Intrasperm Vertical Symbiont Transmission

Intrasperm vertical symbiont transmission

Kenji Watanabea, Fumiko Yukuhiroa, Yu Matsuurab,c,1, Takema Fukatsuc, and Hiroaki Nodaa,2

aNational Institute of Agrobiololgical Sciences, Tsukuba 305-8634, Japan; bGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8577, Japan; and cNational Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan

Edited by Nancy A. Moran, University of Texas, Austin, TX, and approved April 11, 2014 (received for review February 7, 2014) Symbiotic bacteria are commonly associated with cells and tissues infection to the sperm head nucleus is expected to impair genetic of diverse animals and other organisms, which affect hosts’ biol- material and normal functioning of the sperm and, thus, intrasperm ogy in a variety of ways. Most of these symbionts are present in vertical symbiont transmission may seem unlikely to occur. In this the cytoplasm of host cells and maternally transmitted through study, however, we demonstrate that such a case exists in an insect. host generations. The paucity of paternal symbiont transmission The green rice leafhopper Nephotettix cincticeps (Uhler) is likely relevant to the extremely streamlined sperm structure: the (Hemiptera: Cicadellidae) (Fig. 1A), known as a notorious pest head consisting of condensed nucleus and the tail made of micro- of rice in East Asia, is associated with two bacteriome-associated tubule bundles, without the symbiont-harboring cytoplasm that is obligate symbionts, Sulcia and Nasuia, and a facultative symbiont discarded in the process of spermatogenesis. Here, we report a pre- of the genus Rickettsia (22). The Rickettsia symbiont of N. cinc- viously unknown mechanism of paternal symbiont transmission ticeps represents a basal lineage of the genus Rickettsia (Fig. 1B) via an intrasperm passage. In the leafhopper Nephotettix cincti- and exhibits high infection frequencies among N. cincticeps ceps, a facultative Rickettsia symbiont was found not only in the strains established from natural populations in Japan (Table 1). cytoplasm but also in the nucleus of host cells. In male insects, Previous histological studies described that Rickettsia-like bac- strikingly, most sperm heads contained multiple intranuclear Rick- terial cells are present not only in the cytoplasm but also in ettsia cells. The Rickettsia infection scarcely affected the host fitness the nucleus of various cells and tissues of several leafhopper including normal sperm functioning. Mating experiments revealed species including N. cincticeps (23, 24). Here we report that, in both maternal and paternal transmission of the Rickettsia symbiont N. cincticeps, the Rickettsia symbiont efficiently targets and through host generations. When cultured with mosquito and silk- infects the host’s cell nuclei including sperm head nuclei, and Rickettsia

worm cell lines, the symbiont was preferentially localized vertically transmitted to the next host generation not only ma- MICROBIOLOGY within the insect cell nuclei, indicating that the Rickettsia symbiont ternally via ovarial passage but also paternally via intrasperm itself must have a mechanism for targeting nucleus. The mecha- passage with high fidelity. nisms underlying the sperm head infection without disturbing sperm functioning are, although currently unknown, of both basic Results and Discussion and applied interest. When we observed various cells and tissues of our N. cincticeps stocks by transmission electron microscopy, the Rickettsia sym- ndocellular bacterial symbionts are commonly found in di- biont was consistently found not only in the cytoplasm, but verse eukaryotes including animals, plants, fungi, and protists also in the nucleus of Malphigian tubule cells, midgut cells, E A B (1–7). In the majority of these cases, the symbionts are located in and other types of host cells (Fig. 2 and ). In an attempt Rickettsia the cytoplasm of the host cells. Whereas the cytoplasmic sym- to microbiologically characterize the symbiont, asep- N. cincticeps bionts are simply passed to daughter cells through host cell di- tically dissected ovaries of were subjected to culti- vision in unicellular protists (6, 7), sex-related asymmetry in vation with insect cell lines, by which we established continuous vertical symbiont transmission is generally observed in multicellular metazoans with sexual reproduction. Namely, the sym- Significance bionts are transmitted vertically to the next host generation via infection to eggs in the maternal body, but not via infection to Diverse organisms are commonly associated with bacterial endo- sperms (8, 9). Exceptional reports of paternal symbiont trans- symbionts, which often affect hosts’ biology and phenotypes in mission are venereal transmission cases of several symbiotic a variety of ways. The majority of these symbionts are generally bacteria (10, 11) and biparental transmission cases of some sym- present in the host cell cytoplasm and maternally transmitted biotic viruses (12). Oocytes accumulate a large quantity of cyto- through host generations. Here, however, this conventional plasm that provide a room for symbiont infection, whereas knowledge is countered by our discovery of intrasperm vertical sperms discard their cytoplasm (together with inhabiting symbi- transmission of nuclear-targeting bacterial symbiont (Rickettsia) Nephotettix cincticeps otic bacteria) during spermatogenesis and transform into a in an insect (leafhopper ), which poten- streamlined shape with the small head consisting of condensed tially erodes the nuclear-cytoplasmic conflict that governs the nucleus and the slender tail made of microtubule bundles for majority of endosymbiotic associations. The molecular and cel- motility (13, 14). Therefore, if such symbiotic bacterial cells can lular mechanisms underlying the sperm head infection without be transmitted via sperm, a possible target may be the sperm disturbing sperm functioning are of not only basic but also ap- head nucleus. Intranuclear bacterial symbionts, such as Hol- plied interest, which may provide insights into the development of sperm-mediated genetic transformation and/or material de- ospora and Caedibacter, have been relatively well-documented livery technologies. from unicellular ciliates (6, 15), but reported only rarely from

