Molecular Ecology Notes (2004)

4

, 577–579 doi: 10.1111/j.1471-8286.2004.00740.x

© 2004 Blackwell Publishing Ltd

Blackwell Publishing, Ltd.

PRIMER NOTE

Polymorphic microsatellite loci for the trematode

Diplostomum pseudospathaceum

THORSTEN B . H. REUSCH, GISEP RAUCH and MARTIN KALBE

Max Planck Institut für Limnologie, August-Thienemann Strasse 2, 24306 Plön, Germany

Abstract

We present primers for five polymorphic microsatellite loci in the eye fluke

Diplostomumpseudospathaceum

(Trematoda), a widely distributed parasite with a complex life cycle usedas a model for parasitology and fish immunology. The loci were identified using a GA/CT-enriched genomic library by subtractive hybridization with magnetic particles. All five lociwere highly polymorphic, displaying 17 to 61 alleles and heterozygosities ranging from 0.53to 0.92. We isolated populations of parasites within the first (snail) and second (fish) inter-mediate host and found small but significant genetic differentiation (

F

ST

= 0.012) between thetwo life stages of the parasite.

Keywords

:

genetic marker, parasite, three-spined stickleback, trematode

Received 24 March 2004; revision accepted 16 June 2004

The eye fluke

Diplostomum pseudospathaceum

Niewiadomska1984 is a widely distributed parasite of the eye lenses offreshwater fish. This trematode is a known cause of diseasesand mortality both in commercial fish farming and naturalpopulations (Chappell

et al

. 1994). It is therefore widely usedas a model for the epidemiology, ecology and physiologyof digenean trematodes. The life cycle of

D. pseudospathaceum

involves snails (

Lymnaea stagnalis

or

L. palustris

) as first inter-mediate hosts and a wide range of fish species as secondintermediate hosts. Infected fish are eaten by the parasite’sdefinitive hosts, mainly gulls, and sexual reproductiontakes place.

For parasite species with life stages within hosts, it isparticularly useful to develop species-specific primers thatdo not amplify co-occurring host tissue (Binz

et al

. 2000).Here, we report the development of five species-specificprimers for

D. pseudospathaceum

. Correct taxonomic deter-mination is a critical issue within trematodes and thegenus

Diplostomum

in particular. The identification of

D. pseudospathaceum

was performed under phase contrastmicroscopy (

×

1000) of the cercariae, the second larval stagepenetrating a fish, according to Niewiadomska & Kiseliene(1994).

For the genomic library, 100 parasites were isolated fromthe eye lenses of several experimentally infected sticklebacks

(

Gasterosteus aculeatus

). DNA was extracted using the DNeasytissue kit (Qiagen). We used a hybridization selection methodfor enrichment with GA/CT motifs (Kijas

et al

. 1994). Weligated short, self-complementary oligonucleotides to therestriction site of the target DNA fragments (Edwards

et al

.1996) after restriction of the total DNA with

Sau

3A. Double-stranded linker molecules with a 5

′

-GATC overhang wereproduced by denaturing and snap-cooling the oligonucle-otides linker F (5

′

-TTGCTTACGCGTGGACTC-3

′

) andlinker R (5

′

-GATCGAGTCCACGCGTAAGCAA-3

′

). Weamplified 5

µ

L of the ligation product in a 50-

µ

L poly-merase chain reaction (PCR) containing 0.1% bovine serumalbumin, 250

µ

m

of each dNTP, 0.4

µ

m

primer (only linkerF), 1.5 m

m

MgCl

2

, 1

×

PCR buffer (Promega) (10 m

m

Tris-HCl, 50 m

m

HCl, 0.1% Triton X-100) and 2.5 U of

Taq

DNApolymerase (Promega). The thermal profile consisted of aninitial denaturation step of 94

°

C for 4 min and 28 cycles of94

°

C for 30 s, 60

°

C annealing for 1 min and 72

°

C extensionfor 2 min. The PCR-amplified DNA fragments that rangedfrom 400 to 750 bp in length were then subjected to hybrid-ization with a (GA)

