Europ. J. Protisto!. 33, 192-199 (1997)June 30, 1997

European Journal of

PROTISTOLOGY

Molecular Identification of Clones of TestateAmoebae Using Single Nuclei for peR Amplification

Manfred Wanner1, Jbrg M. Nahring2, and Rainer Fischer-

'Institute of Biology II (Zoology), 21nstitute of Biology I (Botany), RWTH, Aachen, Germany

Summary

Culture growth and shell morphology of clonal cultures ofterrestrial testate amoebae are highly variable in alteringenvironmental conditions which is clearly reproducibleand even reversible. Due to these taxonomical and ecologi­cal implications, a genetic approach based on RAPD-PCRwas developed to supplement currently available data.Only when paired random oligonucleotide primers wereused, most analyzed cultures of testate amoebae showedfingerprints that appeared to be taxon-specific. Furtherexperiments demonstrated that a foreign DNA source wasresponsible for these culture-specific RAPD patterns, al­though no eukaryotic contamination was visible. The dataobtained pointed out that this foreign DNA source is dis­tinct from the food or the culture medium componentsand located within the amoebae casing, and therefore wasinevitably transferred with the amoebae to each new sub­culture. This severe problem of coamplifying foreign butculture-specific DNA can be prevented by using singlenuclei of testate amoebae as a reference. In the case of Eug­lypha strigosa, single cells were divided into a nucleus-freeand nucleus-containing part. Comparing one to 63 cellsofdifferent cultures of the same clone of Euglypha strigosa al­ways resulted in one or two primer-dependent, highly re­producible bands of amplified DNA which only appearedin the amoeba-nucleus containing sample. Specificprimersderived from one sequenced RAPD-DNA fragmentdemonstrated its amoeba-specific nuclear origin. TheseRAPD-derived specificprimers allowed highly specificam­plification of testate amoebae DNA derived from one nu­cleus even in the presence of large amounts of contaminat­ing foreign DNA and provided the basis for more detailedmolecular identification studies in the future.

Key words: RAPD; PCR; DNA isolation; Contaminations;Testate amoebae.

Introduction

Testate amoebae play an important role in food andenergy turnover of terrestrial and aquatic ecosystemsand are valuable bioindicators of natural and anthro-

© 1997 by GustavFischer Verlag

pogenic influences [e.g. 6, 9]. Moreover, it has beenshown that population growth as well as shell mor­phology of testate amoebae is highly variable depend­ing on the species and environmental conditions. Fur­thermore, this variability is clearly reproducible andeven reversible [10, 11, 12]. On the one hand, thispoints to a new and fascinating tool in bioindication,but on the other hand to serious taxonomical problems,because classification of closely related testate amoebaeis primarily based on these highly variable shell charac­teristics. Therefore novel approaches like using molec­ular markers are required to support available conven­tional morphological data in order to characterize clon­al populations or closely related taxa of testate amoebaein a quick and reproducible manner. Until recently [4],molecular techniques have not been applied to testateamoebae. Due to severe methodological difficulties inculturing testate amoebae, in most cases neither suffi­cient amounts of uniform genetical material nor se­quence information are available. However, at least oneof these are necessary for RFLP or PCR based analysis.Additionally, contaminated amoebae cultures, due topersistent digestive vacuoles and bacteria sticking to theamoebae shell and impossible to remove, further in­crease these problems. Thus the utilization of randomamplified polymorphic DNA markers, detected bypolymerase chain reaction (RAPD-PCR [14]), can cir­cumvent these obstacles.

RAPD-PCR based approaches have the advantage ofrequiring only small amounts of anonymous genomicDNA to obtain specific genetic fingerprints. Further­more, their competitive nature for priming in eukaryot­ic target DNA allows specific amplification and detec­tion even in a large excess of bacterial DNA [15]. Thusthis technique seemed to be the method of choice foranalyzing testate amoebae.