multicellular metazoans (16, 17). In insects and other arthro- Author contributions: K.W., T.F., and H.N. designed research; K.W., F.Y., Y.M., and H.N. pods, intracellular Rickettsia and Orientia pathogens/symbionts performed research; Y.M. contributed new reagents/analytic tools; K.W. and H.N. ana- are sometimes observed to localize not only to the cytoplasm, but lyzed data; and T.F. and H.N. wrote the paper. also to the nucleus of the host cells (18–21). In bathymodiolin The authors declare no conflict of interest. mussels inhabiting hydrothermal vents and cold seeps, intra- This article is a PNAS Direct Submission. nuclear bacterial parasites “Candidatus Endonucleobacter bath- Freely available online through the PNAS open access option. ymodioli” have been described (16). Thus far, no case of paternal 1Present address: Graduate School of Environmental Science, Hokkaido University, Sapporo, symbiont transmission via intrasperm passage has been reported. Hokkaido 060-0810, Japan. Considering the extremely streamlined sperm structure, bacterial 2To whom correspondence may be addressed. E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1402476111 PNAS | May 20, 2014 | vol. 111 | no. 20 | 7433–7437 Downloaded by guest on September 30, 2021 species, which represent the distinct insect orders Hemiptera, A Diptera, and Lepidoptera, strongly suggests that the Rickettsia symbiont itself must have some mechanism for targeting insect cell nuclei.

A B n 1 mm n

88/68 Rickettsia felis [NC_007109] B 81/* Rickettsia symbiont of psocid Liposcelis bostrychophila [DQ407744] 99/60 Rickettsia akari [CP000847]

Rickettsia symbiont of birch catkin bug Kleidocerys resedae transitional 2 m 92/* [JQ726775] 2 m Rickettsia rickettsii [CP000766] 92/64 spotted fever Rickettsia conorii [NC_003103] 0.02 61/* Rickettsia typhi [AE017197] CD 99/80 typhus Rickettsia prowazekii [CP001584] Rickettsia canadensis [CP00409] canadensis n 93/50 n Rickettsia symbiont of aphid Acyrthosiphon pisum [AB196668] Rickettsia symbiont of weevil Curculio sp. [AB604673] 99/88

Rickettsia symbiont of seed bug Nysius expressus [JQ726774] bellii 93/61 Rickettsia bellii [CP000087] Rickettsia symbiont of ladybird Adalia bipunctata [U04163] adalia 97/80 93/60 Rickettsia symbiont of springtail Onychiurus sinensis [AY712949] onychiurus 2 m Rickettsia symbiont of bean beetle Kytorhinus sharpianus [AB021128] rhizobius 0.3 m Rickettsia symbiont of leafhopper Nephotettix cincticeps [AB702995] 63/* 100/99 Rickettsia symbiont of leech Torix tagoi [AB066351]

torix E F 65/57 Rickettsia symbiont of psocid Cerobasis questfalica [DQ652596] Rickettsia symbiont of ciliate Diophrys sp. [AJ630204] hydra Orientia tsutsugamushi [NC_010793] Wolbachia wMel of fruit fly Drosophila melanogaster [AE017196]