13

oligonucleotide probe that was labelledwith dideoxy-guanin triphosphate at the 3

′

-end to elimin-ate PCR artefacts after the enrichment (Koblizkova

et al

.1998). The probe was 5

′

-bound via biotin-streptavidin toparamagnetic particles (Dynal; Kijas

et al

. 1994). Hybrid-ization reactions (100

µ

L) took place under gentle agitationat 60

°

C in 6

×

standard sodium citrate (SSC) including0.1% sodium dodecyl sulphate. The bead suspension was

Correspondence: Thorsten B. H. Reusch. Fax: + 49-4522-763-310;E-mail: reusch@mpil-ploen.mpg.de

578

P R I M E R N O T E

© 2004 Blackwell Publishing Ltd,

Molecular Ecology

Notes

, 4, 577–579

Tab

le 1

Cha

ract

eriz

atio

n of

five

mic

rosa

telli

te lo

ci fo

r

Dip

lost

omum

pse

udos

path

aceu

m

Prim

er s

eque

nce

(5

′−

3

′

)R

epea

tR

eact

ion

C

prim

n

cyc

nR

H

in s

nail

H

in fi

sh

Gen

Ban

k A

cces

sion

no.

Dip

lo06

F: F

AM

TGCTTACATTGAAGGATGTTCG

(

GA

)

n

A0.

1530

3684

–232

0.65

0.55

AJ6

2925

0R

:

CGCCTTTTAATACGAACCTTG

Dip

lo08

F: H

ex

GCGGTTAGATGGATGGATG

(

GA

)

8

(

GGA

)

2

(

GA

)

16

GG

(

GA

)

10

B0.

4534

6184

–244

0.53

0.61

AJ6

2925

1R

:

TGCTGTTTTGTTTGCCTGAC

Dip

lo09

F: F

AM

CGTCAAAGTGACCAAACTCG

(

GA

)

n

C0.

1330

4116

4–25

80.

920.

87A

J629

252

R:

AAAGCGGATACAAGGAATGC

Dip

lo23

F: H

ex

TTTCGAGTGTCTGTGTGCAA

(

GA

)

n

A0.

1530

1786

–140

0.67

0.73

AJ6

2925

3R

:

AGAACAAATGCCGTTTTCAA

Dip

lo29

F: H

ex

AAGCTGCTTTGTTGTCCAATC

(

GA

)

n

C0.

230

4313

8–25

60.

710.

61A

J629

255

R:

TGTTGTGTCGAACATTTGCTTAG

Rea

ctio

n le

tter

ref

ers

to th

e po

oled

pol

ymer

ase

chai

n re

acti

on (P

CR

);

C

prim

, pri

mer

con

cent

rati

on (

µ

m

);

n

cyc

, num

ber

of P

CR

cyc

les;

n

, num

ber

of a

llele

s ob

serv

ed;

R

, alle

le s

ize

rang

e (b

p);

H

, obs

erve

d he

tero

zygo

sity

in p

aras

ites

from

sna

ils (

n

= 5

0) a

nd fi

sh (

n

= 2

19).

See

text

for

addi

tion

al d

etai

ls o

f the

ther

moc

ycle

r re

acti

on p

rofi

les.

washed at 60

°

C with volumes of 500

µ

L SSC in triplicatewith increasing stringency up to 0.5

×

SSC. Initial testsrevealed that the fraction containing the highest proportionof microsatellites would elute at 75

°

C with 0.2

×

SSC. Theeluted microsatellite-containing fraction was desaltedand PCR amplified as above. The fresh PCR product wasligated and cloned into competent

Escherichia coli

strainsusing the TOPO-TA cloning kit (Invitrogen) following themanufacturer’s protocol. A high percentage of the bacterialplasmid clones contained DNA fragments with micro-satellites (> 60%).

