Dynamics of an Ongoing Wolbachia Spread in the European Cherry Fruit Fly, Rhagoletis cerasi (Diptera: Tephritidae)

Abstract

Numerous terrestrial arthropods are infected with the alphaproteobacterium Wolbachia. This endosymbiont is usually transmitted vertically from infected females to their offspring and can alter the reproduction of hosts through various manipulations, like cytoplasmic incompatibility (CI), enhancing its spread in new host populations. Studies on the spatial and temporal dynamics of Wolbachia under natural conditions are scarce. Here, we analyzed Wolbachia infection frequencies in populations of the European cherry fruit fly, Rhagoletis cerasi (L.), in central Germany—an area of an ongoing spread of the CI-inducing strain wCer2. In total, 295 individuals from 19 populations were PCR-screened for the presence of wCer2 and their mitochondrial haplotype. Results were compared with historic data to understand the infection dynamics of the ongoing wCer2 invasion. An overall wCer2 infection frequency of about 30% was found, ranging from 0% to 100% per population. In contrast to an expected smooth transition from wCer2-infected to completely wCer2-uninfected populations, a relatively scattered infection pattern across geography was observed. Moreover, a strong Wolbachia-haplotype association was detected, with only a few rare misassociations. Our results show a complex dynamic of an ongoing Wolbachia spread in natural field populations of R. cerasi.

1. Introduction

Heritable bacterial endosymbionts are present in a broad range of arthropods with intriguing effects on the ecology and evolution of numerous species [1,2]. Endosymbionts are usually transmitted vertically from infected females to their offspring. By manipulating the reproduction through the induction of cytoplasmic incompatibility (CI), parthenogenesis, feminization, and male-killing, bacterial endosymbionts increase the fitness of infected females and enhance their frequency in host populations [3,4]. Certain endosymbionts are also able to provide fitness benefits protecting their host against RNA viruses or natural enemies [5,6]. Most endosymbionts are able to invade and adapt to new species by interspecific horizontal transmission [7,8,9,10].

One of the most common bacterial endosymbionts that can manipulate arthropod reproduction is Wolbachia [11]. This bacterium has been estimated to infect more than 50% of terrestrial arthropod species [1] and is present in numerous hexapods, crustaceans, chelicerates, and nematodes [11]. The most common reproductive manipulation is CI that is expressed when a Wolbachia-infected male mates with a Wolbachia-uninfected female, or with a female harboring a different Wolbachia strain, resulting in embryonic death of fertilized eggs [12].

A Wolbachia infection can influence the mitochondrial genetic structure of the host [13]. When a Wolbachia strain spreads into a population of uninfected individuals, the mitochondrial haplotype associated with the infected individual will hitchhike with the spreading endosymbiont [13,14,15,16,17]. As a result, an endosymbiont infection can affect the genetic population structure of arthropod species by reducing the diversity of mitochondrial haplotypes when infected individuals—associated with a specific mitochondrial haplotype—eliminate haplotypes associated with uninfected individuals [13].

The European cherry fruit fly, Rhagoletis cerasi (L., 1758) (Diptera: Tephritidae), is a widespread insect with a complex life history. In addition to a broad spectrum of evolutionary research questions [9,16,18,19,20], this tephritid is also of particular interest in applied fields [21]. It infests hosts of two different plant families, that is, Prunus spp. (Rosaceae) and Lonicera spp. (Caprifoliaceae) [18,20] and is therefore of significant human interest, as it is a severe pest of sweet and sour cherries [22,23].

Rhagoletis cerasi has a univoltine life cycle. In spring, prior to the ripening of fruits, adult flies emerge from their overwintering sites next to their natal host and usually do not disperse over long distances [21]. After mating on or close to host plants, usually one egg is deposited into a ripening fruit. These fruits are marked with specific pheromones to avoid an additional oviposition [24,25,26]. After embryonic development, larvae feed on fruits of their natal host plant. Last-instar larvae leave the fruits, dig into the soil, pupate, and overwinter in a diapausing state, completing their univoltine life cycle [21,27].