The objective of the present study was to develop aspecific and reproducible DNA isolation procedureallowing ~APD-PCR analysis from low amounts of

target DNA even in the presence of large amounts ofcontaminating foreign DNA, as it is typical for clonalcultures of terrestrial testate amoebae. Moreover, a pairof testate amoebae-specificprimers had to be designedfrom a cloned RAPD-DNA fragment to provide thebasis for further, more detailed genetic characterizationanswering still unsolved questions concerning taxono­my, ecology or reproduction of testate amoebae.

Material and Methods

Cultures of Testate Amoebae

The species cultivated were Cyclopyxis kahli Deflandre,clones G20 (Ulm, Germany) and H3 (Helsinki, Finland, var.cyclostoma Bonnet and Thomas); Cyclopyxis eurystoma (De­flandre), clones D29 (Ulm) and XP21 (Ochsenhausen, Ger­many); Euglypha strigosa (Ehrenberg), clone a4 (Ulm); andTrinema lineare Penard, clone MTI (Northwest of MexicoCity, Mexico). All amoebae originated from the humus layerof spruce stands. Clonal cultures of testate amoebae weregrown in petri-dishes containing 1% (w/v) agar (Merck,Darmstadt) covered with culture medium ("Volvic" mineralwater + 0.37 mM KH zP04: T lineare, E. strigosa) and werefed regularly on Enterobacter aerogenes Hormaeche and Ed­wards. Other cultures contained only "Volvic" and KHzP04

(Cyclopyxis spp.) and were provided regularly with the yeastSaccharomyces cerevisiae Hansen and shell building material.From time to time, one of the consecutive subcultures wasprovided with an extract prepared from humus layer. Moredetails regarding the cultivation techniques had been de­scribed [11]. All cultures were monitored regularly for visibleeukaryotic contamination and only clean material was usedfor subsequent DNA preparation. The maximum yield of tes­tate amoebae was about 1000 to 2000 cells per petri-dish.

Preparation of Template DNA

Four different DNA-extraction protocols [2, 8] weremodified and used in this study (Table 1).

Protocol 1. According to suggestions and protocols pro­vided by Dr. Steinbriick (Tubingen) and Dr. Bock (Miinchen)who used ciliate or human genomes, respectively, about 1000amoebae shells were washed in TE buffer (pH 7.6) andcrushed twice in a teflon homogenizer. DNA was extracted inTE buffer (50 ul) containing SDS (0.1 to 0,2% (w/v» andtreated with proteinase K (100}lg ml') [2, 8]. About 2 to 15}l1crude extract per 50 pl PCR was used. However, initial results

Molecular Identification of Testate Amoebae 193

indicated that due to the particular shell and culture proper­ties of the testate amoebae, improved DNA-extracting proto­cols had to be developed. The resulting protocol 2 was basedon a more complex cell lysis and debris removal, whereas pro­tocol 3 contained an additional shell crushing and cell lysisstep, and protocol 4 finally used individually prepared amoe­bae shells using the nuclei as a reference for PCR.

Protocol 2. About 1000-2000 cells were thoroughly rinsedin sterile culture medium, dried, subsequently homogenized,resuspended in 1 ml of 5xTE-buffer (50 mM Tris-Cl, pH 8.0;5 mM EDTA), and homogenized again. 100}l1 of 10% (w/v)SDS, 50}l1 of 2 M Tris, and 100}l1 of 0.5 M EDTA were added,and after incubation the solution was mixed with 125 ulof cold 3 M potassium acetate (pH 5.5). Treatment withRNase A, PCI extraction and DNA precipitation followed[2, 8]. Resulting DNA pellets were dissolved in 50-150 plwater and stored in aliquots at -20°C.

Protocol 3. Because testate amoebae have rigid shells andplasmatic properties, different shell crushing and cell lysismethods were tested. a) approximately 1000 amoebae werehomogenized with glass beads (75 to 150 }lm diameter,Sigma), treated with proteinase K as described [2] and 2 to15 }ll used as PCR template. b) 200 to 1000 amoebae werefrozen at -20°C to crack the shells and heated for 20 min at70°C to inactivate DNases. c) The frozen pellet was homo­genized. d) Frozen or fresh amoebae pellets were resuspendedin lysis buffer containing 350 ul of 5xTE, 35 pl of 10% (w/v)SDS, 18}l1 of 2 M Tris, and 35}l1 of 0.5 M EDTA.

