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Year: 2013

Infections with the microbe Cardinium in the Dolichopodidae and other Empidoidea (Diptera)

Martin, Oliver Y ; Puniamoorthy, Nalini ; Gubler, Andrea ; Wimmer, Corinne ; Germann, Christoph ; Bernasconi, Marco V

Abstract: Maternally transmitted reproductive parasites such as and Cardinium can drasti- cally reshape reproduction in their hosts. Beyond skewing sex ratios towards females, these microbes can also cause cytoplasmic incompatibility. Wolbachia probably infects two thirds of insects, but far less is known about the occurrence or action of other with potentially similar effects. In contrast with the two more widespread reproductive parasites, Wolbachia and Spiroplasma, far less is known of infections with Cardinium () and possible consequences in the Diptera. Here, in an ex- tensive survey, 244 dipteran species from 67 genera belonging to the Dolichopodidae, Empididae, and Hybotidae were assessed for the presence of the microbe Cardinium. Although 130 of the species screened tested positive (ca. 53%), the presence of Cardinium could only be confirmed in 10 species (ca. 4%) based on analysis of sequences. Numerous additional sequences were found to be assignable to known or unknown Bacteroidetes. Considering the known issues concerning specificity of Cardinium primers and the phylogenetic uncertainties surrounding this microbe, the actual prevalence of this symbiont is worthy of further scrutiny. Potential directions for future research on Cardinium-host interactions in Diptera and in general are discussed.

DOI: https://doi.org/10.1673/031.013.4701

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-92204 Journal Article Published Version

Originally published at: Martin, Oliver Y; Puniamoorthy, Nalini; Gubler, Andrea; Wimmer, Corinne; Germann, Christoph; Bernasconi, Marco V (2013). Infections with the microbe Cardinium in the Dolichopodidae and other Empidoidea (Diptera). Journal of Insect Science, 13:47. DOI: https://doi.org/10.1673/031.013.4701

Journal of Insect Science: Vol. 13 | Article 47 Martin et al.

Infections with the microbe Cardinium in the Dolichopodidae and other Empidoidea

Oliver Y. Martin1a*+, Nalini Puniamoorthy2,3, Andrea Gubler2, Corinne Wimmer2, Chris- toph Germann2, Marco V. Bernasconi2,4b+

+ these authors contributed equally

1ETH Zürich, Experimental Ecology, Institute for Integrative Biology, CHN J 11, Universitätsstrasse 16, CH-8092 Zürich, Switzerland 2Institute of Evolutionary Biology and Environmental Studies, University of Zurich, Winterthurerstrasse 190, CH- 8057 Zürich, Switzerland 3Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singa- pore 4Natur-Museum Luzern, Kasernenplatz 6, CH-6003 Luzern, Switzerland

Abstract Maternally transmitted reproductive parasites such as Wolbachia and Cardinium can drastically reshape reproduction in their hosts. Beyond skewing sex ratios towards females, these microbes can also cause cytoplasmic incompatibility. Wolbachia probably infects two thirds of insects, but far less is known about the occurrence or action of other bacteria with potentially similar effects. In contrast with the two more widespread reproductive parasites, Wolbachia and Spiroplasma, far less is known of infections with Cardinium (Bacteroidetes) and possible consequences in the Dip- tera. Here, in an extensive survey, 244 dipteran species from 67 genera belonging to the Dolichopodidae, Empididae, and Hybotidae were assessed for the presence of the microbe Car- dinium. Although 130 of the species screened tested positive (ca. 53%), the presence of Cardinium could only be confirmed in 10 species (ca. 4%) based on analysis of sequences. Nu- merous additional sequences were found to be assignable to known or unknown Bacteroidetes. Considering the known issues concerning specificity of Cardinium primers and the phylogenetic uncertainties surrounding this microbe, the actual prevalence of this symbiont is worthy of further scrutiny. Potential directions for future research on Cardinium-host interactions in Diptera and in general are discussed.