Moreover, R. cerasi has an intriguing reproductive biology. Crossing studies among European populations revealed strong incompatibility patterns from up to 98% when males from Southern and Central Europe mated with females from other European regions [18,19]. After the detection of Rickettsia-like organisms (RLOs) in reproductive organs [28], Riegler & Stauffer [29] described the presence of Wolbachia in this tephritid. While all individuals are infected by the same Wolbachia strain wCer1, only populations from Southern and Central Europe are additionally infected by a second strain, wCer2 [16,29]. The distribution of Wolbachia strains matches the incompatibility patterns reported by Boller et al. [19], which suggests that wCer2 is causing this reproductive alteration [29].

Between southern and northern R. cerasi populations, there is a shift from completely wCer2-infected populations to populations not infected with wCer2 [29]. In these transitional zones, wCer2-infected and wCer2-uninfected flies coexist with gradients in infection frequencies (Figure 1a) [16,29,30]. Empirical studies combined with mathematic modelling showed that wCer2 is currently invading wCer2-uninfected R. cerasi populations [16,30]. A recent study of two R. cerasi transects in the Czech Republic and Hungary showed a smooth gradient from completely wCer2-infected populations in the south to uninfected populations in the north and east, documenting a spatial spread of 1–2 km per generation [30].

Sequencing a part of the mitochondrial COI gene showed that R. cerasi exhibits a low genetic diversity in its European range, with only two haplotypes [16]. Comparison of haplotype of the fly and its Wolbachia infection status revealed a strong association between the endosymbiont and the mitochondrial haplotype—individuals of haplotype 1 (HT1) are wCer2-uninfected, whereas wCer2-infected flies are associated with haplotype 2 (HT2). Occasional misassociations have been reported from transitional zones as a result of intraspecific horizontal transfer and imperfect vertical transmission, highlighting the complex spread of this endosymbiont [16].

The invasion dynamics of Wolbachia in natural field populations are rarely studied [16,30,31,32,33]. To get detailed insights into the temporal and spatial spread of Wolbachia in R. cerasi, we performed a fine-scale sampling of 19 populations in Central Germany, where wCer2 is currently spreading. By studying Wolbachia infection frequencies, assessing mitochondrial genotypes of the host, and comparing these results with historic data [16], we aim to get new insights into the dynamics of an ongoing Wolbachia spread under natural conditions.

2. Materials and Methods

2.1. Sampling and DNA Extraction

Samples were collected from infested fruits of Prunus avium and Lonicera xylosteum in July 2016. In total, specimens from 19 German locations were sampled: One site from the northern part of Baden-Württemberg, i.e., 16/1, and 18 populations from central-south Hesse, i.e., 16/2–16/19 (

Table 1. Infection frequencies of wCer2 across different R. cerasi populations and ratios of wCer2-infected flies associated with haplotype 2 (HT2) per population (* = populations with Wolbachia-haplotype misassociations).

Population #	Location	% wCer2	% wCer2/HT2
16/1	Dossenheim	100	100
16/2	Bensheim	6.3	6.3
16/3	Stockstadt	18.8	18.8
16/4	Ober-Ramstadt	6.3	6.3
16/5	Erbenheim	6.7	6.7
16/6	Idstein	10.0	10.0
16/7	Rosbach	0.0	0.0
16/8	Hailer	100	100
16/9	Weckesheim	25.0	25.0
16/10	Utphe	12.5	12.5
16/11	Langsdorf	18.8	18.8
16/12	Lich	0.0	0.0
16/13	Gießen/Lonicera	75.0	62.5 *
16/14	Gießen/Prunus	100	68.8 *
16/15	Lahnau	18.8	0.0 *
16/16	Alsfeld	12.5	12.5
16/17	Wallenrod	0.0	0.0
16/18	Schlüchtern	37.5	37.5
16/19	Grossenmoor	14.3	14.3

, Figure 1, Table S1). Individuals were collected from a single plant per location, either as larvae from infested fruits in the field or as pupae after emerging from cherries in the laboratory. To avoid the analysis of siblings, fruits from different parts of one tree/shrub were taken. Samples were stored in absolute ethanol at −20 °C. DNA was extracted from 16 individuals per location, except for the populations 16/5 (n = 15), 16/6 (n = 10), and 16/19 (n = 14; Table S1), using the GenElute Mammalian Genomic DNA miniprep kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions.

