MOLECULAR ENTOMOLOGY Expression of Cytoplasmic Incompatibility and Host Fitness Effects in Field Populations of Sogatella furcifera Infected With Cardinium

1 1 2 XIANG-FEI ZHANG, DONG-XIAO ZHAO, HAO-SEN LI, AND XIAO-YUE HONG

Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China Downloaded from https://academic.oup.com/jee/article/105/6/2161/797780 by guest on 23 September 2021 J. Econ. Entomol. 105(6): 2161Ð2166 (2012); DOI: http://dx.doi.org/10.1603/EC12268 ABSTRACT ÔCandidatus CardiniumÕ is a maternally inherited intracellular bacterium that infects a wide range of and causes diverse reproductive alterations of its hosts, including cytoplasmic incompatibility (CI), parthenogenesis and feminization. CI is the most common effect and is expressed as a reduction in the number of offspring in crosses between infected males and uninfected females (or females infected with a different bacterial strain). The white-backed Sogatella furcifera (Horva´th) is a major rice pest in Asia and is a vector of the Southern rice black-streaked dwarf virus (SRBSDV). In this study, we found severe CI expression induced by Cardinium in Yunnan and Guangxi populations of S. furcifera by performing four crosses of Cardinium-infected (C) and uninfected (U) : &U ϫ (U, &U ϫ (C, &C ϫ (U, &C ϫ (C. Investigations of the Þtness costs and beneÞts of Cardinium infection in these populations showed that it had no effect on the fecundity of females, but could shorten the developmental time of nymphs.

KEY WORDS Cardinium, Sogatella furcifera, CI, sex ratio, Þtness

Cardinium (Flexibacteriaceae, Class Sphingobacteria, mon effect associated with endosymbiont infection Phylum ) is a bacterial endosymbiont (Stouthamer et al. 1999, OÕNeill et al. 1997), and has that was Þrst found in cell cultures established from been observed in crosses between infected males the tick Ixodes scapularis Say (Kurtti et al. 1996). and either uninfected females (unidirectional CI) Compared with , which have been or females infected by a different strain (bidirec- known to manipulate arthropod reproduction since tional CI). Cardinium-induced CI has been found in 1970s (Werren et al. 2008), Cardinium bacteria are the spider mite Eotetranychus suginamensis (Gotoh relatively new to biological study. Cardinium has been et al. 2007), the red poultry mite Dermanyssus gal- detected in Hymenoptera (Hunter et al. 2003, Matalon linae (De Luna et al. 2009), the parasitoid wasp et al. 2007, Weeks et al. 2003, ZchoriÐFein et al. 2001, Encarsia pergandiella (Hunter et al. 2003), and the ZchoriÐFein and Perlman 2004), (Weeks sexual spider mite Bryobia sarothamni (Ros and et al. 2003, ZchoriÐFein et al. 2004, Bigliardi et al. 2006, Breeuwer 2009). Perlman et al. 2006), Acari (Enigl and Schausberger The whitebacked planthopper Sogatella furcifera 2007, Gotoh et al. 2007, Groot and Breeuwer 2006, Hoy (Horva´th) (Hemiptera: ) is a major rice et al. 2008, Weeks et al. 2001), and Areneae (Duron et pest in Asia. The nymphs and adults suck sap from rice al. 2008). Cardinium was found in Þve of Þve species plants, resulting in leaf yellowing, reduced tillering, (100%) of spider mites (Gotoh et al. 2007), and in 9 of stunting, unÞlled grains and hopperburn. S. furcifera 22 species (22%) of spider mites, in 4 of 25 species (16%) of biting midges Culicoides (Diptera: Cera- has caused intermittent famines in eastern Asia after topogonidae) and in 27 of 57 species (47.4%) of pl- the so-called Green Revolution of the 1960s (Noda et anthoppers (Nakamura et al. 2009, Zhang et al. 2012a). al. 2008). Particularly, S. furcifera is an important vec- Cardinium can induce a number of reproductive tor of the Southern rice black-streaked dwarf virus manipulations in arthropods similar to those in- (SRBSDV). We previously showed that the infection duced by Wolbachia to optimize its transmission. frequency of Cardinium was high in Chinese popula- Cardinium can induce parthenogenesis in scale in- tions of S. furcifera (Zhang et al. 2012b). Here, we sects (Provencher et al. 2005) and Encarsia hispida, investigated the reproductive manipulations and Þt- and feminization in Brevipalpus phoenicis (Weeks et ness costs and beneÞts by Cardinium in S. furcifera. We al. 2001, Groot et al. 2006). Cardinium can also induce determined the phylogenetic relationships of the Car- cytoplasmic incompatibility (CI). CI is the most com- dinium strains found in two S. furcifera populations and measured their effects on CI, the female ratio, 1 These authors contribute equally. fecundity, survival, and development times under lab- 2 Corresponding author, e-mail: [email protected]. oratory conditions.