Keywords: reproductive parasite, Rickettsia, Spiroplasma, symbiont, Wolbachia Correspondence: a [email protected], b [email protected], *Corresponding author Editor: Michael Strand was editor of this paper. Received: 9 January 2012 Accepted: 5 September 2012 Copyright : This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unre- stricted use, provided that the paper is properly attributed. ISSN: 1536-2442 | Vol. 13, Number 47 Cite this paper as: Martin OY, Puniamoorthy N, Gubler A, Wimmer C, Germann C, Bernasconi MV. 2013. Infections with the microbe Cardinium in the Dolichopodidae and other Empidoidea. Journal of Insect Science 13:47. Available online: http://www.insectscience.org/13.47

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Journal of Insect Science: Vol. 13 | Article 47 Martin et al.

Introduction Surveys for infections with the two more common reproductive parasites, Wolbachia Maternally inherited reproductive parasites and Spiroplasma, are available for many ar- such as Wolbachia, Rickettsia, and Spiro- thropods (e.g., for spiders: Goodacre et al. plasma species are known to have profound 2006). Within the Diptera, previous extensive effects on reproduction and behavior (e.g., surveys have focused on the genus Drosophila Goodacre et al. 2009) of their hosts (reviewed (Clark et al. 2005; Mateos et al. 2006), the in Engelstädter and Hurst 2008; Goodacre and Empidoidea (Martin et al. 2013), and the Martin 2012). More recently, a further repro- Muscoidea, including the yellow dung fly, ductive parasite, Cardinium hertigii Scathophaga stercoraria (Martin et al. 2012). (Bacteroidetes) (Zchori-Fein et al. 2004), has Both these symbionts have been shown to been described. Cardinium infections were manipulate host reproduction in flies. Wolba- first observed in ticks (Kurtti et al. 1996), and chia causes cytoplasmic incompatibility in are found in a wide range of spiders, mites, Culex pipiens (Yen and Barr 1971), and Spi- and other arachnids (Zchori-Fein and Perlman roplasma is associated with male-killing in 2004; Chigira and Miura 2005; Groot and Drosophila willistoni (Hackett et al. 1986). Breeuwer 2006; Enigl and Schausberger 2007; Wolbachia has also been shown to affect other Duron et al. 2008b; Gruwell et al. 2009; Mar- reproductive traits in Drosophila. Specifically, tin and Goodacre 2009; Chang et al. 2010; the parasite increases male mating rate Breeuwer et al. 2012). Although this microbe (Champion de Crespigny et al. 2006) and de- seems to be more common in arachnids, it is creases sperm competitive ability (Champion also known to infect insects (Zchori-Fein and de Crespigny and Wedell 2006). However, Perlman 2004; Provencher et al. 2005; knowledge of infections with Cardinium and Bigliardi et al. 2006; Marzorati et al. 2006; possible consequences in the Diptera lags far Sirviö and Pamilo 2010). behind.

Cardinium is now known to cause three of the As outlined above, Diptera, and especially four classic phenotypes often associated with Drosophila flies, have been instrumental in reproductive parasites: cytoplasmic incom- furthering the understanding of host- patibility in the parasitoid wasp Encarsia reproductive parasite interactions. With over pergendiella (Hunter et al. 2003), the spider 7,100 species worldwide, the Dolichopodidae, mite Bryobia sarothamni (Ros and Breeuwer long-legged flies, represent one of the most 2009), and the tetranychid mites Eotetrany- speciose families in the Diptera (Pape et al. chus suginamensis (Gotoh et al. 2006) and 2009). The actual number of taxa is presuma- Tetranychus cinnabarinus (Xie et al. 2010); bly higher, especially in the tropics, where feminization in Brevipalpus mites (Weeks et dozens of new taxa are discovered every year al. 2001); and parthenogenesis in the hemip- (e.g., Bickel 2009). The greatest diversity and teran Aspidiotus nerii (Provencher et al. 2005) abundance of long-legged flies are found at and in Encarsia species (Zchori-Fein et al. humid sites, and the flies show high potential 2001; 2004). In contrast to the better-known as bio-indicators for natural quality assess- symbionts Wolbachia and Spiroplasma, the ments of biotopes due to their specific habitat parasite Cardinium has so far not been found requirements (Pollet 2009). Adult and larval to be associated with male-killing. stages of most species feed on small soft-