2.2. Wolbachia Screening

Samples were PCR-screened for the presence of wCer1 and wCer2 using strain-specific primers targeting a part of the Wolbachia surface protein (wsp) [29,34,35]. PCR reactions were performed in a total volume of 10 µL, containing 1 mg/mL BSA, 2 mM Y-buffer (PeqLab/VWR, Erlangen, Germany), 800 µM dNTPs, 0.2 µM forward and reverse primer each, 0.5 U Taq polymerase (PeqLab/VWR), and 1 µL of template DNA. PCR conditions were 2 min at 94 °C, followed by 35 cycles of 94 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min, followed by a final extension at 72 °C for 10 min.

PCR-amplified fragments were electrophoretically separated on a 2% agarose gel stained with GelRed Nucleic Acid Dye (Biotum, Hayward, CA, USA). As wCer1 is fixed in European R. cerasi [16,29,30,35], screenings for this strain were performed to control for sufficient DNA quality. In order to avoid false-negatives, all results were confirmed by two independent PCR runs.

2.3. Mitochondrial Genotyping of R. cerasi

To study the association of R. cerasi mitochondrial haplotypes with the Wolbachia infection status, genotyping of the flies was performed by applying restriction fragment length polymorphism (RFLP) as used in [16]. In brief, a part of the mitochondrial COI gene was PCR-amplified using the primers Pat and Dick [36]. Following PCR (conditions were the same as described above), 10 µL of the PCR product was incubated with 2 U of HaeIII (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 3 h. Fragments were separated on a 2% agarose gel—HT2 is cut into a 342 bp and a 204 bp fragment, while HT1 remains undigested [16].

2.4. Comparison of Our Results with Historic Data

Eight out of the 19 R. cerasi populations studied here were already Wolbachia-screened and genotyped between 1999 and 2014 [16]: Dossenheim (16/1), Stockstadt (16/3), Ober-Ramstadt (16/4), Lich (16/12), Gießen (16/13, 16/14), Lahnau (16/15), and Alsfeld (16/16). To assess the spatial and temporal dynamics of wCer2, infection frequencies of this strain of the various years were compared.

3. Results

3.1. Wolbachia Infection Frequencies

Screening of R. cerasi for the presence of wCer2 revealed that 30.2% of samples (89 out of 295) were infected with this strain (Table 1, Figure 1c, Table S1). In three out of 19 locations, wCer2 was fixed: In the southernmost site Dossenheim (16/1), and in two central locations Hailer (16/8) and Gießen/Prunus (16/14). In contrast, the three populations Rosbach (16/7), Lich (16/12), and Wallenrod (16/17) were completely wCer2-uninfected. wCer2 infection frequencies in the other 13 sites ranged from 6.3% in Bensheim (16/2) and Ober-Ramstadt (16/4) to 75.0% in Gießen/Lonicera (16/13) (Table 1, Figure 1c, Table S1).

wCer2 frequencies of the R. cerasi populations studied here exhibit no distinct transitional infection pattern with smooth gradients from completely wCer2-infected to entirely wCer2-uninfected populations [30] (Figure 1c). The southernmost site Dossenheim (16/1) was completely wCer2-infected, whereas the population in Bensheim (16/2), less than 30 km further north, showed a low infection frequency of 6.3%. wCer2 frequencies in the adjacent locations Stockstadt (16/3) and Ober-Ramstadt (16/4), 14 km north-west and 17 km further north-east, respectively, ranged from 6.3% to 18.8% (Figure 1c). The wCer2 infection frequency was similar in Erbenheim (16/5) and Idstein (16/6), 30 km and 50 km further north, with 6.7% and 10%, respectively. The population in Rosbach (16/7), however, was already completely wCer2-uninfected. In contrast, 25% of the individuals from the population in Weckesheim (16/9), 13 km further north-west, were wCer2-infected, whereas all individuals from Hailer (16/8), 30 km further south-east, were infected by wCer2 (Figure 1c).

A similar pattern was found in the northern locations where a wCer2-uninfected population (Lich, 16/12) was surrounded by two transitional populations 5 and 12 km apart, and just 14 km distant from the completely wCer2-infected population in Gießen/Prunus (16/14) (Figure 1c).

3.2. Mitochondrial Genotyping of R. cerasi and Haplotype-Wolbachia Associations

As only two mitochondrial R. cerasi haplotypes across Europe have been described previously, we used an RFLP approach to determine the haplotype affiliation of each individual. Genotyping of flies showed that the Wolbachia infection status was strongly associated with the two mitochondrial haplotypes of the host (Figure 2b). Almost all wCer2-uninfected individuals were associated with HT1 (205 out of 206). Just one individual from Alsfeld (16/16) was wCer2-uninfected but associated with HT2. In contrast, 88.8% of wCer2-infected R. cerasi were associated with HT2, whereas 10 wCer2-infected individuals from three populations were associated with HT1 (Figure 2b).

