not observed in our procedure, as we used a phenolchloroform step after enzyme/SDS-extraction to get rid of these substances.

Therefore, from our investigations, we conclude that the lysozyme/SDS phenolchloroform-DNA extraction method is the one best-suited, allowing even for the analysis of single copepod individuals.

The band intensities of PCR-products on ethidium bromide stained agarose gels of phenol-chloroform extracts retrieved from laboratory-grown and field-caught Acartia sp. and field-caught Temora sp. were compared. Based on strong PCR product signals on an agarose gel, it can be assumed that field-caught Temora sp. may harbour more bacteria than field-caught and laboratory-grown Acartia sp. (Fig. 2).

Since the copepods were not measured or weighed in this study, nor were the attached bacteria counted, we only presume that Temora sp. harbour more bacteria than Acartia sp. because of their larger size. However, this will need further investigations.

The DGGE fingerprinting analysis revealed clear differences among the investigated DNA extraction techniques. The banding patterns varied both in band numbers and band intensities. This might be due to low template DNA concentrations, resulting in too low template concentrations for production of an adequate amount of PCR product. If the quantities of PCR product are too low, no DGGE-band can be formed.

Therefore, it has to be considered that DGGE bands of a single copepod species can vary in number, although they harbour the identical bacterial community. These results underline the importance of a highly efficient DNA-extraction-technique, which is essential for investigating the bacterial communities not only of copepods.

Distinct banding patterns are evident for each of the three copepod groups subjected to phenol-chloroform-DNA extraction treatment and PCR-DGGE (Fig. 3). Hence, it is probable that specific copepod genera harbour different bacteria communities.

Additionally, there were differences in the DGGE-banding patterns of intact and ground copepods extracted with phenol-chloroform, clearly suggesting that there are differences between the bacteria populations in-and outside of the copepods. To gain more information about this topic, it would be necessary to investigate, e.g., the gut of the copepods separately, because this should be the body part inside the copepods which harbours the most bacteria. That work would also contribute to answer the question of whether the gut microflora of these important links of the marine food web is transient or permanent [START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF].

For sequencing, the PCR products of the appropriate size were excised from the agarose gel to exclude unspecific by-products (approx. 100 bp) (Fig. 2). After sequencing the most prominent DGGE bands of the investigated copepod groups two bacterial phyla were found by sequence analysis. The identified phylotypes in this study belonged to the Alphaproteobacteria, Deltaproteobacteria and the Bacteroidetes. The phylotypes were related to marine surface-water bacteria as well as to surface attached bacteria. One DGGE band (Tem_104/51) was related with a bacterium associated with larval stages of the tropical rock lobster (Panulirus ornatus) [START_REF] Payne | Microbial diversity within early-stage cultured panulirus ornatus phyllosomas[END_REF].

Four identified sequences of Temora. sp.-associated bacteria (Tem_104/52, Tem_104/39, Tem_104/45 and Tem_104/56) (Tab. 1) were strongly related to two sequences of attached bacteria of the copepod Calanus sp. found by [START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF]. These bacteria were members of the Roseobacter lineage of the Alphaproteobacteria.

The finding that copepod attached bacterial phylotypes were related to the Roseobacter lineage is not surprising, considering that members of the Roseobacter lineage are among the most predominant bacterial phylotypes recovered from marine plankton clone libraries and have been found to be dominant in the North Sea [START_REF] Eilers | Isolation of novel pelagic bacteria from the german bight and their seasonal contributions to surface picoplankton[END_REF]. Some excised bands (Aca_104/21, Aca_104/22 and Aca_104/48) result in the same sequence although they were excised from different gel positions (Tab. 1). This is probably due to microheterogeneity of different rRNA-operons present in this bacterial species, as was reported previously [START_REF] Nübel | Sequence heterogeneities of genes encoding 16s rrnas in paenibacillus polymyxa detected by temperature gradient gel electrophoresis[END_REF]v. Wintzingerode et al. 1997).

Although several other studies observed members of the genus Vibrio among the attached bacteria on copepods [START_REF] Sochard | Bacteria associated with the surface and gut of marine copepods[END_REF][START_REF] Huq | Ecological relationships between vibrio cholerae and planktonic crustacean copepods[END_REF][START_REF] Tamplin | Attachment of vibrio cholerae serogroup o1 to zooplankton and phytoplankton of bangladesh waters[END_REF], no members of this genus were found in our study. Members of the bacterial genus Vibrio seem to have a particularly strong affinity for all marine surfaces, especially under nutrient-limiting conditions [START_REF] Dawson | Adhesion: A tactic in the survival strategy of a marine vibrio during starvation[END_REF][START_REF] Tamplin | Attachment of vibrio cholerae serogroup o1 to zooplankton and phytoplankton of bangladesh waters[END_REF][START_REF] Carman | Epibiotic microorganisms on copepods and other marine crustaceans[END_REF]. Nalin and co-workers [START_REF] Nalin | Adsorption and growth of vibrio cholerae on chitin[END_REF]) found a connection between chitinous surfaces and Vibrios. Huq and co-workers [START_REF] Huq | Ecological relationships between vibrio cholerae and planktonic crustacean copepods[END_REF][START_REF] Huq | Influence of water temperature, salinity, and ph on survival and growth of toxigenic vibrio cholerae serovar 01 associated with live copepods in laboratory microcosms[END_REF], using culture techniques, detected both V. cholerae and V. parahaemolyticus preferentially attached to planktonic copepods relative to other bacteria (e.g. Escherichia coli and Pseudomonas sp.). The absence of Vibrio sp. in the present study cannot be explained yet. However, maybe the DGGE-bands which represented Vibrio sp. were not excised because they were too faint or the sequencing of the excised band failed. Muyzer et al. and Murray et al. (Muyzer et al. 1993;[START_REF] Murray | Phylogenetic compositions of bacterioplankton from two california estuaries compared by denaturing gradient gel electrophoresis of 16s rdna fragments[END_REF] reported that PCR-DGGE is sensitive enough to detect bacteria which make up only 1 to 2 % of bacterial populations in the mixed assemblage of selected bacterial strains. Thus, the PCR-DGGE method tends to bias towards the predominant groups within a community (v. Wintzingerode et al. 1997;[START_REF] Casamayor | Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: Comparison by microscopy and denaturing gradient gel electrophoresis[END_REF]. Moreover, the detection limit of the PCR-DGGE method is affected by the relative abundance of a population as well as by ribosomal RNA (rrn) operon copy numbers [START_REF] Kan | Temporal variation and detection limit of an estuarine bacterioplankton community analyzed by denaturing gradient gel electrophoresis (dgge)[END_REF]). Nonetheless, substantial information about the species composition can be obtained from complex microbial communities by DGGE analysis [START_REF] Muyzer | Application of denaturing gradient gel electrophoresis (dgge) and temperature gradient electrophoresis (tgge) in microbial ecology[END_REF].