0022-0493/12/2161Ð2166$04.00/0 ᭧ 2012 Entomological Society of America 2162 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 6

Materials and Methods Sample Collection. S. furcifera were collected from Guangxi Zhuang Autonomous Region (GX) and Yun- nan Province (YN) in south China in July 2010. Field- collected planthoppers were reared on rice seedlings in a climate-controlled room (25ЊC, 60% relative hu- midity [RH], and a photoperiod of 16:8 [L:D]). Preparation of a Singly Cardinium-Infected Strain. Single pairs of male and female adult planthoppers of a natural population were placed in plastic cups (120 mm in height and 80 mm in diameter) containing rice

seedlings. The test pairs laid eggs in the rice seedlings Downloaded from https://academic.oup.com/jee/article/105/6/2161/797780 by guest on 23 September 2021 and were removed from the cups after 2 wk. The females were checked for Wolbachia and Cardinium infection by polymerase chain reaction (PCR) ampli- Þcation. The offspring from the singly Cardinium- infected (Wolbachia-free) mothers were separately reared in plastic cups. For each group, pairs of males and females were bred as in the Þrst generation. Off- spring from females that did not have the same infec- tion status as their mothers were discarded. This se- lection regime was maintained for Þve successive generations until 100% Cardinium-infected popula- Fig. 1. Phylogenetic tree based on 16S rDNA sequences tions were obtained. of Cardinium, constructed by using a neighbor-joining pro- cedure in CLUSTAL X. Each Cardinium is shown by its host PCR and Cloning. DNA was extracted from single name. Numbers on the nodes indicate bootstrap values (%). planthopper according to the method of OÕNeill et al. S. furcifera populations were collected from Guangxi Zhuang (1992). DNA was extracted by homogenizing a single Autonomous region (GX) and Yunnan Province (YN) in individual in a 40 ␮l mixture of STE buffer (100 mM south China, respectively. Asterisks designate symbiont se- NaCl, 10 mM Tris-HCl, 1 mM EDTA, and pH 8.0) in quences obtained in this study. a 1.5 ml Eppendorf tube and incubated with 2.5 ␮l proteinase K (10 mg/ml, 2.5 ␮l) at 37ЊC for 30 min, then heated at 95ЊC for 5 min. The samples were then removed from the tube. After another week, centrifuged brießy, then used at once for the PCR newly hatched nymphs by this time were counted, and reactions or stored at Ϫ20Њ. PCR detection of Wolba- the remaining deposited eggs in the seedlings were chia was performed using primers wsp-81 F and wsp- dissected to microscopically observe the develop- 691R (Braig et al. 1998). Thermal cycles were as fol- ment. Eye pigmentation (red in color) was examined lows: 95ЊC for 3 min; followed by 35 cycles of 95ЊC for as an indicator of egg development (Noda et al. 2001). 