Journal of Invertebrate Pathology 88 (2005) 257–260

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Short communication

DNA isolation from museum and type collection slides of microsporidia �

Miroslav Hylin a,¤, Jaroslav Weiser b, Miroslav Oborník c, Jilí Vávra a,c

a Department of Parasitology and Laboratory of Electron Microscopy, Faculty of Science, Charles University, Prague, Czech Republicb Institute of Entomology, Academy of Sciences of the Czech Republic, Beské Bud jovice, Czech Republicc Institute of Parasitology, Academy of Sciences of the Czech Republic, Beské Bud jovice, Czech Republic

Received 4 October 2004; accepted 16 February 2005Available online 23 March 2005

Abstract

DNA from 19 species of microsporidia was isolated and ampliWed from infected host tissue that were originally prepared betweenthe years 1946 and 1996. The smears, on glass microscope slides, were either Giemsa-stained or unstained. Methanol-Wxed, Giemsa-stained smears proved to be suitable for DNA isolation; DNA was ampliWed from only two of 14 unstained slides. The isolated DNAwas successfully ampliWed in PCRs using small subunit and large subunit rDNA primers and sequenced. The high eYciency of DNAisolation demonstrates the usefulness of archival and type collection slides for some molecular biology and molecular taxonomystudies. 2005 Elsevier Inc. All rights reserved.

Keywords: Microsporidia; Lepidoptera; Trichoptera; DNA isolation; Type collection; Phylogeny

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1. Introduction

Microsporidia have been collected, described, andclassiWed for more than 100 years. Historically, thedescriptions of genera and species were based on charac-teristics of spores and developmental forms that couldbe observed under light microscopy in fresh tissuesmears and Giemsa-stained tissue smears, as well ascharacteristics of infection and host and tissue speciWc-ity. The deposition of type slide preparations in individ-ual or museum collections has been generally required tovalidate descriptions of genera and species. In the late1960s, ultrastructural studies added to the number ofstructural characteristics available to identify and

� Presented at the NATO Advanced Research Workshop “EmergentPathogens in the 21st Century: First United Workshop on Microspor-idia from Invertebrate and Vertebrate Hosts,” Beské Bud jovice,Czech Republic, July 12–15, 2004.

¤ Corresponding author.E-mail address: mirekhylis@volny.cz (M. Hylin).

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0022-2011/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2005.02.004

describe microsporidia and older descriptions that didnot include ultrastructural characters were considered tohave less value. New species and genera were, in manycases, described mainly on ultrastructural characterswith little eVort to compare critically the new Wndingswith information preserved in old type slide collections.This situation has escalated in the present era, whenmolecular data can be used successfully in microsporidiaclassiWcation. However, in this study we show thatmolecular biology methods oVer the opportunity toextract valuable information from old slide preparationsand thus to critically compare and evaluate previousmicrosporidia descriptions with those currently beinggenerated.

2. Materials and methods

We selected 40 permanent slides from the microspo-ridian slide and type slide collection of one of the

258 M. Hylin et al. / Journal of Invertebrate Pathology 88 (2005) 257–260

authors (J.W.). These slides were prepared during theyears 1946–1996 and represented 25 microsporidianspecies. Twenty-six of the smears were stained withGiemsa and 14 were unstained smears. The stainedsmears were prepared using standard methods (Undeenand Vávra, 1997), which included smearing the infectedhost tissue on a slide, allowing the smear to dry, Xood-ing the smear with undiluted methanol, and allowing itto dry again. Various brands of Giemsa stain were usedfor staining. Four of the smears were mounted in theCedax mounting medium and several smears were sul-lied by immersion oil. Smears covered by mountingmedium or remnants of immersion oil were cleanedwith xylene before isolation. Each slide made ready forisolation was carefully inspected under 400£ lightmicroscopy to ascertain that only a single microsporid-ian species was present (using spore morphology) andto estimate the number and type of developmentalstages in the smear. The smear material was liberatedby applying several drops (about 80 �l) of TE buVer(10 mM Tris, 1 mM EDTA) pH 8.0 or BuVer No. 1from the DNeasy Tissue Kit (Qiagen, Germantown,Maryland, USA) to an area of approximately18 £ 18 mm of the slide and allowed to stand for 5 min.The suspension obtained was wiped from the slide witha clean Eppendorf pipette tip into a 1.5-ml Eppendorftube and was shaken with an equal volume of 0.5 diam-eter glass beads for 20 s at maximum speed in the Mini-beadbeater (Biospec Products). DNA was isolatedeither by the DNeasy Tissue Kit (Protocol for AnimalTissues) or by the phenol/chloroform method (Baker etal., 1994) in which TE buVer instead BBS buVer wasused. Isolated DNA was examined by electrophoresisand used for PCR (95 °C for 2 min initial denaturation,30 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for2 min, and 72 °C for 10 min termination cycle) withSSU and LSU rDNA primers (ss530f GTG CCA GCAGCC GCG G; ss1047r AAC GGC CAT GCA CCACC; ss1537f GAA CCA GCA GCA GGA TCA TAA;ls26f GCA TAT CAA TAA GCG GAG GAA AAG;and ls580r GGT CCG TGT TTC AAG ACG G)