The present and other similar studies allow only a small insight into the identity of the bacteria colonising the exoskeleton and the intestine of marine copepods. Hence, for further insights into the bacterial communities of different copepod genera, or even species, as well as into the copepod gut microflora and faecal pellet associated bacteria, more studies have to be conducted. The outcomes of these studies strongly

  As a standard for all DGGE gels the combined PCR-amplicons (P3-clamp / 907r) of five bacteria (Polaribacter filamentus DSM 13964, Sulfitobacter mediterraneus DSM 12244, Arthrobacter agilis DSM 20550, Microbacterium maritypicum DSM 12512, Leifsonia aquatica DSM 20146) with different GC-contents were used.

  Commercial kit combination: Combining the BIO101 FastDNA Kit to extract DNA from copepod tissue and the silica-based DNA purification in spin-columns from the Blood and Tissue Kit successfully retrieved PCR-amplifiable DNA. The amounts of DNA and PCR product detected were comparable to Lyse-N-Go treatment with pellet pestle. Incubation with proteinase K yielded lower quantities of PCR product. Lysozyme/SDS: The highest amount of PCR product for all investigated copepod groups was achieved using the lysozyme/SDS treatment in combination with phenolchloroform-DNA extraction together with pellet pestle homogenisation. PCR amplification was consistent for copepod suspensions with ten, five or one individual(s). PCR product quality checks revealed that amplification with DNA from phenol-chloroform-DNA extracts of laboratory-grown and field-caught Acartia sp. resulted in notably less amount of PCR product compared to those of field-caught Temora sp. (Fig. 2). DGGE Bacteria-specific PCR products obtained from DNA extracted with different methods generated notably different DGGE banding patterns. A lower number of DGGE bands were observed with PCR products received from the Lyse-N-Go procedure compared to banding patterns obtained with PCR products achieved from phenol-chloroform-DNA extraction.

  sequences of DGGE bands showed high similarity with sequences of Alphaproteobacteria, only one with Deltaproteobacteria, and two with Bacteroidetes. No Gammaproteobacteria were observed. Comparison of sequence data of excised bands appearing at the same position in DGGE gels revealed identical closest relatives in most cases (e.g. Tem_104/59, Tem_104/57, Tem_104/5, Tem_104/46, Tem_104/30, Tem_104/53 and Tem_104/40). Moreover, bands Aca_104/21, Aca_104/22 and Aca_104/48 resulted in the same sequence although they were excised at different gel positions (Tab. 1).A neighbour-joining tree (Fig.5) of selected members of the Alphaproteobacteria, Deltaproteobacteria and Bacteroidetes shows that the excised bands sequences Tem_104/59 (Tem_104/57, Tem_104/5, Tem_104/46, Tem_104/30, Tem_104/53 and Tem_104/40 are not shown in the tree) cluster with a sequence of a surface attached marine bacterium of the Roseobacter clade (EU005307)[START_REF] Dang | Cross-ocean distribution of rhodobacterales bacteria as primary surface colonizers in temperate coastal marine waters[END_REF]).

  Therefore, one of the aims of our study was to develop an extraction protocol suited for the analysis of copepod-associated bacterial DNA. This optimised DNA extraction technique is absolutely essential for molecular biology, since a modification of the extraction method is required for each different sample and each tissue[START_REF] Roose-Amsaleg | Extraction and purification of microbial DNA from soil and sediment samples[END_REF]. Two previous studies on the extraction of DNA from copepod associated bacteria for PCR-DGGE analysis have been published so far(Tang et al. 2006b;[START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF]). Tang and co-workers used a treatment with zirconium beads and hot phenol-chloroform-isoamylalcohol, whereas Møller et al. used two other different methods (see above). This shows that so far no standard DNAextraction technique has been established.

Fig. 1

 1 Figures:

Fig. 4

 4 Fig. 4 Differences in the DGGE banding pattern of intact or ground copepods. White boxes highlight the differences.

Fig. 5

 5 Fig. 5 Phylogenetic tree of Alphaproteobacteria, Deltaproteobacteria and members of the Bacteroidetes. GenBank accession numbers are given in parentheses. Bootstrap

Fig. 1 Fig. 2 A+B

 12 Figures:

Fig. 4

 4 Fig. 4 Differences in the DGGE banding pattern of intact or ground copepods. White boxes highlight the differences.

Fig. 5

 5 Fig. 5 Phylogenetic tree of Alphaproteobacteria, Deltaproteobacteria and members of the Bacteroidetes. GenBank accession numbers are given in parentheses. Bootstrap values above 50% are displayed. Marked with stars: Accession numbers of copepod associated bacteria found by[START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF]

  Figure

  Figure

  Figure

Figure

  Figure

  Fig.5

Figure

  Figure

Table

  Closest relatives to the sequenced DGGE bands. Marked with stars: Sequence accession number of copepod associated bacteria found by[START_REF] Møller | Bacteria associated with copepods: Abundance, activity and community composition[END_REF]

	Band	Host	% Similarity	Alignment length	Accession number	Closest relative	Taxonomic affiliation as reported in GenBank
	Tem_104/59		99	510/511
	Tem_104/57		99	510/511
	Tem_104/5		99	473/474
	Tem_104/46	Temora sp. Field	99	460/461	EU005307	Clone G3-45	uncultured Alphaproteobacterium
	Tem_104/30		99	476/477
	Tem_104/53		100	435/435
	Tem_104/40		100	463/463
	Tem_104/52		100	465/465
	Tem_104/39 Tem_104/45	Temora sp. field	99 99	459/462 460/464	DQ839261*	DGGE band NS16	uncultured Alphaproteobacterium
	Tem_104/56		98	484/490
	Tem_104/44 Temora sp. field	97	439/451	DQ839261*	DGGE band NS16	uncultured Alphaproteobacterium
	Tem_104/50 Temora sp. field	97	510/522	EF215784	Clone PV2-37	uncultured Bacteroidetes
	Tem_104/51 Temora sp. field	91	496/540	DQ831068	Isolate LP-OTU63	uncultured Bacteroidetes
	Aca_104/21	Acartia sp. lab	98	452/459	DQ372849	Clone NH10_29	uncultured Alphaproteobacterium
	Aca_104/22	Acartia sp. lab	97	442/453	DQ372849	Clone NH10_29	uncultured Alphaproteobacterium
	Aca_104/48	Acartia sp. lab	96	412/426	DQ372849	Clone NH10_29	uncultured Alphaproteobacterium
	Aca_104/36	Acartia sp. lab	95	402/420	DQ395758	Clone ctg_CGOF389	uncultured Alphaproteobacterium
	Aca_104/1	Acartia sp. field	96	440/454	DQ234217	Clone DS134	uncultured Alphaproteobacterium
	Aca_104/37	Acartia sp. field	93	443/472	DQ911789	DGGE band GB03-a10-p uncultured Deltaproteobacterium

Tab. 1

ind. 5 ind.1 ind.