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Journal of Insect Science: Vol. 13 | Article 47 Martin et al. bodied (Ulrich 2004), and because families, namely Empididae and Hybotidae, of this they are considered to be of potential for infection with Cardinium via PCR (see use in biocontrol. Finally, their elaborate Martin et al. 2013 for data on three other courtship behavior and their conspicuous male symbionts). DNA was extracted from whole secondary sexual characters further provide fly specimens using DNeasy Tissue kits excellent opportunities for studying sexual (Qiagen AG, www.qiagen.com) according to selection and speciation processes (Germann the manufacturer’s instructions. Whole speci- et al. 2010; Martin et al. 2013). This is par- mens were triturated mechanically in ticularly relevant, as reproductive parasites microtubes using a TissueLyser (Mixer Mill have been shown to have profound effects on MM 300, Qiagen AG). Following digestion reproductive traits and potential involvement with Proteinase K (2 µg/mL), samples were in reproductive isolation (Wade and Stevens applied to the columns for DNA absorption 1985), with obvious direct links to the study and washing. Finally, DNA was eluted in 200 of sexual selection (discussed in Martin et al. µl of the buffer from the kit and stored at -20° 2012; 2013). C. All the extracted fly specimens were de- posited at the Zoological Museum, Institute of Considering the dramatic effects the microbe Evolutionary Biology and Environmental Cardinium can have on its hosts, it Studies, University of Zurich. Standard PCR is important to acquire further knowledge, reactions were performed with 2 µl of the ex- both concerning the range of hosts affected tracted DNA as template, 1 µl of each primer and the precise consequences of infection. (10 µM), 12.5 µl Master Mix (250 units, Moreover, it has been suggested that a greater HotStarTaq Master Mix Kit, Qiagen AG), and range of arthropods should be screened in or- 8.5 µl distilled H2O, for a total volume of 25 der to achieve a better understanding of µl (manufacturer’s buffer). The following spe- Cardinium phylogenetic diversity (Nakamura cific primers (Microsynth GmbH, et al. 2009). Surprisingly, knowledge of Car- www.microsynth.ch) were used: Cardinium dinium infections within the Diptera remains (16S rDNA gene), CardiniumCh-F: particularly hazy. Here, 244 fly species from TACTGTAAGAATAAGCACCGGC, and 67 genera belonging to the Dolichopodidae, CardiniumCh-R: Empididae, and Hybotidae (all superfamily GTGGATCACTTAACGCTTTCG (Zchori- Empidoidea) were surveyed for the presence Fein and Perlman 2004). The PCR reaction of this reproductive parasite (as also done for mixtures were subjected to 10 min DNA de- Wolbachia, Spiroplasma, and Rickettsia in naturation at 95° C, 50 cycles of denaturation Martin et al. 2013). at 94° C for 30 sec, annealing at 50° C for 20 sec, and elongation at 72° C for 30 sec. Elon- Methods gation was completed by a further 7 min step at 72° C. PCR reactions were performed in a Samples, DNA extraction, amplification DNA Thermal Cycler (Perkin-Elmer Applied Extensive DNA samples from previous stud- Biosystems, www.perkinelmer.com). Purifica- ies (Bernasconi et al. 2007a; 2007b; Germann tion of PCR products for direct sequencing et al. 2010; 2011ab; Pollet et al. 2010; 2011) was performed by adding 0.5 µl (1 U/µl) and a few additional fly samples were used to Shrimp Alkaline Phosphatase (Promega AG, assess a range of representatives of the Doli- www.promega.com), 0.25 µl (20 U/µl) Exon- chopodidae and species from other empidoid culease I (New England Biolabs (Bioconcept),