Haplotype-Wolbachia misassociations were found in populations in geographically close proximity, i.e., Gießen/Lonicera (16/13), Gießen/Prunus (16/14), and Lahnau (16/15). In Gießen/Lonicera (16/13), two of the 12 wCer2-infected samples were associated with HT1, whereas in Lahnau all three wCer2-infected flies were associated with HT1. The most significant deviation was found in Gießen/Prunus (16/14), where all 16 individuals were wCer2-infected, but only 11 (68.8%) were associated with HT2.

3.3. Wolbachia Dynamics and Haplotype Associations in Time and Space

Out of the 19 R. cerasi populations studied here, eight had already been screened for their Wolbachia infection status and mitochondrial haplotypes between 1999 and 2014 [16]. We compared our results with these historic data to infer the dynamics of Wolbachia infection and the associated mitochondrial haplotype of the fly. Rhagoletis cerasi collected from Dossenheim (16/1)—where all individuals screened were infected by wCer2—was already completely invaded by this strain in 1999 and 2008 (

Table 2. Comparison of wCer2 infection frequencies among the years 1999 (99), 2001 (01), 2008 (08), and 2016 (16).

Location	Population #	Host	Year	n	% wCer2
Dossenheim	99/1	Lonicera	1999	10	100
	08/1	Prunus	2008	16	100
	16/1	Prunus	2016	16	100
Ober-Ramstadt	08/3	Lonicera	2008	15	0
	08/4	Prunus	2008	15	6.7
	16/4	Prunus	2016	16	6.3
Stockstadt	08/2	Prunus	2008	16	12.5
	16/3	Prunus	2016	16	18.8
Lich	08/7	Prunus	2008	16	6.3
	14/1	Prunus	2014	10	0
	16/12	Prunus	2016	16	0
Gießen	01/4	Lonicera	2001	10	10
	08/8	Lonicera	2008	16	68.8
	16/13	Lonicera	2016	16	75
	16/14	Prunus	2016	16	100
Lahnau	08/9	Lonicera	2008	16	12.5
	16/15	Prunus	2016	16	18.8
Alsfeld	01/5	Lonicera	2001	10	50
	16/16	Lonicera	2016	16	12.5

). In Stockstadt (16/3) and Lahnau (16/15), wCer2 infection frequencies increased from 12.5% in 2008 to 18.8% in 2016 in both locations, however, wCer2 frequencies did not differ significantly among these years (both locations: χ² = 0.237, p = 0.434). In Ober-Ramstadt (16/4), the ratio of infected individuals remained constant—in 2008, one out of 15 flies (6.7%), and in 2016, one out of 16 flies (6.3%) was wCer2-infected (χ² = 0.002, p = 0.499). In 2008, the population in Lich (16/12) had a wCer2 infection frequency of 6.3%, however, none of the flies were wCer2-infected in 2014 (χ² = 0.650, p = 0.339) and in 2016 (χ² = 1.032, p = 0.297). A significant decrease of wCer2 occurred in the R. cerasi population of Alsfeld (16/16), where 50% of individuals were wCer2-infected in 2000, while in 2016, just 12.5% of the samples were infected by this strain (χ² = 4.398, p = 0.036). Moreover, we found a potential effect of the host plant of the fly on its Wolbachia infection. In Gießen (16/13 and 16/14), flies collected from Lonicera had a significant increase of wCer2 from 10% in 2001 to 68.8% in 2014 (χ² = 8.547, p = 0.003). Comparing infection frequencies from the same site (16/13) in 2016 revealed a further increase in infection frequencies to 75% (χ² = 0.155, p = 0.456). Rhagoletis cerasi collected from Prunus (16/14), however, was already completely wCer2-infected.

Comparing Wolbachia-haplotype associations between different years revealed that wCer2-infected flies associated with HT1 occurred in the same populations before (Figure 2a). Overall, the number of wCer2-infected R. cerasi associated with HT1 increased in all three populations. In contrast, the population in Alsfeld (16/16), where one wCer2-uninfected individual was associated with HT2, did not show this pattern in 2001. However, two out of five wCer2-infected flies were associated with HT1 in 2001, a pattern that was not confirmed in 2016 (Table S2).