Acknowledgements

This study is part of the Helgoland Foodweb project. We would like to thank Katja Kreisel for her helping hands, Martin Löder, Christina Gebühr, Florian Hantzsche and Katherina Schoo for stimulating discussions and the latter one for introduction into the world of copepods and the donation of copepod eggs.


Antoni Margalida
email: margalida@inf.entorno.es

C Gortázar

Supplementary feeding during the chick-rearing period is ineffective in increasing the breeding success in the bearded vulture (Gypaetus barbatus)

Keywords: Bearded vulture, Breeding success, Gypaetus barbatus, Supplementary feeding

Supplementary feeding during the chick-rearing period is ineffective in increasing the breeding success in the bearded vulture ()

Introduction

In several facultatively siblicidal species, supplementary feeding during the nestling period increases fledging success, suggesting that food requirements of nestlings limit the number of offspring that can be raised [START_REF] Lack | The significance of clutch size. I. Intraspecific variation[END_REF][START_REF] Lack | The natural regulation of animal numbers[END_REF][START_REF] Wiehn | Food limitation on brood size: experimental evidence in the Eurasian kestrel[END_REF][START_REF] González | Supplementary feeding as an effective tool for improving breeding success in the Spanish imperial eagle (Aquila adalberti)[END_REF]). However, in obligately siblicidal species, it has been proposed that aggressiveness is not related to food availability [START_REF] Forbes | Food, information and avian brood reduction[END_REF]. Thus, although in obligate brood reducers it is not possible to increase the fledgling success through supplementary feeding, this management technique may help ensure the rearing of the only brood and increase the global breeding success of a population. Until now, there have been no empirical data on the effectiveness of supplementary feeding in increasing breeding success in an obligately siblicidal species. To test this, we applied an experimental supplementary feeding programme during the chick-rearing period in the bearded vulture Gypaetus barbatus, a typical obligate brood reducer [START_REF] Simmons | Offspring quality and the evolution of cainism[END_REF][START_REF] Margalida | Hatching asynchrony, sibling aggression and cannibalism in the bearded vulture Gypaetus barbatus[END_REF]). This species is considered endangered in Europe with 160 territories in the European Union in 2009 and with Spain holding, the most important population with 98 territories (63% of the European population; Donázar et al. 2009b). One of the management measures proposed by the National Strategy for the Conservation of the Vulture in Spain (CNPN 2002) is to 'utilize specific food for breeding pairs through the application of a program that delivers meat remains during the critical period of hatching and first weeks of life of the chick'. In this sense, taking into account that most breeding failures take place during hatching and chick-rearing periods, the quality of food and the breeding experience are considered possible factors that influence breeding failures (see Margalida et al. 2003).

One of the most widespread tools in the conservation of this and other vulture species is the creation of feeding stations or vulture restaurants [START_REF] Brown | an evaluation of supplementary feeding for bearded vultures and other avian scavengers in the Natal Drakensberg[END_REF][START_REF] Mundy | The vultures of Africa[END_REF]Gilbert et al. 2007;Donázar et al. 2009b). Although feeding stations (FS) for the non-breeding population of bearded vultures have proven useful in increasing pre-adult survival [START_REF] Oro | Testing the goodness of supplementary feeding to enhance population viability in an endangered vulture[END_REF], the effectiveness of specific supplementary feeding sites (SSFS) close to nesting sites for the breeding population has not been tested. Thus, managers and policy-makers must have objective data to implement priority conservation measures successfully, based on experimental studies that show the utility or lack thereof of specific conservation tools. In this sense, it has been hypothesised that if food quality (as a consequence of its spatial and temporal unpredictability) is a limiting factor for the breeding success of several pairs with less breeding experience or inhabiting low quality territories limited by food availability (Margalida et al. 2003), then SSFS could be useful in increasing breeding performance. In addition, if the ingestion of poisoned baits is the most important potential mortality factor that principally affects the breeding population (Margalida et al. 2008b;[START_REF] Oro | Testing the goodness of supplementary feeding to enhance population viability in an endangered vulture[END_REF]) and other vulture species (see Hernández andMargalida 2008, 2009), SSFS could contribute to reducing or eliminating the effects of this potential threat mitigating breeding failures as a consequence of adult or chick mortality.

Here, we present the first results of an experimental specific supplementary feeding programme for the Spanish breeding population of bearded vultures applied between 2002 and 2009. We discuss the effectiveness of this experimental supplementary feeding programme in order to improve future management conservation measures and optimise the resources invested in conservation programmes.

Materials and methods

The study species

The bearded vulture is a large, territorial bird of prey with a long lifespan, which nests in rocky cliffs [START_REF] Brown | an evaluation of supplementary feeding for bearded vultures and other avian scavengers in the Natal Drakensberg[END_REF][START_REF] Margalida | Territorial defence behaviour of bearded vulture Gypaetus barbatus against conspecifics and heterospecifics[END_REF]. In monogamous pairs, both sexes invest equally in rearing the offspring, although males play a more active part in nest building and territorial defence, while tending the nest is more pronounced in females [START_REF] Margalida | Breeding behaviour of the bearded vulture (Gypaetus barbatus): minimal sexual differences in parental activities[END_REF]. Although the species' diet is largely based on the bones of domestic and wild ungulates, they also feed on wild animals such as birds, small carnivores or micro-mammals during the breeding period to feed the nestlings [START_REF] Margalida | Diet and food preferences of the endangered bearded vulture Gypaetus barbatus: a basis for their conservation[END_REF].

Study area and methods

The study was carried out in the Catalan Pyrenees in NE Spain between 2002 and 2009. The area contains 35 Bearded Vulture territories, and breeding regularly occurs in 29 of those territories. In this area, during the breeding season, there are seven FS at which supplementary food (principally bone remains and sheep limbs) is provided from November to April. These FS are situated at distances of 4-10 km from the nesting areas and are mainly used by the non-breeding population (see [START_REF] Oro | Testing the goodness of supplementary feeding to enhance population viability in an endangered vulture[END_REF]. Taking into account the characteristics of food provided in the FS (bone remains) and the differences in the diet between the chick-rearing period and the rest of the year (Margalida et al. 2009), their use by breeding adults to feed their chicks is low (author's unpublished data). From 2002 to 2009, during the application of the SSFS, we supplemented 20 pairs (n=52 breeding attempts) with food in order to document whether food delivered increased breeding success. During the same period, non-supplemented nests (n=20 of which ten territories were also controlled as supplemented) in adjacent areas and in which hatching was also documented were considered as control nests (n=52 breeding attempts). The mean altitude of the nests studied was 1,450±230 m (range, 1,100-1,700). Prey remains were delivered from the hatching of the first chick until the first 2 months of chick-rearing and were placed at the top of the cliff in which the nest was situated or in a 500-m radius (area defended against conspecifics and heterospecifics; Margalida and Bertran 2005) around the nest. Prey items were delivered in the morning (9.00-11.00 a.m.) every 2 days and consisted of meat remains (generally rabbit remains). The quantity of food provided varied between 2 and 3 kg per delivery (Table 1) and consisted of small pieces of food scattered across a 5-m radius in order to make it easier for the bearded vultures to eat some of it in the presence of other species (e.g. the common raven Corvus corax and the griffon vulture Gyps fulvus). Observations carried out during the monitoring of the nests suggest that most of the pairs accepted the food supplied, although the way they used it was only quantified in a territory monitored by video cameras.

