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Wolbachia Infection in the Walnut-Husk Fly Rhagoletis completa Cresson 1929 (Diptera: Tephritidae)

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Zusammenfassung Wir berichten über eine Wolbachia-Infektion in einer österreichischen Population der Walnussfruchtfliege Rhagoletis completa. Erste Untersuchungen mit konventioneller PCR zeigten keine sichtbaren Banden. In Folge wurden zwei Techniken zum Nachweis von Niedrigtiter-Infektionen-Southern Blot und nested-PCR – angewandt, mit denen Wolbachia gefunden werden konnte. Die Sequenzierung von Klonen des wsp Amplikons zeigte die Präsenz von sehr unterschiedlichen Wolbachia Stämmen in der Walnussfruchtfliege.
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Mi t t . Dt s c h . Ge s . a l l G . a n G e w . en t . 18ha l l e (sa a l e ) 2012
243
Wolbachia Infection in the Walnut-Husk Fly Rhagoletis completa Cr e s s o n 1929
(Diptera: Tephritidae)
Hannes Schuler
1, Wolfgang Arthofer
2, Susanne Krumböck
1, Coralie Bertheau
1 &
Christian Stauffer1
1 Institute of Forest Entomology, Forest Pathology & Forest Protection,
Boku, University of Natural Resources and Life Sciences, Vienna, Austria
2 Molecular Ecology Group, Institute of Ecology, University of Innsbruck, Austria
Zusammenfassung Wir berichten über eine Wolbachia-Infektion in einer österreichischen
Population der Walnussfruchtiege Rhagoletis completa. Erste Untersuchungen mit konventioneller
PCR zeigten keine sichtbaren Banden. In Folge wurden zwei Techniken zum Nachweis von
Niedrigtiter-Infektionen - Southern Blot und nested-PCR – angewandt, mit denen Wolbachia
gefunden werden konnte. Die Sequenzierung von Klonen des wsp Amplikons zeigte die Präsenz
von sehr unterschiedlichen Wolbachia Stämmen in der Walnussfruchtiege.
Key Words: Rhagoletis completa, Wolbachia, invasive species
Hannes Schuler, Institute of Forest Entomology, Forest Pathology and Forest Protection,
Hasenauerstraße 38, A-1190 Wien, E-Mail: hannes.schuler@boku.ac.at
Introduction
The Walnut Husk Fly, Rhagoletis completa (Diptera: Tephritidae), is a pest in Juglans spp.. Native to
Midwestern United States, it was introduced to Europe in the early 1990s (Me r z 1991). Since then the
spread of this species was quite rapid and today R. completa has been described in several countries in
Central Europe (a
l u j a
& al. 2011). Unlike other Rhagoletis species, R. completa oviposits a batch of
eggs into the walnut mesocarp. Larvae feed in the walnut husk tissue causing shell staining and darkened
stones (Bo y c e 1934).
Wolbachia is an endosymbiotic bacterium present in up to 65% of insects. It manipulates the host’s
reproduction by causing male-killing, parthenogenesis, feminization and cytoplasmic incompatibility (CI)
(we r r e n & al. 2008).
Several Rhagoletis-species like R. cerasi (ri e G l e r & sta u f f e r 2002, arthofer & al. 2009B),
R. cingulata (sc h u l e r & al. 2009) and R. pomonella (sc h u l e r & al. 2011) are described to be infected
by Wolbachia. Wolbachia-infections in low-titer, not detectable by conventional PCRs, are supposed
to be more prevalent than assumed so far (a
rthofer
& al. 2009
a
,
B
, h
uGhes
& al. 2011). Although
R. completa is considered to be Wolbachia free (Dr o s o p o u l o u & al. 2010), this study was designed to
detect Wolbachia in low-titer using two independent techniques: Southern blot and nested PCR.
Materials & Methods
In 2008, larvae of R. completa were collected in the Institute’s garden in Vienna and stored in ethanol
at - 20 °C. DNA was extracted and PCR using the general wsp primers for Wolbachia detection (B
r a i G
& al.
1998) was applied. Subsequently a Southern hybridization of the PCR amplicons (a
r t h o f e r
& al. 2009
B
)
and a nested PCR with internal wspif and wspir primers (a
rthofer
& al. 2009
a
) were performed. An
aliquot of the nested PCR amplicon was ligated into a pTZ57R/T vector (Fermentas) and transformed
into competent JM109 Escherichia coli cells. Twelve white colonies were picked and Sanger sequencing
was performed using M13 primers.
Mi t t . Dt s c h . Ge s . a l l G . a n G e w . en t . 18 ha l l e (sa a l e ) 2012
244
Results & Discussion
PCR with general wsp primers resulted in no visible amplicons on the agarose gel. Both Southern
hybridization and nested PCR revealed positive Wolbachia bands (Fig. 1), indicating that R. completa
is infected with Wolbachia. Fourteen plasmid sequences from a total of three individuals revealed four
different sequence types. In a BLAST search and a phylogenetic analysis (data not shown), one sequence
grouped in the wMel clade together with wCer1 and wCer2 of R. cerasi, and a second sequence is
identical to that of the B group strain wCer5 of R. cerasi. The other two sequences had no exact matches
in Genbank. The short 317bp wsp amplicon hinders a more detailed strain characterization.
Low-titer Wolbachia are difcult to characterize genetically and phenotypically and are considered
to not induce reproductive phenotypes like CI (i
keDa
& al. 2003). Phenotypic effects of such cryptic
strains are not known in R. completa, and a study including more individuals from different European
and American populations would be necessary to obtain deeper knowledge on the genetic characteristics
and the phenotype of the diverse strains.
Acknowledgements
We wish to thank the Austrian Science Fund FWF for nancial support.
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