Protocol 4. All lysis and incubation steps were carried out.. in the PCR tube. a) 20 to 100 amoebae shells were opened in­

dividually with a micromanipulator. The PCR reaction con­taining the opened amoebae shells, 2 to 10}l1 culture medium,0.5 volumes proteinase K (1 mg ml:"), and 25 ul of mineral oilwas incubated for 45 minutes at 55°C and inactivated for 10minutes at 95 0C. Microscopical analysis of treated testateamoebae on a heated slide confirmed the lysis of the cells andthe nuclear envelope. b) One to six single nuclei of Cyclopyx­is kahli and Euglypha strigosa were isolated using a microma­nipulator, transferred into the PCR tube and digested by pro­teinase K. However, the isolated nuclei stuck to the surfacesof all materials used and got lost frequently. Therefore the fol­lowing technique was developed: c) Euglypha strigosa cellswere washed in freshly prepared culture medium and thencarefully squeezed using a micromanipulator needle. As aconsequence, the plasmatic content passed the shell throughthe pseudostome except for the nugeUs, which adhered to theinner shell surface. Then, eitherrlle whole shell with the nu­cleus inside or a shell fragment ~ith the adhering nucleus wastransferred into the PCR tube and digested with proteinase Kas described above. The remaining, nucleus-free amoebaeplasma served as a control. d) Finally, 1 to 63 Euglypha stri-

Table 1. DNA - extraction protocols used for testate amoebae. G =amoebae shells ground with a teflon homogenizer; GB =shells homogenized with glass beads; I =amoebae shells opened individually; '; =amoebae shells crushed by freezing; PK =pro­teinase K digest; SDS =lysis buffer containing SDS; PCI =debris removal by phenol! chloroform/ isoamylalcohol steps.

Protocol material shell crushing lysis debris removal cells

1 shells G SDSandPK 10002 shells G SDS PCI 1000 -20003 shells G,GB,'; SDSorPK 200-10004 nuclei I PK 1-63

194 M. Wanner, J. M. Nahring, and R. Fischer

gosa cells were split into two parts only one of which con­tained the clearly visible nucleus. Thus two PCR reactionswere carried out in parallel, both containing amoebae shellsand plasmatic contents, but only one reaction contained theamoebae nuclei. Nucleus-free amoebae fragments as well aspure culture medium served as controls (freshly prepared ordirectly from the respective culture with agar fragments orother amoebae-freeculture debris).

RAPD-PCR Amplification and AnalysisThe amplification conditions used was a slightly modified

version of Williamset al. [14].Briefly, the reactions were performed in a volume of 25 pl

containing l x PCR buffer (Goldstar, Eurogentec), 100)lM ofeach dNTP, 1.5 mM of MgClz, 0.2 to 0.8)lM of 10mer oligo­nucleotide primers (Operon, OPG 1-20),various amounts ofgenomic DNA (depending on the extraction protocol; referto Table 1), and 0,5 units ofTaq DNA polymerase (Goldstar,Eurogentec). Amplification of DNA was conducted in athermocycler (TCV516, Vers GmbH, Hannover) using thefollowing PCR conditions: denaturation for 5 min at 94°C;45 cyclesconsisting each of 1 min at 36°C, 2 min at 72°C and1 min at 94 °C; and a final extension step of 1 min at 36°Cand 10 min at 72 °C, using the quickest available transitionsbetween each step. Aliquots of amplified DNA fragmentswere subjected to agarose gel electrophoresis in 1,2% or1,5% (w/v) TBE-gels containing ethidium bromide (0.5pg/ml).