The age of the nestlings and causes of breeding during the chick-rearing period were determined by accessing the nest immediately after the confirmation of nest abandonment (generally 1-6 days after breeding failure) with the help of specialist climbers from the Government of Catalonia's Rural Agents Corps and video cameras [START_REF] Margalida | A solar-powered transmitting video camera for monitoring cliff-nesting raptors[END_REF]. First, we determined the age at which the chick died, taking into account the date of hatching or the date of egg laying and 53 days of incubation [START_REF] Margalida | Hatching asynchrony, sibling aggression and cannibalism in the bearded vulture Gypaetus barbatus[END_REF]). Subsequently, we determined the causes of breeding failure by analysing the remains found in the nest establishing two categories: natural mortality (e.g. adverse weather, predation) and non-natural mortality (e.g. poisoning). We considered adverse weather to be when the period of breeding failure coincided with persistent snow or rain, and it could be seen that the wool lining the nest was completely soaked. The breeding failure was then attributed to possible hypothermia. The remains found were examined and analysed by the Forensic Wildlife Laboratory (LFVS, Madrid). For a detailed description of the methods and techniques used to determine the cause of death, see Hernández andMargalida (2008, 2009) and Margalida et al. (2008b).

Statistical analyses

Using the Wilcoxon test for matched pairs, we began by comparing the average breeding success between supplemented territories in terms of controlling territories during the period in which the supplementary feeding programme (2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009) was applied. We then compared the breeding success of nine focal territories in which we obtained data of breeding success during the supplementary period for years in which no supplementation occurred (see Table 2). For these analyses, we only used territories supplemented for at least 3 years. In order to increase the sample size of control data (without supplementary food), we also used breeding attempts carried out during the period 2000-2001. The statistical significance was set at P<0.05. Values are presented as means ± SD.

Results

Remains provided were observed delivered to the nest or present in the nest of most of the pairs monitored intensively (with telescopes and/or video cameras) confirming their consumption by breeding bearded vultures. In the only nest monitored with video cameras during 14 days of observations, a total of 34 prey remains were delivered to the nest of which 26 (74.5%) pertained to the remains provided by the SSFP.

Other species as the griffon vulture, common raven, red kite (Milvus milvus), golden eagle (Aquila chrysaetos) and Egyptian vulture (Neophron percnopterus) were also observed eating the remains provided.

No relationship was found between the average number of days of food delivered (Table 1) and the breeding success (r s =0.30, P>0.05, n=8).

Differences in breeding success

The average breeding success for the 20 territories supplemented with food was 0.793±0.193 chicks/pairs 1). When we compared the breeding success obtained in nine supplemented territories (n=33 breeding attempts) to results obtained in the same territories during the feeding period with no supplementation (n = 34 breeding attempts), the differences were not significant (supplementary period, 0.712± 0.307 chicks/pairs with clutch hatched; no supplementary period, 0.642±0.311 chicks/pairs with clutch hatched; Wilcoxon test, z=0.297, P=0.766; Table 2). As shown in Table 2, although the sample size was small, breeding success was improved in six (66.7%) territories.

Age and causes of breeding failure

The age at which the chick died was determined in 13 cases in supplemented nests and nine cases in non-supplemented nests. The age of the chick in failed nests that had received supplementary food was 20.96±20.78 days (range 5-85, n=13) whereas in non-supplemented nests, the average age was 28.61 ± 18.23 days (range 3-60, n = 9, Wilcoxon signed-rank test, z=1.96, P=0.050). Concerning the possible causes of breeding failure, only on three occasions was it possible to access supplemented nests to determine the cause, and in another three attempts, non-supplemented nests were accessed. Of these, the probable causes of the death of the chicks in the supplemented nests were poisoning (Endosulfan), adverse weather plus predation by ravens and colibacillosis (produced by Escherichia coli).

With regards the non-supplemented nests, in three cases, the most probable cause of death was adverse weather, which probably caused hypothermia.

Discussion

Although SSFS for breeding bearded vulture territories slightly increases the global breeding success and the individual breeding performance of the territories, the values obtained were very similar. This suggests that the SSFS programme applied after hatching is apparently not a useful measure for increasing breeding success, at least in the current study area. Thus, factors that provoke breeding failure after hatching continue to operate independently from the supplementary feeding programme. In fact, contrary to what was expected, breeding failures occurred earlier in supplemented nests than in non-supplemented nests. We have no concluding explanation for this trend, and more data are necessary to confirm this as a consequence of the low sample size. A possible explanation for why the SSFS were unsuccessful could be related to the period of supplementation. The latter was carried out during the first 2 months of the chick's life, whereas-in non-supplemented nests-the chick-rearing period for this species is on average 4 months (see Margalida et al. 2003). Thus, covering the total chickrearing period could reduce the number of breeding failures. However, our results suggest that the average time at which the chicks died was during the first 3 to 4 weeks, and the supplementary feeding programme ended during the seventh or eighth week, which implied that the most critical period was covered. Previous results about the age of breeding failures in the study area during the period 1992-1999 confirm that the most critical breeding stage is the hatching period (51.3%, n = 39, with this period including the end of incubation and <7 days of chick's life; Margalida et al. 2003). During the chick-rearing period (considering the nests with chicks with >7 days), the age of the chick in failed nests was 37 days (range 10-74 days). Thus, the SSFP seems to adequately cover the critical period of chick rearing, when the diet of the chick is more dependent on high-quality food with meat remains [START_REF] Margalida | Diet and food preferences of the endangered bearded vulture Gypaetus barbatus: a basis for their conservation[END_REF]). On the other hand, the aim of the SSFS programme during the first 2 months after hatching is to avoid the dependency of breeding pairs on this management technique, as this action can have detrimental effects, such as changes in foraging behaviour [START_REF] Anderson | The advantages and disadvantages of vulture restaurants versus simply leaving livestock (and game) carcasses in the veldt[END_REF][START_REF] Piper | Supplementary feeding programs: how necessary are for the maintenance of numerous and healthy vulture populations[END_REF]). Finally, another goal is to avoid the attraction and habituation of other scavengers that could provoke inter and intra-specific conflicts during the breeding process, which may have a negative effect on breeding success or increase the risk of predation (see, for example, [START_REF] Bertran | Interactive behaviour between bearded vultures Gypaetus barbatus and common ravens Corvus corax: predation risk and kleptoparasitism[END_REF]). Thus, it seems that factors that cause breeding failure are not avoided with SSFP.