(slightly modiWed primers of Weiss and Vossbrinck,1999). The PCRs were carried out in 25 �l tubes with50–100 ng of each DNA isolation, 25 pmol of eachprimer (in set ss530f:ss1047r; ss1537f:ls580r; andls26f:ls580r), 1 U Taq polymerase (Takara Bio, Otsu,Shiga, Japan) and buVer/dNTP (TaKaRa) accordingTaKaRa set protocol. PCR products were puriWed,cloned (TOPO TA Cloning Kits, Invitrogen, Carlsbad,California, USA), and sequenced on an automaticsequencer (Beckman CEQ 2000 XL).

3. Results and discussion

We obtained DNA suitable for PCR ampliWcationand sequencing from 17 of 26 methanol-Wxed andGiemsa-stained slides. DNA was isolated from only twounstained smears; no DNA was obtained from theremaining 12 unWxed, non-stained smears. The isolatedDNA (representing microsporidian DNA and that ofhost tissues, bacteria, and other contaminants) was suc-cessfully ampliWed using microsporidia speciWc primers(Fig. 1). Partial sequences of SSU and LSU rDNA of upto 594 bp were obtained.

There was no obvious correlation between the successof the DNA isolation, the age of the smear or the micro-sporidian species involved. DNA was successfully iso-lated from slides with smears of microsporidia fromboth the terrestrial and aquatic environments. Terres-trial microsporidia included 11 species from Lepidop-tera, representing (according to the original label) thegenera Nosema, Octosporea, Larssoniella, and Endoreti-culatus. Aquatic microsporidia included six species fromTrichoptera, representing the genera Glugea, Thelohania,Cougourdella, Pyrotheca, Stempellia, and two speciesfrom crustacean Gammarus pulex L., representing generaThelohania. There was no correlation between successfulDNA isolation and the host species and tissue fromwhich the smear was made. The oldest isolation wasmade from a smear of Thelohania plectrocnemiae pre-pared in 1946 (Table 1).

Fig. 1. (A) Agarose gel electrophoresis of DNA isolated from slides as compared to DNA isolated from fresh spores. Lanes 2, 10, and 11: DNA iso-lated from slides. Remaining lanes: DNA isolated from fresh microsporidian spores. Lane S, standard. (B) Agarose gel electrophoresis of PCR ampli-Wcation products from microsporidian DNA isolated from slides. Lanes 1–6, lsu rRNA primers ls26f:ls580r. Lanes 7–12, ssu rDNA primersss530f:ss1047r. Lane S: standard. Asterisk required amplicons.

M. Hylin et al. / Journal of Invertebrate Pathology 88 (2005) 257–260 259

Among the factors inXuencing DNA isolation fromold slides, the amount of material present on the slide,the microsporidian stages present, and slide preparationand handling were considered. The amount of parasitematerial in the smears was less important than we antici-pated. For example, only 1–2 spores per microscope Weldat 400£ magniWcation (<500 spores/�l) were observed ina smear of Larssoniella resinellae Weiser and David,1997, yet suYcient DNA was obtained for the PCR.Developmental stages and spores were present in allsmears. We were not able to determine whether theDNA was preferentially isolated from the spores or fromthe developmental stages; however, very low qualityDNA was produced from a smear of Nosema sp. fromLymantria monacha L. with numerous developmentalstages but only a few spores present. Conversely, Wveother smears of Nosema sp. microsporidia containingmostly spores yielded the highest DNA quantity of allisolations. It is evident that methanol-Wxed, Giemsa-stained smears gave the best results, however, not allsuch smears allowed DNA isolation. The reason for thefailed isolations is not known, but the methods by whichindividual smears were originally prepared and handled

(e.g., time period between the smearing, Wxation, andstaining steps, length of time the methanol Wxation wasperformed, storage conditions of individual slides, etc.)might be involved. Smears that were not stained provedmostly unsuitable for DNA isolation. The two smears(out of 14) from which DNA was isolated were excep-tions, but it is possible that these smears were methanol-Wxed and left unstained.