Fig. 1. The green rice leafhopper N. cincticeps and its Rickettsia symbiont. (A) An adult male of N. cincticeps.(B) Phylogenetic placement of the Rick- ettsia symbiont of N. cincticeps on the basis of 16S rRNA gene sequence. A Bayesian phylogeny inferred from 1,296 aligned nucleotide sites is shown. Posterior probabilities for the Bayesian phylogeny and bootstrap probabilities for the maximum likelihood phylogeny at 50% or higher are shown at 0.5 m 2 m the nodes, whereas asterisks indicate support values lower than 50%. Se- quence accession numbers are shown in brackets. Major Rickettsia groups G H (40) are indicated on the right side. st

Rickettsia cultures with the AeAl2 mosquito cell line and the aff3 silkworm cell line. In these heterospecific host cells, strikingly, the Rickettsia symbiont exhibited localizations not mv only to the cytoplasm but also to the nucleus (Fig. 2 C and sh D ). The consistent nuclear localization in the different host *

Table 1. Rickettsia infection frequencies in laboratory strains of 0.3 m 1 m N. cincticeps derived from different natural populations in Japan Strain Origin Year Infection rate,* % Fig. 2. Transmission electron microscopy of nuclear localization of the Rickettsia symbiont in tissues of N. cincticeps and cell lines of other insects. Tsukuba-A Yawara, Ibaraki 1988 100 (96/96) (A) Malphigian tubule cell of N. cincticeps.(B)MidgutcellofN. cincticeps. Tsukuba-B Tsukuba, Ibaraki 2006 100 (96/96) (C) Cell line aff3 derived from the silkworm B. mori.(D) Cell line AeAl-2 Jyouetsu Jyouetsu, Niigata 1993 100 (96/96) derived from the mosquito A. albopictus.(E) Cross-section of sperm heads Kagoshima Kagoshima, Kagoshima 2001 100 (96/96) in testis of N. cincticeps.(F) Longitudinal section of sperm heads in testis of Total ——100 (384/384) N. cincticeps.(G) Magnified image of the sperm heads. (H) Spermatheca of Rickettsia-uninfected female of N. cincticeps after mating with Rickettsia- *Diagnostic PCR was performed by using the primers NcRic_16S/f1 (5′-TGA infected male. mv, microvilli on the epithelium of spermatheca; n, nucleus; CGG TAC CTG ACC AAG A-3′) and NcRic_16S/r1 (5′-AAG GGA TAC ATC TCT sh, sperm head; st, sperm tail. Asterisk in H highlights an intrasperm GCT T-3′) as described (22). Rickettsia cell.

7434 | www.pnas.org/cgi/doi/10.1073/pnas.1402476111 Watanabe et al. Downloaded by guest on September 30, 2021 ABC

5 m

Fig. 3. In situ hybridization of the Rickettsia symbiont in mature sperm heads obtained from seminal vesicles of Rickettsia-infected males of N. cincticeps.(A) Red hybridization signals due to 16S rRNA of the Rickettsia symbiont. (B) Blue signals due to DNA staining of sperm heads. Note the unstained areas within the sperm heads, reflecting endonuclear localization of the Rickettsia symbiont cells. (C) Merged image. Note that a number of Rickettsia cells are arranged in a row within each sperm head.