4. Discussion

Here, we studied the frequency of wCer2 in R. cerasi in the central part of Germany, a region where this strain is currently spreading. Compared to previous work in this area [16], our fine-scale sampling and screening of 19 populations allowed us to characterize the spatial distribution of wCer2 in this transition zone. Furthermore, comparing our results with historic data from the last two decades [16] allowed us to assess the temporal dynamics of this Wolbachia strain in certain populations. In contrast to our expectation of a transitional zone with smooth gradients from completely infected to entirely uninfected flies, as described in other parts of the species’ range [30], we found a surprisingly scattered pattern of wCer2 infection frequencies across different populations. Comparison of the Wolbachia infection status and the mitochondrial haplotype of individuals showed a strong relationship, however, occasional misassociations suggest events of intraspecific horizontal transfer. Taken together, our data provide new insights into the ongoing Wolbachia spread in European R. cerasi.

4.1. Wolbachia Infection Frequencies in Time and Space

At least five Wolbachia strains have been described from R. cerasi, and one individual fly can harbor various strains [29,35]. The spread of the strain wCer2 in Central Europe from south to north represents a rare event of an ongoing Wolbachia invasion in natural field populations [16,29,30]. By screening 295 individuals from 19 populations, we found an overall wCer2 infection frequency of about 30%, ranging from 0% to 100% per population. Instead of gradual transitional zones between completely wCer2-infected and entirely wCer2-uninfected R. cerasi populations [30], a relatively scattered infection pattern was found. Populations completely infected with wCer2 were in close proximity to locations with low wCer2 infection frequencies. Our results, together with previous data [16,29,30], provide a comprehensive picture of an ongoing Wolbachia spread in natural field populations of R. cerasi and suggest that the spatial pattern of this endosymbiont infection is influenced by various factors, such as long distance migration of flies and/or passive movement with infested cherries.

Although Wolbachia is one of the best-studied bacterial endosymbionts, only a limited number of studies have given insight into its spatial dynamics under natural field conditions [16,30,31,37,38]. In addition to empirical observations, theoretical modelling can help to provide a basic understanding on the mode of Wolbachia spread [14,39,40]. One of the best studied examples of an ongoing Wolbachia spread in the field is the mosquito Aedes aegypti, artificially infected with the strain wMel, that naturally occurs in Drosophila melanogaster [41,42]. wMel was found to spread relatively slowly in release areas with a rate of 100–200 m per year [41]. Major factors influencing this spread are deceased fitness of transinfected mosquitoes and a low dispersal rate of the host [41]. This is in contrast to the estimated spread of wCer2 in central Europe of 1–1.9 km per year [30], a spread that might benefit from the long adaptation of wCer2 to its host—with expected low fitness costs—and the higher dispersal capacity of the fly. Both systems showed that human-mediated dispersal of insects can influence their long-range migration and can result in unexpected spatial patterns of host organisms and their associated symbionts [16,43].

Generally, Wolbachia that causes fitness costs in its host needs to reach a sufficiently high equilibrium frequency to get established in a host population [41]. Thus, infections in low frequencies do not result in an establishment of the bacterium [40]. In contrast, Wolbachia can be established in a host population even from very low initial infection frequencies by providing positive fitness effects to their host, as it was reported from Drosophila simulans in Australia [37] and California [31]. Although the spatial pattern of wCer2 infection rates in Germany is different from other regions in the range of R. cerasi [16,29,30], our data suggest that a certain equilibrium infection frequency in a population is necessary for this strain to become established [16,30]. This was shown by the comparison of Wolbachia infection frequencies at the same sites among different years. For example, in the locations Ober-Ramstadt (16/3), Stockstadt (16/4), Idstein (16/6), Lich (16/12), and Lahnau (16/15), infection frequencies remained at low levels over a period of eight to 15 years. In contrast, in Gießen/Lonicera (16/13), wCer2 infection frequencies of R. cerasi collected from honeysuckle increased rapidly from 10% in 2001, to 68.8% in 2008 [16], and to 75% in 2016. We assume that wCer2 reached a sufficiently high infection frequency over time, and we expect that this strain will get fixed in the following years. The importance of an equilibrium infection frequency is further supported by the absence of intermediate infection frequencies over the sampling years, as it was found in almost all locations.