Cloning of a RAPD-PCR Fragment for Designof Testate Amoebae-Specific PCR Primers

Cloning procedures were carried out as described [2, 8].To show the specificity of DNA-extraction protocol4d andto design a probe for identification and characterization ofsingle contaminated amoebae cells, a specific 400 bp DNAfragment from Euglypha strigosa (amplified twice using 2and 9 amoebae nuclei, primers OPG 4+5) was purified usingthe QIAEX protocol. The PCR product was ligated into thepUC 18 plasmid (Sma I digested, BAP treated) and subse­quently transformed into Escherichia coli (strain HB 101).Recombinant plasmids were identified by TELT miniprepa­ration [2] and high quality DNA for sequencing was ob­tained by alkaline lysis. Sequencing was performed accord­ing to the dideoxy method [2] on an.Al.Fcauromared DNAsequencer using 2 pUC l S-specific fluorescent labelledprimers (Pharmacia) binding to both DNA strands. A re­gion of 140 base pairs gave identical sequences in both theplus as well as the minus strand which was used for design­ing two primers (P1, P2) for specific amplification of this140 bp DNA region of Euglypha strigosa, clone a4 (P1: 5'­ACGAATTTAAACTAACTCAA-3' and P2: 5'-GGGT­CTCATAGGTAGTCCTC-3'; TIB MOLBIOL). PCRconditions were as follows: denaturation for 5 min at 94°C;35 cycles consisting each of 30 sec at 58 °C, 1 min at 72 °Cand 30 sec at 94°C; and a final extension step of 10 min at72°C, using 5 pmoles of each primer and 2 pl template (5nuclei of Euglypha strigosa) in a 25 pl reaction. Aliquots ofthe amplified DNA fragment were analyzed on 2% (w/v)agarose gels, using the PCR amplified 150 bp region of thepUC 18 polylinker (primers M13, M13RS; Boehringer,Mannheim) as a reference.

Results

The establishment of RAPD-PCR based finger­prints for the characterization of related taxa of testateamoebae first required the development of a reliableDNA-isolation procedure. If paired primers were usedin RAPD-PCR, substantially more reproducible fin­gerprints were obtained as compared to single randomprimers. This was tested for different primer combina­tions (OPG 2+3, 4+5, 5+6, 6+7,10+18,17+18) and canbe attributed to the competitive nature of the amplifica­tion reaction and the reduced background [13, 15]. Be­cause of the difficult cultivation conditions, it was notpossible to omit the food organisms, Enterobacteraerogenes or Saccharomyces cerevisiae. Exponentiallygrowing amoebae usually contain large amounts offood organisms, while starving, "clean" amoebae growextremely slowly, encyst themselves, or die. Substrac­tion of food organism-specific bands (which were themost reproducible ones) resulted in only 1 to 3 repro­ducible bands in most cultures of the same amoebaclone even when using different DNA concentrationsas a PCR template (Fig. 1).

Although these primary data indicated that protocol2 seemed to be suitable for characterizing clonal cul­tures of testate amobae by RAPD-PCR (protocol 1produced less consistent results), further experimentsshowed several ambiguities. For .exarnple yeast DNAplus "amoeba"DNA resulted in the same pattern aspure yeast DNA wheras an equal mixture of bacterialand "amoebal" DNA produced only "amoeba]" DNApatterns (perhaps the bacterial genome is considerablysmaller than the "amoebae" genome [14, 15]). Addi­tionally, the DNA preparations of two different clonesof the same taxon (c. eurystoma, clones D29 and XP21)resulted in nearly identical patterns. These findingswere quite unexpected, since clones D29 and XP21have had significant differences in shell size since sever­al years ago. More surprising, the same result could beobtained with template containing the amoebae as wellas with amoeba-free template, containing only the cul­ture medium (Fig. 2). Finally, the DNA amounts of theextracted 1000 to 2000 amoebae cells were about 0.5 to3.0 pg, which is more than expected. Assuming that anamoeba cell contains comparable amounts of DNA asmost other protozoan and animal cells do, only 1 to2 ng DNA should be obtained.

