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The bacterial endosymbiont Wolbachia in the invasive cherry fruit fly Rhagoletis cingulata (Diptera, Tephritidae)

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Abstract

Wir berichten über zwei Wolbachia Isolate in einer europäischen Population der ame-rikanischen Kirschfruchtfliege Rhagoletis cingulata. Die Isolate wCin1 und wCin2 wurden durch Amplifikation, Klonierung und Sequenzierung des Wolbachia surface protein (wsp) Gens identifiziert. Eine phlyogenetische Analyse der wsp Region ergab, dass wCin1 und wCin2 ident mit wCer1 und wCer2 sind, welche in der Europäischen Kirschfruchtfliege, R. cerasi, gefunden wurden. Potentieller horizontaler Wolbachia Transfer und mögliche Folgearbeiten werden diskutiert.
Mi t t . Dt s c h . Ge s . a l l G . a n G e w . en t . 17
h
a l l e (sa a l e ) 2009
99
The bacterial endosymbiont Wolbachia
in the invasive cherry fruit y Rhagoletis cingulata (Diptera, Tephritidae)
Hannes Schuler
1
, Wolfgang Arthofer
1*
, Susanne Krumböck
1
, Kirsten Köppler
2
,
Heidrun Vogt
2
, Luis A.F. Teixeira
3
, Markus Riegler
4
& Christian Stauffer
1
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 Kirschfruchtiege Rhagoletis cingulata. Die Isolate wCin1 und wCin2 wurden durch
Amplikation, Klonierung und Sequenzierung des Wolbachia surface protein (wsp) Gens identiziert.
Eine phlyogenetische Analyse der wsp Region ergab, dass wCin1 und wCin2 ident mit wCer1 und
wCer2 sind, welche in der Europäischen Kirschfruchtiege, 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
l ü M e l
& r
u s s
(1989) detected Rickettsia Like Organisms (RLOs) in the
ovaries of individuals in all populations. By applying PCR techniques with Wolbachia specic 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 MgCl
2
, 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 amplication 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 specic 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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... Rhagoletis pomonella is infected with the wPom strain, which seems to be very similar (or identical) to wCer2, while other sequence variants for different MLST genes (not attributed to specific strains) have also been reported (Schuler et al. 2011). Rhagoletis cingulata is infected with at least two strains very similar (or identical) to wCer1 and wCer2 (Schuler et al. 2009; Drosopoulou et al. 2011), named wCin1 and 2 (Schuler et al. 2009). Wolbachia strains derived from R. cerasi have been recently used for the development of an alternative and environment-friendly strategy to control two major agricultural pests: the Mediterranean fruit fly, C. capitata (Zabalou et al. 2004Zabalou et al. , 2009) and the olive fly, Bactrocera oleae (Apostolaki et al. 2011 ). ...
... Rhagoletis pomonella is infected with the wPom strain, which seems to be very similar (or identical) to wCer2, while other sequence variants for different MLST genes (not attributed to specific strains) have also been reported (Schuler et al. 2011). Rhagoletis cingulata is infected with at least two strains very similar (or identical) to wCer1 and wCer2 (Schuler et al. 2009; Drosopoulou et al. 2011), named wCin1 and 2 (Schuler et al. 2009). Wolbachia strains derived from R. cerasi have been recently used for the development of an alternative and environment-friendly strategy to control two major agricultural pests: the Mediterranean fruit fly, C. capitata (Zabalou et al. 2004Zabalou et al. , 2009) and the olive fly, Bactrocera oleae (Apostolaki et al. 2011 ). ...
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Rhagoletis cerasi (Diptera: Tephritidae) is a major pest of sweet and sour cherries in Europe and parts of Asia. Despite its economic significance, there is a lack of studies on the genetic structure of R. cerasi populations. Elucidating the genetic structure of insects of economic importance is crucial for developing phenological-predictive models and environmental friendly control methods. All natural populations of R. cerasi have been found to harbor the endosymbiont Wolbachia pipientis, which widely affects multiple biological traits contributing to the evolution of its hosts, and has been suggested as a tool for the biological control of insect pests and disease vectors. In the current study, the analysis of 18 R. cerasi populations collected in Greece, Germany, and Russia using 13 microsatellite markers revealed structuring of R. cerasi natural populations, even at close geographic range. We also analyzed the Wolbachia infection status of these populations using 16S rRNA-, MLST- and wsp-based approaches. All 244 individuals screened were positive for Wolbachia. Our results suggest the fixation of the wCer1 strain in Greece while wCer2, wCer4, wCer5, and probably other uncharacterized strains were also detected in multiply infected individuals. The role of Wolbachia and its potential extended phenotypes needs a thorough investigation in R. cerasi. Our data suggest an involvement of this symbiont in the observed restriction in the gene flow in addition to a number of different ecological factors.