Another possible factor than can affect the results is that not all the pairs make use of the food delivered in the same way. Observations of specialist and facultative scavengers eating the remains delivered suggest that, in several territories, the effectiveness of the SSFS can be by the presence of these species. If fact, inter-and intraspecific kleptoparasitic interactions in bearded vultures are common, and in 96.3% of all interactions observed in the study area (n=54), breeding bearded vultures acted as hosts and only in two did they act as kleptoparasites (Margalida and Bertran 2003). In addition, the low quantities of remains provided (2 to 3 kg) made it easy for the food to be consumed quickly by other species thus leaving none for the bearded vultures.

One example of the difficulty in ensuring breeding success when non-natural mortality factors occur is the case of the death of a 85-day-old chick in a nest supplemented with food, as a consequence of illegal poisoning. On the other hand, natural mortality factors such as the effects of adverse weather that provoked several failures were not avoided by the supplementation of food. Thus, although the SSFS may help to maintain or slightly increase breeding success during its application, if non-mortality factors (as illegal poisoning) or natural factors (adverse weather) continue to occur, the effectiveness of this conservation measure is limited. Indeed, in an experimental study using vulture restaurants against vulture mortality through diclofenac exposure in Pakistan (Gilbert et al. 2007), the results showed that this tool can reduce, but not eliminate, mortality related to the ingestion of this toxic substance.

Our results also show a decreasing trend in breeding success during the study period. One possible explanation for this could be related to the food regulations involved in sanitary policies implemented in 2003 and applied after 2006 as a consequence of bovine spongiform encephalopathy. In other vulture species, the decrease in breeding success has also been documented (see review in Donázar et al. 2009a), and in the study area, the breeding parameters of the Egyptian vulture and the griffon vulture have also decreased progressively over the last 10 years [START_REF] García | Vultures, feeding stations and sanitary legislation: a conflict and its consequences from the perspective of the conservation biology[END_REF]. Although the SSFS for breeding adults and the presence of FS may counterbalance the low food availability, it seems insufficient to avoid the effects of illegal poisoning (and other mortality factors that can lead to breeding failure) and to provide the food resources necessary for the scavenger population. In the bearded vulture, food quality (i.e. meat remains and nutritious bones) is important and selected during the first few weeks of chick's life [START_REF] Margalida | Bearded vultures (Gypaetus barbatus) prefer fatty bones[END_REF][START_REF] Margalida | Diet and food preferences of the endangered bearded vulture Gypaetus barbatus: a basis for their conservation[END_REF], and the temporal variation in food quality may influence breeding success (see Margalida et al. 2003). In addition, the low availability of food increases resource competition, favouring species such as the griffon vulture, which monopolises the resource (Cortés-Avizanda 2006;Donázar et al. 2009a). Thus, this period of low availability could contribute to the increase in breeding failure, as has been documented in other vulture species if the food availability situation is not reversed (see review in Donázar et al. 2009b). Nevertheless, several confounding factors such as density dependence of demographic parameters in this population as a consequence of the combined effects of the shrinkage of territories and the presence of floaters around supplementary feeding points (Carrete et al. 2006a;Margalida et al. 2008a), the increase of polyandrous trios that implies social interactions and a reduction in productivity (Carrete et al. 2006b;[START_REF] Bertran | Agonistic behaviour and sexual conflict in atypical reproductive groups: the case of bearded vulture polyandrous trios[END_REF]) are also influencing breeding parameters. Thus, additional research on this subject is necessary in order to determine the real effects of artificial feeding on breeding success.

Management measures

Our preliminary results suggest that the provision of supplementary food during the chick-rearing period aimed at increasing breeding success does not seem to be effective. The supplementary feeding programme apparently failed to mitigate the effects that influenced breeding failure, suggesting that in an obligate brood reducer, supplementary feeding to increase breeding success may be an ineffective conservation tool. The effects of the illegal poisoning on the breeding and population dynamics of this species (see [START_REF] Oro | Testing the goodness of supplementary feeding to enhance population viability in an endangered vulture[END_REF]) are troubling, and until this activity has been reduced or eliminated, any management measure will probably be unsuccessful. Taking into account the fact that this factor led to the extinction of most populations of the species in Europe [START_REF] Mingozzi | Analysis of a historical extirpation of the bearded vulture Gypaetus barbatus (L) in the western Alps (France-Italy): former distribution and causes of extirpation[END_REF], this should be a focus of managers in optimising and to prioritising the investment of economic resources in conservation actions. Finally, from a conservation point of view, taking into account the possible detrimental effects of artificial feeding (see review in Donázar et al. 2009b), it may be more effective to invest in habitat management (i.e. increasing the carrion provided by extensive livestock or game species) and to provide natural food resources from extensive grazing or hunting practices to guarantee feeding resources and increase breeding success (see Donázar et al. 2009b;[START_REF] Olea | The role of traditional farming practices in ecosystem conservation: the case of transhumance and vultures[END_REF][START_REF] Olea | When hunting benefits raptors: a case study of game species and vultures[END_REF]. This has been put forward as the most inexpensive and efficient management method for the conservation of scavengers without modifying habitat quality artificially (Donázar et al. 2009c).

determine causes of breeding failure. This study was funded by the Ministerio de Medio Ambiente, Medio Rural y Marino and Departament de Medi Ambient i Habitatge of Generalitat Catalunya. The experiments comply with the laws currently in force in Spain, the country in which they were carried out.