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www.neb.com), and 24.25 µl distilled H2O Results (ratio of PCR product and ExoSap-mix 1:1) to each PCR product. The ExoSAP protocol con- Out of the 244 species screened, over half of sisted of 45 min incubation at 37° C and 15 the species tested positive (130/244, ca. 53%; min deactivation at 80° C. Cycle sequencing see results of PCR survey displayed in Table reactions were performed in total volumes of 1), with positives found across all three fami- 10 µl using an ABI Prism Big Dye Terminator lies surveyed. At the genus level, the Cycle Sequencing Kit (Perkin-Elmer Applied following patterns were observed: in the Em- Biosystems) on an ABI 3730 DNA Analyser pididae, five out of seven genera (ca. 71%) (Perkin-Elmer Applied Biosystems), again tested positive, compared with 4/9 (ca. 44%) following the manufacturer’s instructions. in the Hybotidae and 35/51 (ca. 69%) in the Dolichopodidae (sensu lato + sensu stricto; Negative and positive controls were used dur- see Pollet and Brooks 2008). However, subse- ing the amplification procedures. The negative quent sequencing of amplicons, and searching controls consisted of micro-tubes/positions in for most closely related bacterial sequences, the reaction plates containing all the necessary indicated that not all of these positives can be reagents, except that the extracted genomic assigned to Cardinium. Of the 169 sequences DNA to be amplified was substituted with dis- amplified, 15 had to be discarded because the tilled H2O. Positive controls consisted of quality was too poor to allow analysis. Of the extracted genomic DNA of samples infected remaining 154, only 10 of the sequences came with the microorganism under study (for the out as clearly assignable to Cardinium (see PCR) or in purified PCR products (for the di- overview in Table 2). These sequences stem rect sequence). Results of PCR amplification from species from five subfamilies within the were visualized via Gel Electrophoresis. Dolichopodidae s. stricto representing the genera Argyra, Chrysotus, Medetera, Peler- DNA sequence analyses opeodes, Sciapus, and Tachytrechus. In six Gene sequences were handled and stored us- cases, the closest matches were sequences ob- ing the Lasergene program Editseq (DNAstar tained from three hemipterans (Table 2). The Inc., www.dnastar.com). Sequence alignment other sequences matched either the hymenop- was performed using the Clustal W method as teran Plagiomerus diaspidis, the copepod implemented in Megalign (DNAstar Inc.) us- Nitocra spinipes, or the spider mite Tetrany- ing the default multiple alignment parameters chus cinnabarinus (Table 2). (gap penalty = 15; gap length penalty = 6.66; delay divergent sqs (%) = 30; DNA transition A further 50 sequences could be assigned to weight = 0.50). All obtained microbial se- other Bacteroidetes as either known or un- quences were checked in GenBank using the known microbes (N = 27) (Table 2). The BLAST tool to verify their identity, i.e., to remaining sequences belonged to the Pro- determine their similarity to known Cardi- teobacteria, including 13 matching the nium sequences or, conversely, to find out opportunistic pathogen Pseudomonas (see their possible identity as other Bacteroidetes D’Argenio et al. 2001). A sequence obtained or unknown (taxonomically undescribed) bac- from one Dolichopus plumipes sample closely teria. matched the pathogen Rickettsiella tipulae (Gammaproteobacteria), known to infect the crane fly, Tipula paludosa (Leclerque and