... American populations of R. cingulata are only infected by one Wolbachia strain, wCin2, a strain that, based on available sequence information, appears identical to the wCer2 strain of R. cerasi. However, European populations of R. cingulata also have a second strain, wCin1 (Schuler et al. 2009. Characterization of the five multi locus sequence typing (MLST) genes and the Wolbachia surface protein (wsp) gene showed that this strain is identical to wCer1, a strain that is omnipresent in R. cerasi (Riegler and Stauffer 2002, Arthofer et al. 2011. ...
... This strain was named wRha according to . BLAST search of the wsp fragment revealed identical sequences for wRha and wCer2 in R. cerasi (Riegler and Stauffer 2002) and wCin2 in R. cingulata (Schuler et al. 2009; Fig. 2a). However, all wRha MLST alleles were different to the MLST alleles of wCer2 from R. cerasi (Arthofer et al. 2011) and wCin2 from R. cingulata , resulting in different phylogenetic placements of the strains (Fig. 2b). ...
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... Thus, R. cingulata and R. cerasi fulfill some of the important host criteria conducive to the horizontal transfer of Wolbachia, as discussed above. Previous studies based on MLST sequencing identified two Wolbachia strains in R. cingulata (Drosopoulou et al., 2011;Schuler et al., 2009). The strain wCin1 in R. cingulata was found to be essentially identical to wCer1 in R. cerasi based on MLST markers and wsp and is present at different frequencies only in invasive R. cingulata populations in Europe and not found in its native range in North America ( Figure 1a). ...
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... Based on multilocus sequence typing (MLST) and wsp gene analysis, all populations of R. cingulata studied so far have been 100% infected with the Wolbachia wCin2 strain (table 2), which is identical to wCer2 of R. cerasi (Schuler et al., 2009Drosopoulou et al., 2011). In addition, Schuler et al. (2013) demonstrated that several European populations of R. cingulata are infected also with the wCin1 strain (identical to wCer1 of R. cerasi, again based on MLST and wsp genes analysis), but not in any individual studied from the USA (table 2). ...
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... Among the frugivorous tephritid flies, Wolbachia was found to infect species of the genera Rhagoletis (Riegler and Stauffer, 2002;Schuler et al., 2009Schuler et al., , 2011Schuler et al., , 2013Arthofer et al., 2009;Drosopoulou et al., 2011;Augustinos et al., 2014), Bactrocera (Kittayapong et al., 2000;Jamnongluk et al., 2002;Liu et al., 2006;Sun et al., 2007;Morrow et al., 2014Morrow et al., , 2015, Dacus (Kittayapong et al., 2000), Ceratitis (Rocha et al., 2005), and Anastrepha (Werren et al., 1995;Selivon et al., 2002;Coscrato, et al., 2009;Cáceres et al., 2009;Marcon et al., 2011;Martínez et al., 2012). Like in other cases of Wolbachia infections, a non-congruence of the endosymbiont phylogenies and their hosts was also observed in fruit flies, suggesting the occurrence of horizontal transmission events (Jamnongluk et al., 2002;Sun et al., 2007;Coscrato et al., 2009). ...
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... A second possibility is that Mexican and US populations possess different communities of endosymbiotic bacteria (e.g., different Wolbachia strains) that cause cytoplasmic incompatibilities between flies. Rhagoletis do harbor Wolbachia and different strains have been documented in the fly (Schuler et al., 2009. Such an explanation could account for the rapid appearance of post-zygotic isolation among certain R. cingulata populations. ...
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... Tephritids appear to be exceptional in their potential for co-infections by multiple Wolbachia strains, with reported incidences of five strains in individuals of Bactrocera ascita (Jamnongluk et al., 2002) and Rhagoletis cerasi (Arthofer et al., 2009b). According to sequence analyses of the wsp gene, some tephritid species share identical (Schuler et al., 2009) or similar alleles (Coscrato et al., 2009;Schuler et al., 2011). However, characterization of just a single Wolbachia locus such as wsp lacks strain resolution because of the high recombination rate documented for Wolbachia genomes (Baldo et al., 2006a) and may lead to an overestimation of horizontal transmission rates. ...
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Wolbachia are endosymbiotic bacteria that infect 40 to 65% of arthropod species. They are primarily maternally inherited with occasional horizontal transmission for which limited direct ecological evidence exists. Previously, we detected Wolbachia in eight out of 24 Australian tephritid species. Here, we have used Multilocus Sequence Typing (MLST) to further characterise these Wolbachia strains, plus a novel quantitative PCR method for allele assignment in multiple infections. Based on five MLST loci and the Wolbachia surface protein gene (wsp), five Bactrocera and one Dacus species harboured two identical strains as double infections; furthermore, Bactrocera neohumeralis harboured both of these as single or double infections, and sibling species Bactrocera tryoni harboured one. Two Bactrocera species contained Wolbachia pseudogenes, potentially within the fruit fly genomes. A fruit fly parasitoid, Fopius arisanus shared identical alleles with two Wolbachia strains detected in one Bactrocera frauenfeldi individual. We report an unprecedented high incidence of four shared Wolbachia strains in eight host species from two trophic levels. This suggests frequent exposure to Wolbachia in this tropical tephritid community that shares host plant and parasitoid species, and also includes species that hybridise. Such insect communities may act as horizontal transmission platforms that contribute to the ubiquity of the otherwise maternally inherited Wolbachia.