30 s, 55ЊC for 45 s, 72ЊC for 1 min, and 72ЊC for 5 min Emerging adult offspring (sons and daughters) were as a Þnal extension after the last cycle. PCR detection scored per cross to determine the sex ratio (the num- for Cardinium was performed using primers CLOf and ber of daughter offspring). Data were analyzed with CLOr (Weeks et al. 2003). Thermal cycles were as one-way analysis of variance (ANOVA) and means follows: 94ЊC for 2 min, 35 cycles at 94ЊC for 30 s, 57ЊC were compared using a Tukey-honestly signiÞcant dif- for 30 s, 72ЊC for 30 s, and 72ЊC for 5 min as a Þnal ference (HSD) test (SPSS 17.0). To normalize the extension after the last cycle. The sequences were data, an arcsine square-root transformation was used determined for at least three clones having opposite for egg hatchability and sex ratio. orientations. The PCR products were cloned into a Fitness Effects. The Þtness costs and/or beneÞts of pEASY-T3 Cloning Vector (TransGen, Beijing, the different infection statuses (Cardinium-infected China), sequenced in both directions using the uni- and uninfected) in S. furcifera were investigated by versal primer pairs M13, m13R. The sequence was analyzing the fecundity and the developmental dura- determined by the Dye Terminator Sequencing tion of females. The effects of different infection types method with a DNA Sequencer (model 377 and 3700, on female fecundity were tested by comparing the Applied Biosystems). number of eggs laid in 15 d by infected and uninfected Cross Experiments. The effects of crosses: &U ϫ females in crosses involving infected and uninfected (U, &U ϫ (C, &C ϫ (U, &C ϫ (C. Singly Car- males. dinium-infected planthopper on CI were determined Development time was measured by placing Ϸ150 (Fig. 1). For each cross, nymphal planthoppers were newly hatched nymphs into individual tubs containing individually reared in a test tube containing rice seed- rice seedlings. They were moved to fresh rice seed- lings and checked daily for emergence (to ensure they lings every 5 d until hatching. Hatching was checked were virgins). Upon emergence a female and a male daily. Data were analyzed with one-way ANOVA and were introduced into a test tube and allowed to lay means were compared using a Tukey-HSD test (SPSS eggs into the rice seedlings at 25Ð26ЊCfor2wkand 17.0). The number of eggs laid per female and the days December 2012 ZHANG ET AL.: CARDINIUM IN Sogatella furcifera 2163 Downloaded from https://academic.oup.com/jee/article/105/6/2161/797780 by guest on 23 September 2021

Fig. 2. CI resulting from crosses of the uninfected (U), Cardinium-infected (C) strains in YN and GX populations. Results are mean percent embryo hatch ϮSEM. F, female; M, male. Number of replicates for each of the nine cross types are shown in parentheses. of nymph stage were normalized by ln transforma- tween uninfected females and infected males (Table tions. 1), indicating that Cardinium induced high levels of CI in S. furcifera of YN and GX populations. Results Effects of Cardinium on Development Time of Nymphs. In both GX and YN populations, Cardinium Sequences of Cardinium Strains and Phylogenetic shortened the time to adulthood of infected females Analysis. We ampliÞed a 406 bp (bp) fragment of the (Fig. 3). In the GX population, the uninfected females 16S rDNA gene from Cardinium infecting S. furcifera. took (17.7 