In Gießen/Prunus (16/14), however, R. cerasi was already completely wCer2 infected. This suggests an influence of the fly’s host plant on the Wolbachia infection dynamics. Some lines of evidence propose host plant-related differentiation patterns in R. cerasi. For example, individuals from Prunus and Lonicera show slightly different eclosion times in spring, maybe a response to a differing fruiting phenology of the host [44]. Potential ecological differentiation between host ecotypes of R. cerasi infesting cherry or honeysuckle [18,20] might reduce gene flow between different populations, influencing the Wolbachia infection dynamics. A contrasting pattern was observed in the northernmost location Alsfeld (16/16) where the ratio of wCer2 infections decreased significantly from 50% in 2000 to 12.5% in 2016. Since none of the wCer2-uninfected individuals had the mitochondrial haplotype HT2, we exclude events of occasional loss of wCer2 as a reason for the decrease of this strain over time. The accidental anthropogenic introduction of wCer2-uninfected flies into this region, for example, via trade with R. cerasi-infested cherries, could explain this finding.

4.2. Wolbachia Infection and Mitochondrial Haplotype of the Host

The genetic structure of European R. cerasi on the mitochondrial level is generally low, reflected by the presence of only two haplotypes [16]. This low genetic diversity might be the result of a previous Wolbachia sweep by the strain wCer1. Schuler et al. [16] hypothesized that a few individuals of R. cerasi with HT1 got previously infected by wCer1 via horizontal transmission. This strain might have provided fitness benefits to the fly and swept through host populations, replacing all other haplotypes. Later, HT2 evolved, acquired wCer2 horizontally, and is now hitchhiking through European populations [16].

The mitochondrial haplotypes are tightly associated with Wolbachia, where wCer2-uninfected flies are associated with HT1 and wCer2-infected individuals with HT2. In our study, out of 295 samples, only 11 had a different Wolbachia-haplotype association. In 10 cases, these misassociations represented a wCer2-infection in individuals with HT1. This finding can be a result of intraspecific horizontal transfer [45]. For example, parasitoid wasps are known to be a potential source of horizontal transmission among species [16,45,46,47,48]. Parasitoid species of R. cerasi were found to be infected with Wolbachia. Their role in horizontal transmission between and within fly species, however, has yet to be studied [45]. An additional mechanism of horizontal transfer might be via cannibalism among R. cerasi larvae [45]. Although multiple larvae within one cherry are uncommon [26], under certain environmental conditions [26] or when resources for oviposition are scarce, several larvae might develop in one fruit. In this case, a larva could acquire Wolbachia by feeding on a co-occurring larva but would remain associated with its mitochondrial haplotype.

Since all cases of a wCer2-infection in HT1 flies were found exclusively in transitional populations (where flies with both Wolbachia infection types are present), this phenomenon was interpreted as transient, where HT1 is assumed to be lost in populations completely invaded by wCer2 [16]. We found just three populations in a restricted area—in Gießen (16/13 and 16/14) and Lahnau (16/15)—with individuals belonging to HT1 harboring wCer2. The comparison with historic data from 2001, 2008, and 2014 [16], however, showed a general increase of individuals with these misassociations. In the location Gießen/Prunus (16/14) where R. cerasi is completely wCer2-infected, 31% of the individuals were associated with HT1. Subsequent studies are needed to understand if these flies can form a stable co-existence of wCer2 and HT1 or if they will be lost in future generations, as simulated by Schuler et al. [16].

Finally, one single wCer2-uninfected individual associated with HT2 was found. This suggests a potential case of unsuccessful maternal transmission of Wolbachia. The low occurrence of these misassociations reflects a strong CI-inducing effect of wCer2 with nearly perfect transmission from females to their offspring [16,19].

5. Conclusions

Our screening of the European cherry fruit fly with focus on the CI-inducing strain wCer2 provides new insights into this unique endosymbiont-host system. In contrast to an expected smooth transition and continuous gradient of infection frequencies from wCer2-infected to completely uninfected R. cerasi populations [30], we found a rather scattered geographic infection pattern of wCer2 infections. Our combined analysis of the Wolbachia infection status and the associated host genotype show a highly complex picture of just partially increasing wCer2 frequencies, possibly shaped by endosymbiont losses, intraspecific horizontal transmission events, and potential anthropogenic effects.