Fig. 1

 1 Fig. 1 Annual variation in breeding success in supplemented (solid line) and non-supplemented (broken line) bearded vulture territories from 2002 to 2009

Table 2

 2 Differences in the breeding performance of nine focal bearded vulture territories with a without the application of the SSFP

	Table 1 Number of bearded
	vulture territories involved in		Territories supplemented (n)	Average number of days invested (range)	Chicks fledged (n)
	the annual SSFP, time duration
	(in days) of the SSFP and the	2002	3		7 (6-8)	3
	total number of young that	2003	5		18 (12-35)	5
	fledged each year	2004	8		11.6 (4-16)	8
		2005	12		10.1 (4-17)	10
	Number of bearded vulture ter-	2006	6		9 (6-15)	4
	ritories involved (n=20) in the annual specific supplementary feeding programme, time (in	2007 2008	6 6		8 (2-14) 8.8 (2-19)	3 4
	days) invested delivering food	2009	6		8.3 (6-13)	4
	and the total number of young	Total	52			41
	that fledged each year
			Territory	With SSFP	Without SSFP
					Success	Failure	Success	Failure
			A		1	3	2	0
			B		3	0	3	1
			C		6	0	3	1
			D		2	2	2	3
			E		3	0	1	2
			F		4	0	3	1
			G		3	0	0	3
			H		2	1	4	0
			I		2	1	4	1
			Total	26	7	22	12

Eur J Wildl Res (2010) 56:673-678

Acknowledgements Thanks to J. Bas, J. Bolado, A. Bonada, J. Canut, E. Edo, M. Gil, P. Gilabert, R. Heredia, L.M. Martín, X. Maurín, J. Mayoral, J. Moga, P. Pelayo, R. Pelayo, J. Perelada, F. Saura and E. Vega for their help during field work. J.A. Donázar, L.M. González and two anonymous referees reviewed a first draft of this manuscript. M. Hernández (LFVS) analysed dead chicks in order to


MD Anders Bjartell
email: anders.bjartell@med.lu.se

Anna Katharina Dahlman

Anders Edsjö

Christer Halldén

Jenny Liao Persson

Samson W Fine

Hans Lilja

William Gerald

Effect of androgen deprivation therapy on the expression of prostate cancer biomarkers MSMB and MSMB-binding protein CRISP3

Keywords: (3-6): microseminoprotein, PSP94, neoadjuvant, castration, tissue biomarker

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Prostate cancer is the most common form of cancer in Western world males, and is also a leading cause of cancer related-deaths in males [START_REF] Jemal | Cancer statistics[END_REF] . Patient prognosis will vary from a rapidly progressing disease with a high probability of death in a minority of patients, to a relatively indolent prostate cancer that can be controlled for the remainder of the patients life with little intervention in a majority of patients. Despite identification of a number of promising new biomarkers, there is still no absolute way of determining disease prognosis at the time of diagnosis. Inevitably, this leads to overtreatment of a large number of men for the benefit of the few who need it.

Attention has been focused on the predictory abilities of β-microseminoprotein (MSMB); also known as prostate specific protein of 94 amino acids (PSP94). This protein is second only to PSA as the most predominant protein in seminal plasma [START_REF] Lilja | Three predominant proteins secreted by the human prostate gland[END_REF][START_REF] Abrahamsson | Radioimmunoassay of beta-microseminoprotein, a prostatic-secreted protein present in sera of both men and women[END_REF] . The function of MSMB is largely unknown, but in vitro and in vivo studies suggest it may be involved in apoptosis, cell mobility, vascularization, and tumor suppressor functions, all recently reviewed by Whitaker [START_REF] Whitaker | The potential value of microseminoprotein-beta as a prostate cancer biomarker and therapeutic target[END_REF] . The suitability of MSMB as a biomarker for prostate cancer has been raised by several groups during the last two decades. It has been reported that MSMB mRNA and protein expression is reduced in malignant prostatic epithelia compared to benign epithelia [START_REF] Tsurusaki | Cellular expression of beta-microseminoprotein (beta-MSP) mRNA and its protein in untreated prostate cancer[END_REF] . In prostate cancer, the MSMB gene may be subjected to transcriptional silencing by methylation, mediated by Enhancer of Zeste Homologue-2 (EZH2), a Polycomb group member which is often overexpressed in CRPC [START_REF] Varambally | The polycomb group protein EZH2 is involved in progression of prostate cancer[END_REF][START_REF] Beke | The gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the Polycomb group protein EZH2[END_REF] .

MSMB expression has been reported by several groups to be negatively associated with disease-free survival 8 and outcome [START_REF] Bjartell | Association of cysteine-rich secretory protein 3 and beta-microseminoprotein with outcome after radical prostatectomy[END_REF] . MSMB expression has also been proposed to be a significant prognostic indicator for prostate cancer progression under endocrine therapy [START_REF] Sakai | Prognostic significance of beta-microseminoprotein mRNA expression in prostate cancer[END_REF] .

Recently, the MSMB gene has gained further attention as one of the primary candidate prostate cancer susceptibility genes [START_REF] Thomas | Multiple loci identified in a genome-wide association study of prostate cancer[END_REF][START_REF] Eeles | Multiple newly identified loci associated with prostate cancer susceptibility[END_REF] , and several causal risk alleles have been identified in the region upstream of the coding sequence [START_REF] Chang | Fine mapping association study and functional analysis implicate a SNP in MSMB at 10q11 as a causal variant for prostate cancer risk[END_REF][START_REF] Lou | Fine mapping and functional analysis of a common variant in MSMB on chromosome 10q11.2 associated with prostate cancer susceptibility[END_REF][START_REF] Kote-Jarai | Mutation analysis of the MSMB gene in familial prostate cancer[END_REF] . Serum levels of MSMB, its binding protein PSP94-binding protein (PSPBP) and the ratio free/bound MSMB, have all been suggested as independent prognostic factors in prostate cancer [START_REF] Wu | Serum bound forms of PSP94 (prostate secretory protein of 94 amino acids) in prostate cancer patients[END_REF][START_REF] Reeves | Identification, purification and characterization of a novel human blood protein with binding affinity for prostate secretory protein of 94 amino acids[END_REF][START_REF] Reeves | Prognostic value of prostate secretory protein of 94 amino acids and its binding protein after radical prostatectomy[END_REF][START_REF] Nam | A novel serum marker, total prostate secretory protein of 94 amino acids, improves prostate cancer detection and helps identify high grade cancers at diagnosis[END_REF] .

We have previously reported that the cysteine-rich secretory family (CRISP) family CRISP 3) also forms a high-affinity complex with MSMB in human seminal plasma [START_REF] Udby | beta-Microseminoprotein binds CRISP-3 in human seminal plasma[END_REF] . CRISP3 has been found to be one of the most upregulated genes in prostate cancer compared to benign tissue [START_REF] Asmann | Identification of differentially expressed genes in normal and malignant prostate by electronic profiling of expressed sequence tags[END_REF][START_REF] Udby | Characterization and localization of cysteine-rich secretory protein 3 (CRISP-3) in the human male reproductive tract[END_REF] , and was proposed to be a useful biomarker for prostate cancer [START_REF] Kosari | Cysteine-rich secretory protein-3: a potential biomarker for prostate cancer[END_REF][START_REF] Bjartell | Immunohistochemical detection of cysteine-rich secretory protein 3 in tissue and in serum from men with cancer or benign enlargement of the prostate gland[END_REF] . Recently we reported that in a tissue microarray (TMA) with samples from 945 prostate cancer patients undergoing radical prostatectomy (RP), high CRISP3 and low MSMB expression were associated with poor outcome [START_REF] Bjartell | Association of cysteine-rich secretory protein 3 and beta-microseminoprotein with outcome after radical prostatectomy[END_REF] .