Ϯ 0.9) days to reach adults. But the Car- Nucleotide sequences of the 16S rDNA genes ampli- dinium-infected females took only (15.3 Ϯ 1.1) days to Þed from the YN and GX populations showed little become adults. In the YN population, signiÞcant dif- variation, with 99.5% similarity (two different nucle- ference was found between the time to adulthood of otides out of 406 nucleotides). The phylogenetic tree uninfected females (18.0 Ϯ 1.0 d) and infected females based on the 16S rDNA genes clearly indicated that Ϯ the microorganisms in the planthoppers were Car- (16.6 1.4 d). Obviously, infected females reached dinium, forming a monophyletic group with other adulthood signiÞcantly earlier than uninfected fe- Ͻ Cardinium. The Cardinium from the white-backed males (P 0.05; t-test) (Fig. 4). planthopper populations seemed to be closely related Effects of Cardinium on Host Fecundity. No differ- to the symbionts from Dicranotropis hamata in the ence in fecundity was observed between infected and phylogenetic tree (Fig. 1). According to the analyses uninfected females in either population (Fig. 4). of 16S rDNA sequences of Cardinium infecting the GX and YN populations using Bioedit, we found that the sequence of the corresponding protein remains un- changed, and thus, the phenotypic differences are not Table 1. Results of sex ratio of crosses between U and C in the expected. Therefore, we conclude that Cardinium in- YN and GX populations of S. furcifera fecting the GX and YN populations belong to the same strain. Cross Sex ratio Population ϫ N Effects of Cardinium on Host Reproduction. The (F M) (% females) proportions of hatched eggs of intra-population GX U ϫ U 17 41.3 Ϯ 2.7a crosses between infected and uninfected individuals U ϫ C 22 19.7 Ϯ 7.2b C ϫ U 23 47.3 Ϯ 2.0a of the YN and GX populations are shown in Fig. 2. Egg C ϫ C 21 43.5 Ϯ 2.9a hatchability was signiÞcantly lower in the incompat- *** ible crosses (U/C, (11.2 Ϯ 3.6) %) than in the com- YN U ϫ U 17 47.7 Ϯ 2.1a patible crosses (U/U, (91.6 Ϯ 7.1) %; C/U, (89.6 Ϯ 1.7) U ϫ C 18 18.1 Ϯ 1.3b Ϯ Ͻ C ϫ U 20 50.8 Ϯ 5.9a %; C/C, (91.6 7.1) %) in the GX population (P C ϫ C 22 46.0 Ϯ 3.3a 0.05). Similar results were found in the YN population *** (U/C, (8.6 Ϯ 7.9) %; U/U, (89.5 Ϯ 3.2) %; C/U, (87.7 Ϯ 1.6) %; C/C, (88.5 Ϯ 5.3) %). C, Cardinium-infected; U, uninfected; N, no. of replicates; NS, not SigniÞcant differences were found in the sex ratio of signiÞcant. Outcomes of statistical analyses are listed for each group of crosses. *P Ͻ 0.05, **P Ͻ 0.01, and ***P Ͻ 0.001 (ANOVA). Values the four crosses in both the GX and YN populations. (mean Ϯ SE) in a column followed by different letters are signiÞ- Male-biased sex ratio was observed in the cross be- cantly different at P Ͻ 0.05 (Tukey HSD test). 2164 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 6 Downloaded from https://academic.oup.com/jee/article/105/6/2161/797780 by guest on 23 September 2021

Fig. 3. Comparisons of development time of nymphs of S. furcifera in YN and GX populations. *P Ͻ 0.05, **P Ͻ 0.01, and ***P Ͻ 0.001 (t-test).