These findings led to the following assumptions: 1)Free amoebae DNA released from dead cells protectedby shell building material had been amplified. 2) Due tothe solid amoeba shell (or other morphological struc­tures), no amoebae-DNA but another highly abundantDNA of yet unknown origin different from the knownfood organism and not microscopically visible as aeukaryotic co~tamination had been amplified. There-

fore it is conceivable that bacteria or small eukaryotes,transferred to the corresponding subcultures over yearsare either attached to the shell surface or located insidethe amoebae.

To elucidate these assumptions, the efficiency ofprotocol 2 with respect to amoeba lysis was investigat­ed. Microscopical examination of thoroughly homoge­nized amoebae showed numerous undamaged shells.Therefore other DNA-extraction techniques as de­scribed in protocol 3 had to be applied to open amoebashells more efficiently. However, in most cases similarresults as in protocol 2 were obtained, or the DNA-pat­tern of the medium plus a few additional bands weredetected, although these bands were not reproducible,stressing the contamination problem(s). This was cor­roborated by peR results using 1) amoebae cells, 2)culture supernatant without solid particles, and 3) cul­ture sediment without living amoebae with decom­posed shell fragments, which gave almost identical pat­terns (e.g. clone H3, Fig. 2). Even if Enterobacter aero­genes was added in surplus to cultures of Euglyphastrigosa (clone a4), obtained DNA-fragment patterns ofthe culture medium differed from the Enterobacterstandard (Fig. 4). These observations led to the assump­tion that some DNA contamination specific for thecorresponding amoebae clone - different from the foodorganism - had been amplified. To eliminate prokary­otic contamination in amoebae cultures, different an­tibiotics were administered at various concentrations(neomycin sulfate, nalidixic acid, kanamycin disulfate),but no consistent results were obtained so far, mainlybecause testate amoebae react often more sensitive tothe tested drugs than the targeted contaminating organ­isms,

To conclude, the problem of low amounts of amoe­bae DNA mixed with large amounts of contaminatingDNA of unknown origin required either an improvedculture technique or another modified DNA isolationprocedure. Because the culturing of terrestrial testateamoebae is often sophisticated and time-consuming, amore suitable DNA-isolation procedure had to be de­veloped first. Isolated amoebae nuclei prepared di­rectly from living cells as depicted in protocol 4seemed to be the only available contamination-freeDNA source, because the amoeba plasma as well asthe shell surface contains numerous small organisms.This approach enabled us to prepare alleast 5 amoe­bae cells within 30 minutes even without a microma­nipulator to provide sufficient template for a routineDNA amplification.

However, single isolated nuclei may contain toolittle DNA amounts for generating reproducibleRAPD-patterns [15] (see also [3]), thus the followingexperimental design was used to test reproducibilityand to eliminate possible artifacts:

Molecular Identification of TestateAmoebae 195

- Euglypha strigosa, divided into numerous cultures ofthe same clone, was chosen because the relatively largenucleus is easily visible within the living, unstainedshell (Fig. 7).- Different amounts of DNA (1 to 63 isolated nucleiper 25 pi reaction) were used to investigate the repro­ducibility of the amplification reaction, and pairedprimers were chosen to increase sensitivity as well asspecificity and to reduce the background [13, 15].- Nucleus-free amoeba plasma and shell fragments,culture medium (freshly prepared or taken from the re­spective culture), and the food organisms served as con­trols.- Specific oligonucleotides derived from sequenceanalysis of an amplified and cloned DNA-fragmentwere designed as taxon-specific primer pairs to detectonly nucleus-containing template, but not the abovementioned controls or other amoebae taxa.