Although the DNA obtained after isolation from oldslides is fragmented, the fragments were usually largerthan the 540 bp needed for distinguishing individual spe-cies. Other favourable factors are the existence of multi-ple rDNA copies in microsporidia and the highspeciWcity of SSU and LSU rDNA primers. The higheYciency of DNA isolation achieved demonstrates thebeneWt of archival and type collection slides for sometypes of molecular taxonomic studies.

Acknowledgment

Thanks are expressed to Dr. Leellen F. Solter, Centerfor Economic Entomology, Illinois Natural History

Table 1Data and characters of slides used for DNA isolation and isolation results

G, Giemsa-stained slides. Uf, UnWxed, unstained slides.a Slides mounted in Cedax.b Slides sullied by immersion oil.c DNA isolated by the phenol/chloroform method, from all other slides DNA was isolated using the DNeasy Tissue Kit (Qiagen).

Original slide label Number and characters

Microsporidian Date, Geographic location Host Slides used Positive isolation

Successful isolationNosema sp. 1990, Varna, Bulgaria Lymantria dispar 1 (1Gb) 1Gb

Nosema kovacevici 1975, Printina, Yougoslavia Euproctis chryssorhoea 1 (1Gb) 1Gb

Nosema sp. 1962, Roztoky, Czech Republic Operophtera brumata 1 (1Ga) 1Ga

Nosema tortricis 1956, Lednice, Czech Republic Tortrix viridana 3 (2Gb,1Uf)c 1Gb,c

Nosema murinanae 1956, Keilovka, Slovakia Choristoneura murinana 1 (1Uf b) 1Uf b

Nosema sp. 1986, Zbiroh, Czech Republic Lymantria monacha 1 (1Gb) 1Gb

Octosporea viridanae 1956, Lednice, Czech Republic Tortrix viridana 1 (1Gb) 1Gb

Thelohania sp. 1968, Pr honice, Czech Republic Tortrix viridana 1 (1Ga) 1Ga

Larssoniella resinellae 1990, Tlebok, Czech Republic Petrova resinella 2 (1Gb,1Uf) 1Gb

Endoreticulatus sp. 1962, Czech Republic Pieris brassicae 1 (1Gb) 1Gb

Endoreticulatus sp. 1953, Czech Republic Mammestra brassicae 1 (1Gb)c 1Gb,c

Glugea trichopterae 1976, Doubravka, Czech Republic Polycentropus Xavomaculatus 2 (1G,1Uf) 1GThelohania plectrocnemiae 1946, Chot bol, Czech Republic Plectrocnemia geniculata 2 (1Ga,1Uf) 1Ga

Thelohania lairdi 1962, Doubravka, Czech Republic Polycentropus Xavomaculatus 3 (3Uf b) 1Uf b

Cougourdella polycentropi 1963, Doubravka, Czech Republic Polycentropus Xavomaculatus 2 (1G,1Uf) 1GPyrotheca sp. 1976, Kokolín, Czech Republic Hydropsyche pellucidula 2 (1G,1Uf) 1GStempellia sp. 1973, Kokolín, Czech Republic Polycentropus Xavomaculatus 1 (1Ga) 1Ga

Thelohania sp. 1996, Eberswalde, Germany Gammarus pulex 1 (1G) 1GThelohania sp. 1996, Prokop.údolí, Czech Republic Gammarus pulex 1 (1G) 1G

Unsuccessful IsolationNosema cacoeciae 1956, Ban.Ktiavnica, Slovakia Choristoneura murinana 2 (1G,1Uf) 0Larssoniella resinellae 1989, Liar, Slovakia Petrova resinella 2 (1Gb,1Uf) 0Endoreticulatus Wdelis 1975, Cuba Leptinotarsa decemlineata 3 (2Gb,1Uf) 0Pleistophora dixippi 1975, France Bacillus rossi 2 (1G,1Uf) 0Pleistophora sp. Krb, Czech Republic Yponomeuta malinellus 1 (1Ga ) 0Pleistophora sp. Ukraine Cydia pomonella 2 (2G) 0

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260 M. Hylin et al. / Journal of Invertebrate Pathology 88 (2005) 257–260

Survey, Champaign, IL, USA for her help in preparingthe article.

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Weiss, L.M., Vossbrinck, C.R., 1999. Molecular biology, molecularphylogeny, and molecular diagnostic approaches to the microspor-idia. In: Wittner, M. (Ed.), The Microsporidia and Microsporidio-sis. AMS Press, Washington, DC, pp. 129–171.