As the impact of androgen availability on the expression of certain tumor biomarkers is currently unclear, we wanted to investigate how the candidate markers MSMB and CRISP3 are regulated by androgens during ADT and in progressive prostate cancer. First, we investigated the effect of short term neoadjuvant ADT on CRISP3 and MSMB gene expression in primary prostate cancer, as well as in a limited set of prostate cancer metastases. CRISP3 and MSMB protein levels were then evaluated in serial tissue samples from a small but unique cohort of men with prostate cancer undergoing transurethral resection of the prostate (TURP) before and during long-term ADT. Finally, as MSMB expression may be differentially regulated in primary prostate cancers and in progressive disease, we also studied whether MSMB expression levels were related to a SNP in the MSMB promotor region and associated with epigenetic silencing of MSMB by EZH2.

MATERIALS AND METHODS

Sample preparation and data analysis of Affymetrix U95 human gene array

Microarray tissue samples were obtained as previously described [START_REF] Holzbeierlein | Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance[END_REF] . In brief, tissue samples were collected from patients undergoing therapeutic or diagnostic procedures at Memorial Sloan-Kettering Cancer Center, New York, NY. All samples were snap-frozen, histologically analyzed, and tissues containing approximately 60-80 % prostate cancer were manually dissected from the frozen block. Samples included 23 primary prostate cancer tissues from patients not receiving adjuvant therapy prior to RP, 17 primary prostate cancer tissues from patients collected at RP after three months of neoadjuvant ADT (monthly injections of 3.6 mg of goserelin and 250 mg flutamide three times daily). A limited set of 9 prostate cancer metastases (the secondary sites were in bone (two samples); lung (one sample) and lymph node (three samples)) and metastatic castration resistant prostate cancer (CRPC) (three samples, sites not specified) were also included. Details on patient characteristics have previously been described [START_REF] Holzbeierlein | Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance[END_REF] .

Gene expression analysis were performed as described previously [START_REF] Holzbeierlein | Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance[END_REF] . In brief, total RNA was extracted from frozen tissue by Trizol (Invitrogen, Carlsbad, CA), purified and evaluated for integrity. Complementary DNA (cDNA) was synthesized from total RNA, and gene expression analysis was performed using Affymetrix U95 human gene arrays with 63 175 probes for genes and expressed sequence tags (ESTs) as described by the manufacturer (Affymetrix, Inc., Santa Clara, CA). Samples were analyzed using Affymetrix Microarray Suite v4.0 as previously published [START_REF] Holzbeierlein | Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance[END_REF] .

Tissue specimens and clinical data from patients undergoing TURP

Formalin-fixed paraffin-embedded human tissue samples were obtained from 16 prostate cancer patients (mean age 70 years, range 52-78) undergoing repeated therapeutic TURP at the University Hospital Malmö, Sweden. Immunohistochemistry was performed as previously described [START_REF] Bjartell | Association of cysteine-rich secretory protein 3 and beta-microseminoprotein with outcome after radical prostatectomy[END_REF] , using antibodies and dilutions as described in Table I. Descriptive characteristics of the patients are given in Table II

Genotype analysis

Paraffin embedded tissue from 13 out of the 16 patients undergoing TURP was available for genotype analysis. Fresh sections were microscopically studied, and tissue was manually punched out of the paraffin block with a 2 mm biopsy needle. DNA was isolated using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The rs10993994 polymorphism was determined using TaqMan primers and probes (Applied Biosystems, Foster City, CA), using 1 ng/µl template DNA in a reaction volume of 25 µl. The analysis was performed on a 7900HT fast real-time PCR system (Applied Biosystems) according to the manufacturer's instructions.

RESULTS

MSMB but not CRISP3 transcript levels decrease in response to short-term neoadjuvant ADT

Changes in expression of MSMB and CRISP3 genes due to androgen deprivation were analysed using data from Affymetrix U95 gene expression microarrays. The clinical and pathological features related to the patients and tissue samples included in the study (patient age, preoperative serum PSA levels, Gleason score, pathological TNM stage, and recurrence) have been described earlier [START_REF] Holzbeierlein | Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance[END_REF] . MSMB expression was found to be high in primary prostate cancer from patients not receiving adjuvant ADT, and expression was significantly decreased upon ADT (Fig. 1; Mann-Whitney, p<0.001). No significant reduction in gene expression levels was seen in either CRISP3 or AR transcript levels in patients receiving neoadjuvant ADT, however both CRISP3 and AR were highly expressed in metastatic prostate cancer lesions (Fig. 1). There was considerable inter-patient variability in expression of both MSMB and CRISP3. Interestingly, the MSMB expression level and decrease upon ADT was equal that of KLK3, a well-known target of androgen signaling (Fig. 1). As expected, KLK3 gene expression levels decreased in the group receiving neoadjuvant ADT compared to the nontreated group (Mann-Whitney, p<0.001). However, as in the case of MSMB, considerable inter-patient variability was detected.

Prostate cancer metastases express low transcript levels of MSMB, and high levels of

CRISP3

A vast majority of prostate cancer patients develop CRPC within a few years following ADT.

Such tumors often exhibit highly upregulated levels of AR though the signaling may be dysfunctional [START_REF] Chen | Molecular determinants of resistance to antiandrogen therapy[END_REF][START_REF] Tran | Development of a second-generation antiandrogen for treatment of advanced prostate cancer[END_REF] . In order to investigate MSMB and CRISP3 transcriptional levels in metastases, we studied gene expression in nine remote tumors, out of which three were from patients with CRPC.

Although caution is a prerequisite when interpreting small sample numbers, we found that MSMB expression is substantially decreased in metastatic tumors compared to primary prostate cancer, whereas CRISP3 is expressed at high levels in metastases (Fig. 1).

Interestingly, CRISP3 expression pattern appears to be somewhat similar to that of AR in that both transcipts are expressed at the same level and neither AR nor CRISP3 is affected by ADT (Fig. 1).

Protein expression of MSMB and CRISP3 during ADT and prostate cancer progression

To evaulate the link between long-term ADT, progressing disease, and the expression levels of MSMB and CRISP3 proteins, we used a small but unique set of serially collected tissue samples from 16 patients undergoing TURP before and during ADT. At the time of the second TURP, all patients were in biochemical progress and considered to be in a CRPC stage (Table II). Tissue sections were immunhistochemically stained for MSMB, CRISP3, and AR, and protein expression was evaluated by a pathologist for both intensity and percentage of positive tumor cells. Representative staining is depicted in Fig. 2.