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The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species boundaries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well‐characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolbachia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in several European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some populations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hungary. In summary, our study provides strong evidence for a very recent inter‐specific Wolbachia transmission with a subsequent spatial spread in field populations.
Article
The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species boundaries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well-characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolbachia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in several European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some populations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hungary. In summary, our study provides strong evidence for a very recent inter-specific Wolbachia transmission with a subsequent spatial spread in field populations.
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Biological control is the purposeful introduction of parasites, predators, and pathogens to reduce or suppress pest populations. Wolbachia are inherited bacteria of arthropods that have recently attracted attention for their potential as new biocontrol agents. Wolbachia manipulate host reproduction by using several strategies, one of which is cytoplasmic incompatibility (CI) [Stouthamer, R., Breeuwer, J. A. J. & Hurst, G. D. D. (1999) Annu. Rev. Microbiol. 53, 71-102]. We established Wolbachia-infected lines of the medfly Ceratitis capitata using the infected cherry fruit fly Rhagoletis cerasi as donor. Wolbachia induced complete CI in the novel host. Laboratory cage populations were completely suppressed by single releases of infected males, suggesting that Wolbachia-induced CI could be used as a novel environmentally friendly tool for the control of medfly populations. The results also encourage the introduction of Wolbachia into pest and vector species of economic and hygenic relevance to suppress or modify natural populations.
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The eubacterial genus Wolbachia comprises one of the most abundant groups of obligate intracellular bacteria, and it has a host range that spans the phyla Arthropoda and Nematoda. Here we developed a multilocus sequence typing (MLST) scheme as a universal genotyping tool for Wolbachia. Internal fragments of five ubiquitous genes (gatB, coxA, hcpA, fbpA, and ftsZ) were chosen, and primers that amplified across the major Wolbachia supergroups found in arthropods, as well as other divergent lineages, were designed. A supplemental typing system using the hypervariable regions of the Wolbachia surface protein (WSP) was also developed. Thirty-seven strains belonging to supergroups A, B, D, and F obtained from singly infected hosts were characterized by using MLST and WSP. The number of alleles per MLST locus ranged from 25 to 31, and the average levels of genetic diversity among alleles were 6.5% to 9.2%. A total of 35 unique allelic profiles were found. The results confirmed that there is a high level of recombination in chromosomal genes. MLST was shown to be effective for detecting diversity among strains within a single host species, as well as for identifying closely related strains found in different arthropod hosts. Identical or similar allelic profiles were obtained for strains harbored by different insect species and causing distinct reproductive phenotypes. Strains with similar WSP sequences can have very different MLST allelic profiles and vice versa, indicating the importance of the MLST approach for strain identification. The MLST system provides a universal and unambiguous tool for strain typing, population genetics, and molecular evolutionary studies. The central database for storing and organizing Wolbachia bacterial and host information can be accessed at http://pubmlst.org/wolbachia/.
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International investigations on the unidirectional incompatibility between two races of Rhagoletis cerasi discovered in 1972 have yielded a new distribution map of the observed phenomenon. Genetic, cytoplasmic and symbiote-induced incompatibility can be proposed as possible mechanisms but increasing evidence indicates that the observed sterility is caused by extra-chromosomal factors. Three hypotheses are suggested to explain the possible origin and mode of action of the observed trait. The potential use of incompatible races of R. cerasi for novel control methods are discussed.
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Wolbachia are common intracellular bacteria that are found in arthropods and nematodes. These alphaproteobacteria endosymbionts are transmitted vertically through host eggs and alter host biology in diverse ways, including the induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm-egg incompatibility. They can also move horizontally across species boundaries, resulting in a widespread and global distribution in diverse invertebrate hosts. Here, we review the basic biology of Wolbachia, with emphasis on recent advances in our understanding of these fascinating endosymbionts.
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Wolbachia is an obligately intracellular, maternally inherited bacterium which has been detected in many arthropods. Wolbachia infections disperse in host populations by mechanisms such as cytoplasmic incompatibility (CI). CI leads to embryonic mortality which occurs when infected males mate with uninfected females or females with a different Wolbachia strain. Populations of the European cherry fruit fly Rhagoletis cerasi (Diptera, Tephritidae) were found to be infected by two different Wolbachia strains, wCer1 and wCer2. Superinfections with both strains occurred throughout southern and central Europe and infections with wCer1 were found in northern, western and eastern Europe. Strong unidirectional CI between European populations of R. cerasi were first reported in the 1970s. From the conformity in the recent geographical distribution of the Wolbachia infections and the CI expression patterns found 25 years ago it was deduced that wCer2 potentially causes CI in R. cerasi. The comparison of the geographical distributions indicated that wCer1 + 2 must have spread into wCer1-infected populations in some areas. In other regions, a spread of wCer1 + 2 was probably prevented by dispersal barriers. There, a sharp transition from infected to superinfected populations suggested regional isolation between wCer1 and wCer1 + 2-infected populations.