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Journal of Insect Science: Vol. 13 | Article 47 Martin et al. Kleespies 2008; Bouchon et al. 2012). The imicola (Ceratopogonidae); Culex pipiens remaining sequences were either unknown (Culicidae); Drosophila melanogaster and D. Proteobacteria (N = 28) or unknown bacteria simulans (Drosophilidae); Musca domestica (N = 38). (Muscidae); and Ceratitis capitata (Tephriti- dae). C. pipiens and the two species of Discussion Drosophila were found to be infected with Wolbachia, but none of the species assessed The species from the Dolichopodidae, Em- were infected with Cardinium. Similarly, no pididae, and Hybotidae assessed here seemed infections with Cardinium were found in a to show a high proportion of infections based survey of 181 strains representing 35 species on PCR results alone. This would be notewor- of Drosophila (Mateos et al. 2006). Duron et thy, as Cardinium is generally thought to be al. (2008) also surveyed 25 dipteran species as less widespread than other symbionts such as part of a larger survey and, again, found no Wolbachia and Spiroplasma. Whereas Wolba- evidence for infections with Cardinium. Fi- chia is thought to infect ca. 66% of insects nally, Nakamura et al. (2009) assessed (Hilgenboeker et al. 2008), previous surveys Culicoides biting midges and described infec- indicated that Cardinium may be much rarer tions with a new Cardinium group in four out overall in arthropods and are perhaps re- of 25 species tested. Here, the first extensive stricted to particular groups, such as survey specifically assessing possible infec- Hemiptera (see Nakamura et al. 2009). For tions with Cardinium in a wider range of fly example, extensive surveys across spiders and species is provided, and the presence of Car- other arachnids indicated that proportions of dinium in 10 species from six genera is infected samples were generally higher than in confirmed. So far, to our knowledge, our sur- insects (Weeks et al. 2003; Duron et al. vey represents the only evidence for infections 2008b; Martin and Goodacre 2009). Neverthe- with Cardinium in Diptera other than Culicoi- less, Zchori-Fein and Perlman (2004) included des (Nakamura et al. 2009). 85 insect species in their study, and found that only 6% of species screened tested positive The majority of sequences could not be as- for Cardinium versus 24% positive for Wol- signed to Cardinium, although numerous bachia. This low prevalence in insects was sequences were assignable to other Bacteroi- mirrored in our study, as a similar prevalence detes within the survey. Some of these of ca. 4% (10/244) was found using only those sequences may well represent Cardinium in- infections that were confirmed via direct se- fections, so this possibility definitely warrants quencing. further scrutiny. However, it must also be not- ed that the situation is complicated by the fact Concerning the more specific situation in Dip- that the phylogenetic relationships within tera, some fly species have been assessed as Cardinium are still confused and hence re- part of more general surveys encompassing main fluid (see Gruwell et al. 2009; Nakamura various arthropod groups. Zchori-Fein and et al. 2009; Perlman et al. 2010; discussed in Perlman (2004) assessed 11 species of Diptera Breeuwer et al. 2012). To a certain extent, this as part of their survey: Lucilia ciricata (Cal- problem also applies to reproductive parasites liphoridae); Asphondylia capparis, in general; for example, whether the far more Dasineuriola sp., and Schizomyia sp. (Ceci- intensively studied Wolbachia represents one domyiidae); Culicoides circumscriptus and C. or two species is still debated (Lo et al. 2007;

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Journal of Insect Science: Vol. 13 | Article 47 Martin et al. Pfarr et al. 2007). This problem may be addi- spider) hosts. tionally compounded by general issues of false negatives inherent to PCR-based screen- Based on work on other symbionts, the conse- ing (see Breeuwer et al. 2012), so overall, it is quences of infections can be extraordinarily likely that frequency of infection with symbi- varied and affect not only reproduction, but onts is being underestimated and novel also immunity or non-reproductive behav- bacteria are being missed (see e.g., Weinert et iours. For example, Wolbachia infections can al. 2007). In our study, the Ch primer set increase resistance to viruses in Aedes aegypti (Zchori-Fein and Perlman 2004) was used, as (Bian et al. 2010), and Rickettsia infection can this primer pair is commonly used for detec- hamper long-range dispersal behavior in a tion of Cardinium (Breeuwer et al. 2012). spider (Goodacre et al. 2009). Another area Further work would be needed to resolve the worthy of investigation is what happens when issue, and one option would be to use more hosts harbor multiple infections (see Table 2). than one primer pair, or supplement molecular Co-infections with different strains of the data with morphological analysis, as done by same bacterium or different reproductive Nakamura et al. (2009). If some of these se- parasites are known to occur across arthro- quences are in fact Cardinium, the microbe pods (Weeks et al. 2003; Zchori-Fein and may be a more widespread symbiont of ar- Perlman 2004; Goodacre et al. 2006; Gotoh et thropods than previously assumed. al. 2006; Duron et al. 2008a; Skaljac et al. 2010; Goodacre and Martin 2012). This prob- Unsurprisingly, considering the glaring pau- lem has been the focus of theoretical (e.g., city of knowledge concerning the range of Engelstädter et al. 2008; Vautrin et al. 2008) Dipteran species infected, so far nothing at all and empirical study focusing specifically on is known of the consequences of infection for interactions between Cardinium and the wide- hosts in this group. Now that it has been es- spread Wolbachia (Gotoh et al. 2006; Ros and tablished that Cardinium can infect additional Breeuwer 2009; White et al. 2009; Sirviö and dipteran hosts from an important group in the Pamilo 2010). context of sexual selection, it would be worthwhile to investigate potential conse- In conclusion, this is the first extensive survey quences. As noted above, Cardinium can specifically assessing possible infections with cause sex ratio distortion or reproductive al- Cardinium in a wide selection of Diptera be- terations in its hosts. So far, research has longing to the empidoid families demonstrated association with parthenogene- Dolichopodidae, Empididae, and Hybotidae sis in Hemiptera, parthenogenesis and (see Martin et al. 2013 for other symbionts). cytoplasmic incompatibility in Hymenoptera, Although a large number of species tested and cytoplasmic incompatibility and feminiza- positive for Cardinium in the PCR survey, tion in Acarida (reviewed in Duron et al. only 10 of the 154 sequences analyzed could 2008a). Effects need not be so drastic though, be assigned with certainty to Cardinium. as Cardinium has also been shown to be asso- These sequences stem from 10 species repre- ciated with increased fecundity in the senting five subfamilies within the predatory mite Metaseiulus occidentalis Dolichopodidae. We suggest that both the is- (Weeks and Stouthamer 2004). It would be sues surrounding specificity of Cardinium highly interesting to assess the effects Cardi- primers (see Breeuwer et al. 2012) and the nium has on its Dipteran (and other, e.g., related question of how commonly this mi-