Our electron microscopic observations of the testis of exhibited high rates of egg development almost comparable to – N. cincticeps revealed that, surprisingly, almost all sperm head those with R males (Table 3), indicating normal functioning of nuclei contained bacterial cells (Fig. 2 E–G). Of 296 sperm head the Rickettsia-infected sperms. Meanwhile, statistical analysis – + nuclei we inspected on electron microscopic images representing showed that the cross between R females and R males pro- a single cross-section of elongate sperm heads, 181 (61.1%) duced significantly less offspring than the other crosses (χ2 test; exhibited one or two bacterial cells on the sectioned plane (Fig. P < 0.001), suggesting slightly but significantly lower performance 2E). Longitudinal sections of the sperm heads revealed that the of the Rickettsia-infected sperms. These patterns may look like a bacterial cells are arranged in a line along the long axis within low level of cytoplasmic incompatibility, but more data should be each sperm head: More than 10 bacterial cells were often ob- accumulated to test this hypothesis. Electron microscopy of dis- served in a sperm head nucleus (Fig. 2 F and G). Fluorescence in sected spermathecae confirmed transfer of intrasperm Rickettsia + – situ hybridization of the sperm heads using specific oligonucle- symbiont cells from R males to R females (Fig. 2H). When all MICROBIOLOGY + – otide probes targeting 16S rRNA of the Rickettsia symbiont possible mating combinations within and between the R and R unequivocally demonstrated that the bacterial cells within the insect strains were examined, the Rickettsia symbiont exhibited sperm head nuclei represent the Rickettsia symbiont (Fig. 3 A–C). 100% maternal transmission and, notably, 61.8% (ranging from Of 1,109 sperm heads inspected by in situ hybridization and 40.0 to 80.0%) paternal transmission (Fig. 4B). This result indicates fluorescence microscopy, 1,026 (92.5%) contained one or more that only a few Rickettsia cells residing in the sperm head nucleus Rickettsia cells, which were on average 5.33 ± 3.52 and ranging are sufficient for establishing the paternal symbiont transmission. from 0 to 23 Rickettsia cells per sperm head (Fig. 4A). The paternally transmitted Rickettsia symbiont exhibited 100% Using a selective symbiont curing technique by rifampicin ad- vertical transmission in subsequent host generations. ministration via rice seedlings (25), we established Rickettsia- In conclusion, we discovered a previously unknown phenom- + – infected (R ) and uninfected (R )strainsofN. cincticeps under the enon: The intranuclear Rickettsia symbiont of N. cincticeps is same genetic background, with the obligate bacteriome symbionts transmitted efficiently through host generations via an intrasperm + – Sulcia and Nasuia remaining intact. These R and R insect passage. In general, endosymbiotic bacteria are maternally trans- strains exhibited similar levels of fecundity, growth, and survival mitted and, thus, potentially incur an evolutionary conflict with (Table 2), indicating no remarkable positive/negative effects of their host organisms whose traits are biparentally inherited, which the symbiont infection on the host fitness at least under our underlies such striking symbiont-mediated host phenotypes as cy- rearing condition, although the possibility cannot be excluded toplasmic incompatibility, male-killing, parthenogenesis induction, that some effects at moderate levels would be detected with and feminization (26). The not only maternal, but also effi- + larger sample sizes. Eggs produced by mating with R males cient, paternal symbiont transmission in N. cincticeps potentially

AB13 pairs 9 pairs 140 n = 187 n = 107 Mean ± SD = 5.33 ± 3.52 100 120 80 100 8 pairs n = 1,109 n = 107 80 60

60 40 40 20 20 rate (%) Transmission 7 pairs n = 94 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 < R+ x!" R+ R+ x#" R R $"x R+ R %"x R

Cumulative number of sperm heads Number of Rickettsia cells per sperm head [Female x Male]

Fig. 4. Number of Rickettsia cells in mature sperm heads and vertical transmission rates of Rickettsia.(A) Number of Rickettsia per sperm head based on in situ hybridization images of 1,109 sperm heads obtained from seminal vesicles of four Rickettsia-infected adult males. (B) Vertical transmission rates of Rickettsia upon all mating combinations within and between the Rickettsia-infected and uninfected host strains. The numbers of parent pairs and those of total offspring are indicated above the columns.