Discussion species, host age, mate frequency, temperature, and endosymbionts interaction. Various levels of CI can be According to phylogenetic analysis of the 16S rDNA sequences, the Cardinium from the white-backed pl- expressed even in different populations of the same anthopper populations seemed to be closely related to species or in the different individuals in the same the symbionts from D. hamata in the phylogenetic population (Gotoh et al. 2007). Phylogenetic analysis tree. The 16S rDNA sequence of the Cardinium strain of the 16S rDNA sequences reveals that S. furcifera of in the GX population had 99.5% similarity (two dif- the GX, YN, and HN populations were infected by the ferent nucleotides out of 406 nucleotides) to the se- same strain of Cardinium because we found that the quence in the YN population. sequence of the corresponding protein remains the Cardinium induced CI in both the GX and YN pop- same by analyzing the 16S rDNA sequences using ulations of S. furcifera. A reduction of egg hatchability Bioedit. Therefore, we infer that the different levels of was observed in the incompatible crosses caused by CI in the three S. furcifera populations result from Cardinium infection. Interestingly, no signiÞcant dif- different genetic backgrounds. From March to July of ference was found in egg hatchability between GX and each year, the S. furcifera of Guangxi and Yunnan YN populations. However, the CI was not as strong as migrate from the Greater Mekong Subregion, such as the CI induced by Cardinium in S. furcifera collected Thailand and Vietnam by the southwest winds. How- from Hainan Province (HN), the egg hatchability be- ever, the S. furcifera in Hainan are native. There are no ing only 3.1% in the HN population (Zhang et al. migrating white-backed planthoppers in the area (Liu 2012b). The strength of CI can be affected by host et al. 2010). Furthermore, endosymbiont effects on its

Fig. 4. Mean fecundity over 15 d for crosses between the uninfected (U), Cardinium-infected (C) strains in YN and GX populations. F, female; M, male. Number of replicates for each of the nine cross types are shown in parentheses. December 2012 ZHANG ET AL.: CARDINIUM IN Sogatella furcifera 2165 host evolve readily in natural populations (Weeks et or the induction of cytoplasmic incompatibility al. 2007). Such rapid evolution also helps to explain the (Bourtzis et al. 2008). Desired genotypes or trans- diversity of Cardinium effects on Þtness of different S. genes related to a Wolbachia infection would be ex- furcifera populations. Further studies are needed to pected to spread into a targeted population following determine the factors that affect the levels of CI be- the seeding of the targeted population with Wolba- tween GX, YN, and HN populations. chia-infected females. The reproductive phenotypes The incompatible crosses produced progeny with a caused by infection with Cardinium are surprisingly male-biased sex ratio, whereas the compatible crosses similar to those induced by Wolbachia. We suggest produced progeny with a balanced sex ratio. The re- that Cardinium has the potential to be used directly or duced number of daughters was mainly the result of as a gene-driving system to prevent S. furcifera from the death of female eggs, which has also been reported transmitting SRBSDV. Further studies are needed to in spider mites (Gotoh et al. 2007). This is concordant determine whether the transmission of SRBSDV by S. with the female mortality type of CI induced by an- furcifera is related to Cardinium infection. Downloaded from https://academic.oup.com/jee/article/105/6/2161/797780 by guest on 23 September 2021 other endosymbiont, Wolbachia (Ros and Breeuwer 2009, Breeuwer 1997, Vavre et al. 