A majority of the patients had very low MSMB levels already at the time of the first TURP, and decreased expression was therefore not readily detected (Fig. 3). In the few patients with more than 50 % tumors cells positive for MSMB at the time of the first TURP, there was a dramatic decrease in expression at the time for the second TURP. No patient had more than 45 % tumor cells positive for MSMB at a CRPC stage. Tumor tissue from these CRPC patients during long-term ADT showed higher levels of CRISP3 and AR compared with tissue from primary tumors from the same patient at the time of diagnosis (Fig. 3).

Increased expression levels of CRISP3 and AR were found in terms of an increased fraction of positive cells. MSMB and CRISP3 staining intensity did not change with progressing disease, but AR staining was stronger at the time for the second TURP. The decreased MSMB protein expression upon long-term ADT is corroborating the studies at the transcriptional level, and is also emphasizing that low MSMB expression is a feature of aggressive disease, whereas CRISP3 and AR show the opposite with high expression levels in CRPC.

Low MSMB protein levels are associated with the TT genotype of SNP rs10993994

The MSMB promoter contains a SNP reported to significantly affect MSMB gene expression.

To investigate the impact of this SNP on MSMB expression in our experimental setup, we performed TaqMan genotyping of the rs10993994 locus on 13 out of the 16 patients previously immunohistochemically examined for MSMB expression. TaqMan genotyping identified the TT genotype previously associated with lower MSMB expression in two patients. Both patients were showing low levels of MSMB. The remaining 11 patients all had CC or CT genotypes, and taken as a group, a higher level of MSMB expression (Fig. 3 and4).

High transcriptional levels of the EZH2 gene are associated with low MSMB levels in progressive disease and metastases

MSMB expression before and during ADT varies considerably between different patients, and can not be explained by either androgen availability or genotype. Previous studies have shown that methylation, mediated by the Polycomb group member EZH2, is yet another way by which MSMB expression may be regulated. Therefore, we analyzed the transcriptional levels of EZH2 in the Affymetrix U95 gene expression microarray described above. Although this is a limited sample set, we found that the EZH2 gene was highly expressed in metastatic lesions from prostate cancer, where MSMB expression levels were very low. EZH2 gene expression does not appear to be affected by neoadjuvant ADT (Fig. 1).

DISCUSSION

In the current study, we wanted to examine impact of short-and longterm ADT on prostate cancer outcome predictors MSMB and the MSMB-binding protein CRISP3. We show that short-term ADT significantly reduces both transcript and protein levels of MSMB, whereas CRISP3 levels do not change. On the other hand, CRISP3 is upregulated in parallell with AR and KLK3 in progressive disease and during long-term ADT.

In concordance with studies conducted by others, our results support that MSMB expression decrease in patients receiving neoadjuvant ADT indicating androgen dependent expression [START_REF] Imasato | PSP94 expression after androgen deprivation therapy: a comparative study with prostate specific antigen in benign prostate and prostate cancer[END_REF] .

Importantly, and in contrast to KLK3, MSMB expression is continuously low in progressive and metastatic disease (Fig. 1). Rising levels of PSA is considered a hallmark for disease progression. This indicates that despite similarities in androgen effect on KLK3 and MSMB expression in the normal prostate and primary prostate cancer, it is obvious that they are differentially regulated in progressive disease. This could be due to silencing of MSMB expression by EZH2 mediated methylation of the MSMB promotor [START_REF] Varambally | The polycomb group protein EZH2 is involved in progression of prostate cancer[END_REF] . We find high levels of EZH2 in metastatic lesions which is consistent of our findings of low MSMB expression in those tumors (Fig. 1). ADT did not appear to affect EZH2 levels, thus methylation is unlikely linked to the rapid MSMB downregulation seen in hormonally treated primay prostate cancer.

Yet another way by which MSMB can be regulated is by a SNP in the promotor region. In our study, the TT allele was only seen in 2 out of 13 patients (Fig. 3 and4). Both patients had very low levels of MSMB, but not lower than many patients carrying the CC or CT alleles.

Despite much recent interest in the rs10993994 SNP genotype, our findings indicate that it does not fully account for the low MSMB levels generally seen in prostate cancer patients with progressive disease.

Our data showed that neoadjuvant therapy does not appear to affect expression of CRISP3. On the other hand, the high CRISP3 expression in metastatic and recurrent tumors may be indicative of a role for CRISP3 in the progression of prostate cancer.

In the present study, we find that MSMB expression may be regulated by several mechanisms, including androgen deprivation. In all, MSMB being suggested to act as a candidate tumor suppressor, it is most thought-provoking to note that according to this study, MSMB is downregulated by hormonal treatment and subsequently it may be silenced by different mechanisms. Our results favor the view of MSMB being a suitable marker for prostate cancer disease progression in CRPC.

  . Samples were collected between 1971 and 2001. From each patient, samples were collected at diagnosis, prior to ADT and from a later TURP due to local tumor progression. Nine patients were subjected to medical castration with GnRH analogue, one patient was treated with flutamide monotherapy and five patients underwent bilateral orchiectomy. Mean follow-up time was 73 months (range 12-167 months) and mean time of hormonal treatment was 37 months (range 3-118 months). Patients were considered to have progressive disease and CRPC at the second TURP date as levels of serum PSA or acid phosphatase (PAP) were rising despite ongoing ADT. The study was approved by the local Ethic's Committee at Lund University, Sweden.

Fig. 1 .

 1 Fig. 1. Transcription profile changes before and after ADT in primary prostate cancer tumors

Fig. 2 .

 2 Fig. 2. Immunohistochemical staining showing expression pattern of MSMB, CRISP3 and

Fig. 3 .

 3 Fig. 3. Bar charts representing the fraction of cancer cells positively immunostained for

Fig. 4 .

 4 Fig. 4. Correlation of the rs10993994 genotype and the corresponding percentage of MSMB

  r e a t e d p r i ma r y p r o s t a t e c a n c e r N e o a d j u v a n t A D T t r e a t e d p r i ma r y p r o s t a t e c a n c e r P r o s t a t e c a n c e r me t a s t a s e s

ACKNOWLEDGEMENTS

We are very grateful to Jeff Holzbeierlein and the American Society for Investigative Pathology for permission to re-print the microarray data on AR expression; to Elise Nilsson for her excellent technical skills in the field of immunohistochemistry; to Lene Udby for providing the CRISP3 antibody; and to Per Fernlund for the MSMB antibody.

The authors declare no conflict of interests.

Supporting grants:

European Union 6th Framework (P-Mark) [grant number LSHC-CT-2004-503011], Swedish Cancer Society, Swedish Research Council (Medicine), Cancer and Research Foundation at University Hospital Malmö, Gunnar Nilsson Cancer Foundation, and the Crafoord Foundation.

CONFLICT OF INTERESTS