Watanabe et al. PNAS | May 20, 2014 | vol. 111 | no. 20 | 7435 Downloaded by guest on September 30, 2021 Table 2. Comparison of fitness parameters between Rickettsia- Rickettsia Cultivation. The Rickettsia symbiont was cultivated with the AeAl-2 infected and uninfected strains of N. cincticeps cell line derived from the mosquito Aedes albopictus (32) by inoculating a † ‡ part of dissected ovary of Rickettsia-infected N. cincticeps into a plastic dish Fecundity* Growth rate Survival, % containing the cells, according to the cultivation procedure for Wolbachia (33). The cultivated Rickettsia symbiont was further transferred to the aff3 Infected 4.5 ± 2.8 (n = 8) 17.1 ± 0.7 (n = 42) 89.9% (89/99) +§ cell line from the silkworm Bombyx mori (34). The AeAl-2 and aff3 cells were strain R grown in medium IPL-41 (GIBCO BRL 11505) supplemented with 5% (vol/vol) ± = ± = Uninfected 6.6 3.0 (n 9) 17.0 0.5 (n 38) 91.5% (86/94) FBS (Sigma) at 26 °C. strain R–§ { P value P = 0.16 P = 0.75 P = 0.70 Electron Microscopy. The tissue and cell samples were prefixed with 0.8% glutaraldehyde and 1% paraformaldehyde in 0.06 M phosphate buffer for *Number of offspring per female in 2 d. 1–2 h on ice, postfixed with 2% (wt/vol) osmium tetroxide for 1 h at room †Nymphal duration in female (d). temperature, and dehydrated through an ethanol series. The dehydrated ‡Adult emergence rate (%). – + samples were embedded in Spurr resin, processed into ultrathin sections, §The uninfected strain R was generated from the infected strain R by an stained with 2% (wt/vol) uranyl acetate and Sato’s lead solution, and ob- antibiotic treatment (Materials and Methods). { served under a transmission electron microscope (JEM-1010; JEOL). P values were estimated by t test for fecundity and growth rate, and by χ2- test for survival. Molecular Phylogenetic Analysis. A multiple alignment of the nucleotide sequences was generated by the program MAFFT version 7.127b (35). The nucleotide substitution model, GTR + I + G, was selected by using the program erodes the nuclear-cytoplasmic conflict that governs the ma- jModelTest 2 (36, 37). The phylogenetic analyses were conducted by Bayesian jority of endosymbiotic associations (27, 28), thereby providing and maximum-likelihood methods using the programs MrBayes v3.2.2 (38) and a unique empirical model that may shed light on the evolu- RAxML version 7.2.6 (39), respectively. Posterior probabilities were calculated tionary aspects of symbiosis. For example, the biparental sym- for each node by statistical evaluation in Bayesian analysis, and bootstrap biont transmission may entail occasional mixing of different values were obtained with 1,000 replications in maximum-likelihood analysis. symbiont lineages, which would potentially lead to the evolution of some virulent phenotypes of the symbiont (29). Considering In Situ Hybridization. By dissecting male seminal vesicles in PBS (137 mM NaCl, that some Rickettsia and allied pathogenic bacteria exhibit nu- 8.1 mM Na2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4), mature sperm suspension clear infections in somatic cells of their hosts (18–21), it seems was prepared, smeared on MAS-coated glass slides (Matsunami Glass Ind., Rickettsia Ltd.), and air-dried. The sperms were fixed with 4% (wt/vol) paraformaldehyde plausible, although speculative, that the symbiont of in PBS for 60 min at room temperature. After rinsing twice with PBS, the N. cincticeps has coopted the pathogenic nuclear infection mech- samples were treated with 0.1 mg/mL pepsin in 0.01 M HCl for 15 min at 37 °C, anism for establishing the sperm head infection and enabling and washed with 100% ethanol twice and air-dried. Approximately 150 μLof the efficient paternal transmission. The molecular and cellular hybridization buffer [20 mM Tris·HCl at pH 8.0, 0.9 M NaCl, 0.01% SDS, and mechanisms underlying the sperm head infection without dis- 30% (wt/vol) formamide] containing 100 nM each of three oligonucleotide turbing sperm functioning are of not only basic but also applied probes specifically targeting 16S rRNA of Rickettsia spp., whose 5′ end was ′ interest, which would potentially provide insights into the de- labeled with AlexaFluor647 dye, namely Apis-Ric16R1 (5 -TCC ACG TCA CCG TCT TGC-3′), Ric-R1071 (5′-CTT ATA GTT CCC GGC ATT AC-3′), and Ric-R1405 velopment of sperm-mediated genetic transformation and/or (5′-ACC CCA GTC GCT AAT TTT AC-3′), was applied onto the samples, covered material delivery technologies that have long been anticipated