2000, Bordenstein et al. 2003, Mouton et al. 2005). In haplodiploid species, female mortality is the only CI type found for Car- Acknowledgments dinium so far (Hunter et al. 2003, Gotoh et al. 2007). We are grateful to Profs. Bao-Ping Zhai and Xia-Nian S. furcifera is the Þrst dipoid species have the reduced Cheng, Nanjing Agricultural University (NJAU), China for female offspring because of CI. However, similarity of help with suggestions and comments. We thank Xiao-Feng CI types between S. furcifera and haplodiploid species Xue and Hui Zhang of NJAU for help with identiÞcation and does not mean that their mechanisms are identical. cross experiments. This study was supported in part by grants Although CI mechanisms of Cardinium in S. furcifera from the Special Fund for Agro-ScientiÞc Research in the are still unclear, this new CI type in dipoid species Public Interest (200803003, 200903051) from the Ministry of Agriculture of China, and a grant-in-aid from the Specialized enlarges the diversity of the effects of Cardinium on its Research Fund for the Doctoral Program of Higher Educa- host and allows the study of the evolutionary history tion (SRFDP) from the Ministry of Education of China (Pri- of the strategies used by Cardinium. Overall, many ority Development Area, 20110097130005). more examples of CI in diploid species are needed to understand the evolution of interaction between en- dosymbionts and their hosts. Besides having strong CI effects, Cardinium also References Cited affected the Þtness of the S. furcifera populations. Bigliardi, E., L. Sacchi, M. Genchi, A. Alma, M. Pajoro, D. Endosymbionts have different effects on the devel- Daffonchio, M. Marzorati, and A. M. Avanzati. 2006. Ul- opment time of hosts. For example, Wolbachia short- trastructure of a novel Cardinium sp. symbiont in Scaph- ened the development time of Tetranychus urticae oideus titanus (Hemiptera: Cicadellidae). Tissue Cell. 38: Koch in Liaoning Province, but prolonged the devel- 257Ð261. opment time of T. urticae in Jiangsu and Hunan Prov- Bordenstein, S. R., J. J. Uy, and J. H. Werren. 2003. Host genotype determines cytoplasmic incompatibility type in inces (Xie et al. 2011). The virulent wDm popcorn the haplodiploid genus Nasonia. Genetics 164: 223Ð233. Wolbachia strain in D. melanogaster can delay the Bourtzis, K. 2008. Wolbachia-based technologies for development time of the host (Reynolds et al. 2003). pest population control. Adv. Exp. Med. Biol. 627: 104Ð However, in the stored-product pest Liposcelis bostry- 113. chophila, the development time of a Cardinium-free Braig, H. R., W.-G. Zhou, S. L. Dobson, and S. L. O’Neill. strain was similar to that of a Cardinium-infected strain 1998. Cloning and characterization of a gene encoding (Wang et al. 2008). Cardinium in both YN and GX S. the major surface protein of the bacterial endosymbiont furcifera populations shortened the development time Wolbachia pipientis. J. Bacteriol. 180: 2373Ð2378. of infected females. Similarly, Cardinium shortened Breeuwer, J.A.J. 1997. Wolbachia and cytoplasmic incom- the development time of nymphs of S. furcifera of the patibility in the spider mites Tetranychus urticae and T. turkestani. Heredity 79: 41Ð47. HN population (Zhang et al. 2012b). Because infected Cao, Y., F. Pan, Q. Zhou, G.-H. Li, S.-Q. Liu, Z.-N. Huang, and females reach adulthood signiÞcantly earlier than un- Y.-Z. Li. 2011. Transmission characteristics of Sogatella infected females, the development advantage pro- furcifera: a vector of the Southern rice black-streaked vided by Cardinium infection should facilitate its dwarf virus. Chin. J. Appl. Entomol. 48: 1314Ð1320. invasion into natural Þeld populations and its persis- De Luna, C. J., C. V. Moro, J. H. Guy, L. Zenner, and O.A.E. tence over time. Cardinium increased in fecundity of Sparagano. 