Referring to protocol 4, the separation of singleamoeba cells into a nucleus-free and a nucleus-contain­ing part was the most promising approach easily usablewithout a micromanipulator. Different primer combi­nations of the OPG-set (Operon) had been tested, butso far only the two combinations OPG 17+18 andOPG 4+5 yielded reproducible patterns with respect toamoebae-nucleus-specific bands (Figs. 3-5). The DNApattern of the nuclei-containing sample showed alwaysone (OPG 4+5) or two (OPG 17+18) additional andtherefore nucleus-specific bands, which became moreintense after increasing the primer concentration from5 to 20 pmoles by using protocol4d (Figs. 4, 5). The re­maining amplified DNA fragments were detectable inall samples, namely the nucleus-containing sample, thenucleus-free plasma and shell particles containing sam­ple, and the culture medium. All DNA-patterns wereclearly distinguishable from the food organism En­terobacter aerogenes, which was added to the tested cul­tures in surplus (Fig. 4). Even in cultures highly contam­inated by numerous small encysted protists (e.g. smallnaked amoebae or flagellates, Fig. ));'it was still possibleto obtain the before mentioned nucleus-specific band,even if various numbers of nuclei were used (Fig. 4) ordifferent cultures of the same amoeba clone were used(Fig. 4) or different cultures of the same amoeba clonewere kept under different conditions (Fig. 3).

As mentioned above, the DNA amount preparedseemed to be far too low to obtain reproducible patterns.Furthermore, the resulting one or two apparently specif­ic bands were not sufficient for a clonal characterizationof testate amoebae. To exclude possible artifacts and totest additional DNA analysis methods, the nucleus-spe­cific DNA fragment generated by the primer pair OPG4+5 was cloned and sequenced to design two testateamoebae specific 20mer primers. These oligonucleotidesproved to be highly specific and were able to detect sin-

196 M. Wanner, J. M. Nahrinq, andR. Fischer

gle copy genes within a large amount of contaminatingDNA, as shown in Fig. 6. Only nucleus-containing sam­ples showed the expected 140 bp band but not the amoe­bae plasma or shell, the culture medium or other taxa oftestate amoebae. The DNA fragment initially generatedby RAPD-PCR could be verified in a subsequent PCRusing the specific primers (PI, 2) resulting in an amoeba­specific band as well. This result was also reproduciblewhen only very low amounts of target DNA (i.e. oneamoeba nucleus) was used in the presence of multiplecontaminations during PCR amplification.

Molecular Identification of Testate Amoebae 197

Discussion

RAPD markers enable fingerprinting of many or­ganisms to study inter- and intraspecific variation ofclosely related species. However, so far this concept hasnot yet been applied to testate amoebae. Although thisapproach seemed feasible using established methods forpreparing template DNA, amoeba clone-specific DNApatterns resulting from intial RAPD-PCR based analy­sis could not be obtained or were ambiguous because ofinevitably contaminated cultures.

Fig. 1. RAPD-PCR based analysis of clonal cultures of testate amoebae. DNA was prepared using protocol 2 and subjected toPCR using different pairs of primers (only OPG 10+18 are shown and analyzed on a 1.5% agarose gel). Each amoebae-clonewas subcultivated three times (a-c) for 1-3 weeks, depending on culture growth to obtain about 1000-2000 cells. Lane 1: lamb­da DNA digested with Pst I (in bp). Lanes 2-4: clone G20a--e (Cyclopyxis kahli), all cultures fed with the yeast Saccharomycescerevisiae. Lanes 5-7: clone D29a--e (Cyclopyxis eurystoma), cultures a and b starved, i.e. without the food yeast, while culture cwas supplemented with yeast in surplus. Lanes 8-10: clone MTla-c (Trinema lineare), and lanes 11-13: clone a4a-c (Euglyphastrigosa), both taxa fed with Enterobacter aerogenes. Enterobaeter aerogenes (E) (lane 14) and Saccharomyces cerevisiae (5) (lane15) served as controls. After subtracting the "food specific bands", only one to three consistent bands remained which are pre­sumably taxon-specific (marked with dots).

