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Mi t t . Dt s c h . Ge s . a l l G . a n G e w . en t . 17 ha l l e (sa a l e ) 2009
99
The bacterial endosymbiont Wolbachia
in the invasive cherry fruit y Rhagoletis cingulata (Diptera, Tephritidae)
Hannes Schuler1, Wolfgang Arthofer1*, Susanne Krumböck1, Kirsten Köppler2,
Heidrun Vogt2, Luis A.F. Teixeira3, Markus Riegler4 & Christian Stauffer1
1 Institute of Forest Entomology, Forest Pathology & Forest Protection, Boku,
University of Applied Life Sciences and Natural Sciences, Vienna, Austria
2 Institute for Plant Protection in Fruit Crops and Viticulture, Julius Kühn-Institute (JKI),
Federal Research Centre for Cultivated Plants, Dossenheim, Germany
3 Department of Entomology, Michigan State University, East Lansing, USA
4 Centre for Plants and the Environment, School of Natural Sciences,
University of Western Sydney, Australia
* Current address: Institute of Ecology, University Innsbruck, Austria
Abstract: Wir berichten über zwei Wolbachia Isolate in einer europäischen Population der ame-
rikanischen Kirschfruchtiege Rhagoletis cingulata. Die Isolate wCin1 und wCin2 wurden durch
Amplikation, Klonierung und Sequenzierung des Wolbachia surface protein (wsp) Gens identiziert.
Eine phlyogenetische Analyse der wsp Region ergab, dass wCin1 und wCin2 ident mit wCer1 und
wCer2 sind, welche in der Europäischen Kirschfruchtiege, R. cerasi, gefunden wurden. Potentieller
horizontaler Wolbachia Transfer und mögliche Folgearbeiten werden diskutiert.
Key words: Rhagoletis, Wolbachia, invasive species
Christian Stauffer, Institute of Forest Entomology, Forest Pathology & Forest Protection, Boku,
University of Applied Life Sciences and Natural Sciences, Vienna, Austria
E-Mail: christian.stauffer@boku.ac.at
Introduction
Wolbachia is a gram negative endosymbiotic bacterium found in up to 65% of insect species (hi l G e n b o e c k e r & al.
2008). Infections have been detected in all major orders of insects and some other arthropod taxa (we r r e n
& al. 2008). Although its main path of transmission is transovarial through the cytoplasm of host eggs
Wolbachia is supposed to occasionally jump horizontally among species (ba l D o & al. 2008). Wolbachia can
change the reproductive traits of its hosts to enhance colonization of the germline and vertical transmission
(we r r e n & al. 2008). Cytoplasmic incompatibility (CI) is the most common phenotype in insects. It leads
to embryonic death of fertilized eggs when infected males mate with uninfected females, while matings
between infected males and females are compatible. This results in a reproductive advantage of infected
over uninfected females and leads to increased infection rates in host populations over generational cycles
(ho f f M a n n & tu r e l l i , 1997).
Based on extensive single pair crossing experiments, b
o l l e r
& al. (1976) concluded that populations of
the European cherry fruit y, Rhagoletis cerasi, are divided into two geographic complexes which exhibit
unidirectional incompatibility. b
lüMel
& r
uss
(1989) detected Rickettsia Like Organisms (RLOs) in the
ovaries of individuals in all populations. By applying PCR techniques with Wolbachia specic primers,
ri e G l e r & st a u f f e r (2002) detected two different Wolbachia strains, wCer1 and wCer2 in cherry fruit y
populations. Transinfection experiments with wCer2 revealed complete CI in the Mediterranean fruit y,
Ceratitis capitata and cage experiments demonstrated that Wolbachia-induced CI could be used as a tool
for population control (Za b a l o u & al. 2004).