One hundred and forty-four colonies were picked andthe plasmid DNA was extracted and sequenced with M13primers using the Big-dye 3.1 sequencing kit (AppliedBiosystems). Electrophoresis and sequence detection tookplace on an ABI 3100 automatic sequencer. Primer pairs weredesigned for 12 candidate loci using the software

primer

3(Rozen & Skaletsky 2000). To determine the microsatellitegenotype of single parasites (cercariae from snails ormetacercariae from the eye lenses of fish) the DNA wasextracted with the DNeasy tissue kit (Qiagen). The PCRs(20

µ

L) contained 3

µ

L DNA extract, 0.1% bovine serumalbumin, 250

µ

m

dNTP, 1.5 m

m

MgCl

2

, 1

×

PCR buffer, 2.5 Uof

Taq

DNA polymerase (Promega) and 0.13–0.45

µ

m

ofeach of two primer pairs (Table 1). The PCR started with3 min at 94

°

C followed by

n

cycles (cf. Table 1) of 94

°

C for1 min, 56

°

C for 1 min and 72

°

C for 1 min, followed by afinal step of 72

°

C for 25 min. PCR reaction A had a touch-down thermal profile for the first 10 cycles starting at 61

°

Cwith a 0.5 °C decrease for 10 cycles until 56 °C was reached;for the remaining cycles and all other reaction profiles (i.e.B and C) the annealing temperature was 56 °C. PCR productswere size separated and scored against an internal standard(Rox 350) on an ABI 3100. Fragment length analysis wasperformed using the software genescan 3.5 (Applied Bio-systems). Of the tested primer pairs, five yielded consistentand polymorphic amplification products. We subsequentlygenotyped a larger array of parasites derived from 10 snails(L. stagnalis, n = 401) and isolated from the eye lenses of10 three-spined stickleback (G. aculeatus, n = 231) from theKleiner Plöner See (Schleswig-Holstein, Germany). AsD. pseudospathaceum reproduces clonally within the snail,identical genotypes (clones) were excluded from the sub-sequent analysis. Locus polymorphism was calculated usingthe software genetix (Belkhir et al. 1996–2002). Allelenumbers and observed heterozygosities were high (Table 1).The locus Diplo08 was particularly polymorphic. Here, theamplification artefacts (‘stutter bands’) of genotypes withboth alleles > 160 bp (i.e. more than 40 dinucleotide repeatunits) did not allow us to distinguish with certainty betweenhomo- and heterozygote individuals. This applied to 14%of the genotypes of Diplo08, resulting in an underestima-tion of heterozygosity. In order to test for Hardy–Weinbergequilibrium we calculated FIS using genetix and found a

P R I M E R N O T E 579

© 2004 Blackwell Publishing Ltd, Molecular Ecology Notes, 4, 577–579

consistent positive deviation of FIS from zero across all lociin both snail and fish stages of the parasite (all P < 0.05;FIS = 0.07–0.51). Linkage disequilibrium was tested usingthe permutation approach implemented in genetix (2000permutations). We detected significant linkage in parasitesfrom the snails only (cercariae) among the locus pairs Diplo23and 29, Diplo08 and 09 and Diplo09 and 29. These locus pairswere clearly unlinked in 219 metacercariae from three-spined stickleback (P > 0.10). All other locus pairs were inlinkage equilibrium in both the snail and fish stages of D.pseudospathaceum (all P > 0.10; Bonferroni correction applied).We found significant genetic differentiation among thepopulations in the first and second intermediate host (FST= 0.012, P < 0.01). Because of their high polymorphism, thepresented primers will be valuable for studying the clonalstructure, population structure and mating system in thisimportant fish parasite.

Acknowledgements

Ilka Dankert and Silke Carstensen were indispensable for devel-oping the genomic library. We thank Sybille Liedtke and TanjaSonntag for help in primer optimization and Gerhard Augustinfor maintaining the aquaria. Filipe Alberto gave advice on theenrichment protocol. This work was jointly funded by DFG (Re1108/4 and -5) and the Max Planck Society.

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