2009. Endosymbiotic bacteria living inside the predatory mite, Metaseiulus occidentalis (Weeks the poultry red mite (Dermanyssus gallinae). Exp. Appl. and Stouthamer 2004), although it had no effect on the Acarol. 48: 105Ð113. fecundity of S. furcifera. Interestingly, crosses of fe- Duron, O., D. Bouchon, S. Boutin, L. Bellamy, L. Zhou, J. males with uninfected males showed that Cardinium Engelstadter, and G. D. Hurst. 2008. The diversity of reproductive parasites among arthropods: Wolbachia do infection did not affect male fertility. not walk alone. BMC Biol. 6: 27. S. furcifera is an important vector of the SRBSDV Enigl, M., and P. Schausberger. 2007. Incidence of the en- (Cao et al. 2011). Several strategies for disease or pest dosymbionts Wolbachia, Cardinium and Spiroplasma in control management have now been proposed, most phytoseiid mites and associated prey. Exp. Appl. Acarol. of which take advantage of Wolbachia virulent strains 42: 75Ð85. 2166 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 6

Gotoh, T., H. Noda, and S. Ito. 2007. Cardinium symbionts Reynolds, K. T., Thompson, L. J., and A. A. Hoffmann. 2003. cause cytoplasmic incompatibility in spider mites. He- The effects of host age, host nuclear background and redity 98: 13Ð20. temperature on phenotypic effects of the virulent Wolba- Groot, T. V., and J. A. Breeuwer. 2006. Cardinium symbionts chia strain popcorn in Drosophila melanogaster. Genetics induce haploid thelytoky in most clones of three closely 164: 1027Ð1034. related Brevipalpus species. Exp. Appl. Acarol. 39: 257Ð Ros, V.I.D., and J.A.J. Breeuwer. 2009. The effects of, and 271. interactions between, Cardinium and Wolbachia in the Hoy, M., and A. Jeyaprakash. 2008. Symbionts, including doubly infected spider mite Bryobia sarothamni. Heredity pathogens, of the predatory mite Metaseiulus occidentalis: 102: 413Ð420. current and future analysis methods. Exp. Appl. Acarol. Stouthamer, R., J.A.J. Breeuwer, and G.D.D. Hurst. 1999. 46: 329Ð347. Wolbachia pipientis: microbial manipulator of arthropod Hunter, M. S., S. J. Perlman, and S. E. Kelly. 2003. A bac- reproduction. Annu. Rev. Microbiol. 53: 71Ð102. terial symbiont in the Bacteriodetes induces cytoplasmic Vavre, F., F. Fleury, J. Varaldi, P. Fouillet, and M. Bou-

incompatibility in the parasitoid wasp Encarsia per- letreau. 2000. Evidence for female mortality in Downloaded from https://academic.oup.com/jee/article/105/6/2161/797780 by guest on 23 September 2021 gandiella. Proc. R. Soc. Lond. B. Biol. Sci. 270: 2185Ð2190. Wolbachia-mediated cytoplasmic incompatibility in Kurtti, T. J., U. G. Munderloh, T. G. Andreadis, L. A. Mag- haplodiploid : epidemiologic and evolutionary narelli, and T. A. Mather. 1996. Tick cell culture isola- consequences. Evolution 54: 191Ð200. tion of an intracellular prokaryote from the tick Ixodes Wang, J.-J., P. Dong, L.-S. Xiao, and W. Dou. 2008. Effects scapularis. J. Invertebr. Pathol. 67: 318Ð321. of removal of Cardinium infection on Þtness of the stored- Liu, J.-N., F.-R. Gua, and Z.-Y. Li. 2010. Genetic diversity of product pest Liposcelis bostrychophila (Psocoptera: Li- the planthopper, Sogatella furcifera in the Greater Me- poscelididae). J. Econ. Entomol. 101: 1711Ð1717. kong Subregion detected by inter-simple sequence re- Weeks, A. R., F, Marec, and J.A.J. Breeuwer. 2001. A mite peats (ISSR) markers. J. Insect Sci. 10: 1Ð14. species that consists entirely of haploid females. Science Matalon, Y., N. Katzir, Y. Gottlieb, V. Portnoy, and E. Zchori- 292: 2479Ð2482. Fein. 2007. Cardinium in Plagiomerus diaspidis (Hyme- Weeks, A. R., R. Velten, and R. Stouthamer. 