Mi t t . Dt s c h . Ge s . a l l G . a n G e w . en t . 17ha l l e (sa a l e ) 2009
100
It has recently been reported that the American cherry fruit y, R. cingulata, is present in Europe. So far,
the species has been found in Austria, Germany, Hungary, Slovenia and Switzerland (bo l l e r 2000, Da n i e l &
wy s s 2007, eG a r t n e r & al. 2008, EPPO 2006, EPPO 2007a, EPPO 2007b, Vo G t & al. 2009). R. cingulata
has a similar biology as R. cerasi with the likely exception of required higher cumulative temperatures for
R. cingulata pupae to reach maturity and delayed emergence of adults from the soil (V
o G t
& al. 2009). R.
cingulata is a serious pest in cherries in Northeast American regions (bu s h 1966, ro t h w e l l & al. 2006).
Here we investigated Wolbachia infections in R. cingulata from a German population. We discuss potential
horizontal Wolbachia transmission between R. cingulata and R. cerasi, as both species might co-occur in the
same cherries. The Wolbachia detection was carried out by PCR using wsp primers and subsequent cloning
and sequencing of the amplicons.
Materials & Methods
R. cingulata ies were collected from yellow sticky traps in Heidesheim, Germany, in 2008 and stored in
absolute ethanol at -20°C. DNA of two individual ies was extracted using the Sigma GenElute Mammalian
DNA extraction Kit following the protocol of the manufacturer. DNA was eluted in 50 µl TE (10 mM Tris,
1 mM EDTA, pH=8.0) and stored at -20°C. All PCR reactions were performed on a 2720 thermal cycler
(Applied Biosystems) in a total volume of 10 µl containing: 1x Mg-free buffer (Fermentas), 2 mM MgCl2, 100
µM dNTPs, 0.2 µM of each primer, 0.25 U Taq polymerase (Fermentas) and 0.8 µl template DNA. Cycling
conditions for universal wsp amplication using the primers wsp81F and wsp691R (b
r a i G
& al. 1998)
were 95°C for 2 min followed by 35 cycles at 94°C for 30 sec, 55°C for 45 sec, 72°C for 1 min and a nal
extension at 72°C for 15 min. For cloning, a 0.8 µl aliquot of PCR product was ligated into the pTZ57R
vector of the InstaClone PCR cloning kit (Fermentas) according to the instructions of the manufacturer.
The ligated plasmids were used for transformation of competent JM109 E. coli cells and after overnight
growth white colonies were picked and transferred to liquid LB medium. Insert size was determined by
PCR with M13 vector primers and plasmid DNA was extracted by alkaline lysis. Sanger sequencing was
performed by a commercial provider. Retrieved sequences were edited manually, aligned using ClustalX
(th o M p s o n & al. 1997) and compared with Wolbachia sequences from GenBank by BLAST analysis.
Results
PCR with the Wolbachia specic primers resulted in positive amplicons in the two analysed German individuals.
These two amplicons were cloned and 21 plasmids were sequenced. Sequence analysis revealed that both
individuals were infested by two Wolbachia variants which were named wCin1 and wCin2. A BLAST search
and subsequent alignment revealed that wsp sequences of wCin1 and wCin2 are identical to those from
wCer1 and wCer2 detected in R. cerasi.
Discussion
We report about two Wolbachia sequence variants in the American cherry fruit y R. cingulata. wsp of
wCin1 and wCin2 are identical to wCer1 and wCer2 detected in R. cerasi. This suggests a horizontal strain
transfer between the two cherry fruit y species. To further test this hypothesis we will need to characterise
more loci of the wCin isolates by Multi Locus Strain Typing (MLST) as described by ba l D o & al. (2006)
and compare with the MLST loci of wCer. This characterization will give deeper insight into the genomes
of the strains and might reveal differences between the Cin and the Cer-strains. In order to interpret the
direction of transfer it has to be tested whether American populations of R. cingulata are also infected by
wCin1 and wCin2.
Acknowledgements
CS and WA thank the Austrian Science Foundation FWF for nancial support.
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