with coverslip, and incubated in a humidified chamber at room temperature but not yet realized (30, 31). overnight. For removing nonspecific probe binding, the samples were washed in the hybridization buffer without the probes for 30 min at 42 °C. After Materials and Methods thorough washing, the samples were mounted in Slowfade antifade solution Insect Rearing. N. cincticeps was reared on rice seedlings at 25–26 °C under (Invitrogen) supplemented with 0.25 μM SYTOX Green (Invitrogen), and ob- a16-hlightand8-hdarkcycleeitherinplasticboxes(30cm× 28 cm × 24 cm) for served under a laser confocal microscope (Pascal 5; Carl Zeiss). stock culturing, in glass bottles (180 mm high and 95 mm diameter) for mass- rearing experiments, or in glass test tubes (130 mm high and 16 mm diameter) Fitness Measurement. Fecundity, nymphal growth, and survival were exam- + – for individual rearing. The insect strain mainly used in this study was originally ined for the Rickettsia-infected (R ) and uninfected (R ) strains of N. cincti- collected from a rice field at Kagoshima, Japan, and harbor the bacteriome ceps. Fecundity was evaluated in terms of the number of offspring produced symbionts Sulcia and Nasuia and the systemic symbiont Rickettsia (22). by a young female in 2 d. In each of glass bottles containing rice seedlings, six adult females and three adult males (3-d-old) were kept for 2 d, and the Antibiotic Treatment. Rice seedlings were grown in glass test tubes with a number of newborn nymphs that emerged in each of the bottles (8 bottles small cotton block at the bottom, to which water containing 200 μg/mL ri- for the infected strain and 9 bottles for the uninfected strain) was counted fampicin was added. Newborn nymphs were introduced into the test tubes 12 d later and divided by 6. Growth rate was evaluated in terms of days of and reared to adulthood. Offspring of the antibiotic-treated insects were nymphal duration to adulthood. Each newborn nymph was reared in a test transferred to new test tubes without the antibiotic, whereby isofemale tube containing rice seedlings, and duration to adult emergence was lines were generated and maintained. After several generations, Rickettsia- recorded individually. Survival was evaluated in terms of percentage of adult negative isofemale lines were screened by diagnostic PCR (22), by which the emergence. Each newborn nymph was reared in a test tube containing rice seedlings until it became an adult or died. Number of the nymphs in- Rickettsia-uninfected strain of N. cincticeps was established. dividually reared in test tubes (#N) and number of the insects that reached adulthood (#A) were recorded, and #A ÷ #N × 100 was calculated. Table 3. Egg development rates in crosses within and between Crossing Experiments. For evaluating egg development rates, each of four Rickettsia-infected and uninfected strains of N. cincticeps + + + – – + mating combinations of 30 females and 15 males (R × R ,R × R ,R × R , – – Females of Females of and R × R ) was reared in each of four plastic boxes containing a rice plant infected strain R+,% uninfected strain R–,% with four or five leaves. After 7 d, the plants were carefully dissected under a binocular microscope to isolate eggs, and the eggs were inspected for red † † Males of 89.5* (196/219) 75.9* (211/278) eyespots as an indicator of embryonic development. For evaluating vertical infected strain R+ transmission rates of the Rickettsia symbiont, four types of mating combi- † † + + + – – + – – Males of uninfected 90.7* (107/118) 94.4* (204/216) nations between a female and a male (R × R ,R × R ,R × R , and R × R ; – strain R 13, 9, 8, and 7 pairs for each type, respectively) were created in glass test tubes containing rice seedlings. From each of the test tubes in which next *Significantly different from expected values (χ2 test; P < 0.001). generation nymphs emerged, 5–28 nymphs were picked up, and 94–187 † Egg development rate (number of eggs with eyespots/total number of eggs nymphs in total for each type were subjected to DNA extraction and di- inspected). agnostic PCR detection of the Rickettsia symbiont.

7436 | www.pnas.org/cgi/doi/10.1073/pnas.1402476111 Watanabe et al. Downloaded by guest on September 30, 2021 ACKNOWLEDGMENTS. We thank Makoto Hattori and Masahiro Hirae for for Promotion of Basic and Applied Researches for Innovations in Bio- providing N. cincticeps strains. This work was supported by Program oriented Industry.

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