Fig.2. Concurring DNA fragment patterns derived from amoebae and the respective amoebae-free culture medium. Clone H3(Cyclopyxis kahli cyclostoma) amplified with primers OPG 2+3 (1.2% agarose gel; only primers OPG 2+3 are shown, but simi­lar results were obtained using the other above mentioned combinations). Lane 1: washed testate amoebae ('I), lane 2: culturemedium supernatant without particles (M), lane 3: culture medium sediment containing decomposed amoebae shells but noliving amoebae cells (5), lane 4: lambda-Pst 1.

Fig. 3. Six different cultures (a-f) of the same clone of E. strigosa (a4), supplemented with Enterobaeter aerogenes in surplus andpartly contaminated with small flagellates (1.2% agarose gel). From each culture, five individuals were taken and divided into anucleus-free (P) and nucleus-containing (N) sample and subjected to RAPD-PCR with primers OPG 4+5. Only the nucleus­containing samples showed a specific 400 bp DNA fragment (marked with a dot).

Fig. 4. Euglypha strigosa, clone a4. Different primers and DNA concentrations (nuclei) were subjected to RAPD-PCR and an­alyzed on a 1.5% agarose gel. Nucleus-containing samples always revealed either two (primers OPG 17+18) or one (primersOPG 4+5) additional, taxon-specific bands (marked with dots) clearly distinguishable from the amoeba plasma, external con­taminations, culture medium or food organism. Lane 1: lambda Pst 1. Lanes 2-7: primers OPG 17+18. Lane 2: five nucleus-con­taining shell fragments (5N), lane3: the respective nucleus-free cell fragments. Lanes 4, 5: repeated amplification with anotherculture, lane 6: "Volvic", freshly prepared culture medium, lane 7: Enterobacter aerogenes (E) which served as food. Lanes 8-15:primers OPG 4+5, different amounts of amoebae nuclei were used as template. Lanes 8, 9: a single amoeba cell was divided intoa nucleus-containing (IN) and nucleus-free (IP) part. Lanes 10-13: Ten amoebae shells were used as template. "'I": completecells; "N" nuclei-containing cell fragments; "P": nuclei-free cell fragments; "C": culture medium containing 100 flagellate cysts.Lane 14: Forty amoebae nuclei (40N) were used for PCR, however, the primer concentrations were reduced to 0.2 pM, resultingin a faint but specific band (see Fig. 5). Lane 15: Enterobacter aerogenes (E).

Fig. 5. Reproducibility of RAPD-PCR using 63 isolated nuclei of Euglypha strigosa. PCR amplifications were carried out with0.2 instead of 0.8 pM primers OPG 4+5 and analyzed on a 1.2% agarose gel. The nucleus-derived band is faint but consistent.Lane 1: lambda Pst I, lane 2: 63 nuclei adhering to shell fragments (63N), lane 3: the culture medium that had been used to pre­pare the 63 individuals of lane 2 containing cell plasma and bacteria (P). Lane 4: amoebae-free culture medium of the respectiveculture (M).

Fig. 6. Two specific 20mer primers (PI, 2), derived from the cloned nucleus-specific PCR band using arbitrary primersOPG 4+5 detect only nuclei from Euglypha strigosa (2% agarose gel). Lane 1: lambda Pst I, lane 2: PCR amplified pUC 18polylinker region (150 bp standard). Lanes 3, 5, 7: five nuclei from three different cultures (Na-c); lanes 4, 6, 8: the respective nu­cleus-free cell fragments. Lanes 9-14: different taxa of testate amoebae and the food organisms showed no amplification with theEuglypha-specific primers. Lanes 9,10: clone MTl, lane 11: clone H3, lane 12: clone XP21, lane 13: yeast (5), lane 14: bacteria(E), lane 15: PCR amplified pUC 18 polylinker region.

Fig. 7. Euglypha strigosa, clone a4. The large nucleus is visible within the living, unstained casing (scale bar = 20 urn).N = nucleus; P = cell plasma. The amoeba shell was divided into a nucleus-free and nucleus-containing part (protocoI4d).