2003. Incidence noptera: Encyrtidae). J. Invertebr. Pathol. 96: 106Ð108. of a new sex ratio-distorting endosymbiotic bacterium Mouton, L., H. Henri, M. Bouletreau, and F. Vavre. 2005. among arthropods. Proc. R. Soc. Lond. B. 270: 1857Ð1865. Multiple infections and diversity of cytoplasmic incom- Weeks, A. R., and R. Stouthamer. 2004. Increased fecundity patibility in a haplodiploid species. Heredity 94: 187Ð192. associated with infection by a Cytophaga-like intracellu- Nakamura, Y., S. Kawai, F. Yukuhiro, S. Ito, T. Gotoh, R. lar bacterium in the predatory mite, Metaseiulus occiden- Kisimoto, T. Yanase, Y. Matsumoto, D. Kageyama, and H. talis. Proc. R. Soc. Lond. B. 271: S193ÐS195. Noda. 2009. Prevalence of Cardinium in planthoppers Weeks, A. R., M. Turelli, W. R. Harcombe, T. K. Reynolds, and spider mites and taxonomic revision of “Candidatus and A. A. Hoffmann. 2007. From parasite to mutualist: Cardinium hertigii” based on detection of a new Car- rapid evolution of Wolbachia in natural populations of dinium group from biting midges. Appl. Environ. Micro- Drosophila. PLoS Biol. 5: 997Ð1005. biol. 75: 6757Ð6763. Werren, J. H., L. Baldo, and M. E. Clark. 2008. Wolbachia: Noda, H., S. Kawai, Y. Koizumi, K. Matsui, Q. Zhang, S. master manipulators of invertebrate biology. Nat. Rev. Furukawa, M. Shimomura, and K. Mita. 2008. Anno- Microbiol. 6: 741Ð751. tated ESTs from various tissues of the brown planthopper Xie, R.-R., X.-L. Chen, and X.-Y. Hong. 2011. Variable Þtness Nilaparvata lugens: a genomic resource for studying ag- and reproductive effects of Wolbachia infection in pop- ricultural pests. BMC Genomics 117: 1Ð18. ulations of the two-spotted spider mite Tetranychus ur- Noda, H., Y. Koizumi, Q. Zhang, and K. J. Deng. 2001. In- ticae Koch in China. Appl. Entomol. Zool. 46: 95Ð102. fection density of Wolbachia and incompatibility level in Zchori–Fein, E., and S. J. Perlman. 2004. Distribution of the two planthopper species, Laodelphax striatellus and Soga- bacterial symbiont Cardinium in arthropods. Mol. Ecol. tella furcifera. Insect Biochem. Mol. Biol. 31: 727Ð737. 13: 2009Ð2016. O’Neill, S. L., R. Giordano, A.M.E. Colbert, T. L. Karr, and Zchori–Fein, E., Y. Gottlieb, S. E. Kelly, J. K. Brown, J. M. H. M. Robertson. 1992. 16S rRNA phylogenetic analysis Wilson, T. L. Karr, and M. S. Hunter. 2001. A newly of the bacterial endosymbionts associated with cytoplas- discovered bacterium associated with parthenogenesis mic incompatibility in insects. Proc. Natl. Acad. Sci. U.S.A. and a change in host selection behaviour in parasitoid 89: 2699Ð2702. wasps. Proc. Natl. Acad. Sci. U.S.A. 98: 1255Ð1260. O’Neill, S. L., A. A. Hoffmann, and J. J. Werren. 1997. In- Zhang, K.-J., X. Han, and X.-Y. Hong. 2012a. Various infec- ßuential passengers. Inherited microorganisms and ar- tion status and molecular evidence for horizontal trans- thropod reproduction. Oxford University Press, New mission and recombination of Wolbachia and Cardinium York, NY. among rice planthoppers and related species. Insect Sci. Perlman, S. J., M. S. Hunter, and E. Zchori–Fein. 2006. The 19. (in press). (doi:10.1111/j.1744-7917.2012.01537.x). emerging diversity of Rickettsia. Proc. R. Soc. Lond. B. Zhang, X.-F., D.-X. Zhao, and X.-Y. Hong. 2012b. Cardinium: 273: 2097Ð2106. the leading factor of cytoplasmic incompatibility in the Provencher, L., G. E. Morse, A. R. Weeks, and B. B. Normark. planthopper Sogatella furcifera doubly infected with 2005. Parthenogenesis in the Aspidiotus nerii complex Wolbachia and Cardinium. Environ. Entomol. 41: 833Ð (Hemiptera: Diaspididae): a single origin of a worldwide 840. polyphagous lineage associated with Cardinium bacteria. Ann. Entomol. Soc. Am. 98: 629Ð635. Received 12 July 2012; accepted 14 September 2012.