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Journal of Insect Science: Vol. 13 | Article 47 Martin et al. crobe might really infect arthopods in toto re- quire further resolution. Finally, little is Bigliardi E, Sacchi L, Genchi M, Alma A, Pa- known of the effects this microbe has in Dip- joro M, Daffonchio D, Marzorati M, Avanzati tera, so possible impacts on its hosts and AM. 2006. Ultrastructure of a novel Cardi- interactions with other endosymbionts in this nium sp. symbiont in Scaphoideus titanus speciose group are worthy of future study. (Hemiptera: Cicadellidae). Tissue & Cell 38: 257–261. Acknowledgements Breeuwer H, Ros VID, Groot TVM. 2012. The authors would like to thank the Swiss Na- Cardinium: The next addition to the family of tional Science Foundation for support reproductive parasites. In: Zchori-Fein E, (Ambizione grants PZ00P3_121777 and Bourtzis K, Editors. Manipulative Tenants: PZ00P3_137514; standard research grant Bacteria Associated with Arthropods. pp. 31003A_125144/1 to O. Y. Martin and 207–224. Taylor & Francis. 31003A-115981 / 47191402 to M. V. Bernas- coni ), N. Puniamoorthy was supported by the Bouchon D, Cordaux R, Grève P. 2012. Rick- National University of Singapore Overseas ettsiella, intracellular pathogens of arthropods. Graduate Scholarship (NUS-OGS). We also In: Zchori-Fein E, Bourtzis K, Editors. Ma- thank Marc Pollet (INBO Brussels) for pro- nipulative Tenants: Bacteria Associated with viding fly samples, and Editor Michael Strand Arthropods. pp. 127–148. Taylor & Francis. for valuable feedback. Champion de Crespigny FEC, Wedell N. References 2006. Wolbachia infection reduces sperm competitive ability in an insect. Proceedings Bernasconi MV, Pollet M, Varini-Ooijen M, of the Royal Society of London B 273: 1455– Ward PI. 2007a. Phylogeny of European Dol- 1458. ichopus and Gymnopternus (Diptera, Dolichopodidae) and the significance of mor- Champion de Crespigny FEC, Pitt TD, Wedell phological characters inferred from molecular N. 2006. Increased male mating rate in Dro- data. European Journal of Entomology 104: sophila is associated with Wolbachia 601–617. infection. Journal of Evolutionary Biology 19: 1964–1972. Bernasconi MV, Pollet M, Ward PI. 2007b. Molecular systematics of Dolichopodidae Chang J, Masters A, Avery A, Werren JH. (Diptera) inferred from COI and 12S rDNA 2010. A divergent Cardinium found in daddy gene sequences based on European exemplars. long-legs (Arachnida: Opiliones). Journal of Invertebrate Systematics 21: 453–470. Invertebrate Pathology 105: 220–227.

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Table 1. Overview of the number of infected species and genera as determined via PCR screens.

*see Pollet and Brooks (2008)

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Table 2. Overview of Cardinium sequences. Evidence for multiple infections when present data is combined with Martin et al. 2013. M. impigra and M. petrophiloides samples were also infected with Spiroplasma, M. saxatilis and Sciapus platypterus additionally harbored Wolbachia, whereas A. vestita and Chrysotus palustris were infected with both Spiroplasma and Rickettsia.

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