198 M. Wanner, J. M. Nahrinq, and R. Fischer

This severe problem is corroborated by a recentpublication on the fine structure of the euglyphid tes­tate amoeba Assulina muscorum [1]. Using scanningand transmission electron microscopy, this study cor­roborates on the one hand that bacteria are attached tothe shell surface, or that digestive vacuoles contain bac­teria. On the other hand, it was shown that the cyto­plasm of Assulina muscorum occasionally containssmall naked amoebae that appear to be commensals.

Pro- or eukaryotic contaminations are usually notdetectable by routine light microscopy particularly ifthey are located inside the amoebae shell or tightly at­tached to the shell surface. In order to surmount theseproblems, an improved method for isolation of tem­plate DNA for subsequent RAPD-PCR was devel­oped. Isolated nuclei treated by proteinase K seemed tobe the method of choice for RAPD-PCR based analysisof clonal cultures from testate amoebae.

Another technique to differentiate between isolateswithin a species is the combination of the polymerasechain reaction with restriction fragment length poly­morphism analysis (PCR RFLP), as recently presentedfor estimation of genetic variation among soil isolatesof the ciliate Colpoda inflata [5]. However, this tech­nique is at least very difficult if not impossible to applyfor testate amoebae which may harbor eukaryotic com­mensals. However, combining both approaches, e.g.applying PCR RFLPs on target DNA for which nucle­ar origin is checked by a taxon-specific probe will allowmore detailed genetic research on testate amoebae.

The results presented indicate that already a few oreven single nuclei (1 to 63) of amoebae cells digested byproteinase K provide adequate DNA template amountsfor RAPD-PCR and the resulting DNA fragment(s) canbe assigned clearly to the respective clone of testateamoebae. This result was additionally corroborated bycomparing sequence data of different amplification reac­tions using 2 or 9 nuclei as a template (data not shown).This may be surprising, because usually much moreDNA is required for reproducible PCR [15]. However,it was also observed that the DNA-pattern obtained by20 ng genomic bacterial DNA (per 25 pi reaction) is re­producible even with 1000-fold less DNA (20 pg) in thecase of procaryotic genomes [3]. Furthermore, usingspecific primers together with more stringent PCR con­ditions appeared to be sensitive enough for amplificationof templates derived from a single cell or genome [7].

Our results indicate that reproducible peR patternsdepend highly on the kind of organism of interest, theamount of template DNA and its genome organization.Fine tuning these methods as well as generating morespecific primer combinations should enable repro­ducible DNA analysis even from field samples (meth­ods in [6, 9, 11]) of testate amoebae thus avoiding time­consuming culture techniques.

Along this line we developed optimized techniquesfor template DNA preparation which enabled us toamplify one or two specific bands from a single nucleusof testate amoebae even in the presence of highamounts of contaminating DNA. However, these dataare not sufficient for a detailed molecular fingerprintanalysis. This will require the development of addition­al specific primer combinations to generate repro­ducible and unambiguous fingerprints for comparisonbetween and within closely related species and moreextensive sequencing of identified PCR products.

Our data clearly demonstrate for the first time a suc­cessful RAPD-PCR-based identification of testateamoebae using template DNA prepared from only afew nuclei or even a single nucleus in the presence ofhighly contaminated target DNA. Futhermore, cloningand sequencing of a testate amoebae-specific RAPD­PCR product enabled us to design a pair of highly spe­cific PCR primers as a first step towards a more detailedmolecular analysis of testate amoebae.

Acknowledgements: We are grateful to Dr. S. Bock(Klinikum der Universitat Miinchen) and to Dr. G. Stein­bruck (Universitat Tubingen) for their hospitality and adviceconcerning PCR. We would also like to thank Ms. S. EBer fortechnical assistance and two anonymous referees for provid­ing helpful comments. Dr. R. Meisterfeld (RWTH Aachen) isacknowledged for his help in culturing testate amoebae andproviding Figure 7.

References

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Address for correspondence: Manfred Wanner, StaatlichesMuseum fur Naturkunde Garlitz, PO box 300154, D -02806Garlitz, Germany. Fax +49 3581401742