Intrasperm vertical symbiont transmissionKenji 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 tissuesof diverse animals and other organisms, which affect hosts’ biol-ogy in a variety of ways. Most of these symbionts are present inthe cytoplasm of host cells and maternally transmitted throughhost generations. The paucity of paternal symbiont transmissionis likely relevant to the extremely streamlined sperm structure: thehead consisting of condensed nucleus and the tail made of micro-tubule bundles, without the symbiont-harboring cytoplasm that isdiscarded in the process of spermatogenesis. Here, we report a pre-viously unknown mechanism of paternal symbiont transmissionvia an intrasperm passage. In the leafhopper Nephotettix cincti-ceps, a facultative Rickettsia symbiont was found not only in thecytoplasm but also in the nucleus of host cells. In male insects,strikingly, most sperm heads contained multiple intranuclear Rick-ettsia cells. The Rickettsia infection scarcely affected the host fitnessincluding normal sperm functioning. Mating experiments revealedboth maternal and paternal transmission of the Rickettsia symbiontthrough host generations. When cultured with mosquito and silk-worm cell lines, the Rickettsia symbiont was preferentially localizedwithin the insect cell nuclei, indicating that the Rickettsia symbiontitself must have a mechanism for targeting nucleus. The mecha-nisms underlying the sperm head infection without disturbingsperm functioning are, although currently unknown, of both basicand applied interest.

Endocellular bacterial symbionts are commonly found in di-verse eukaryotes including animals, plants, fungi, and protists

(1–7). In the majority of these cases, the symbionts are located inthe cytoplasm of the host cells. Whereas the cytoplasmic sym-bionts are simply passed to daughter cells through host cell di-vision in unicellular protists (6, 7), sex-related asymmetry invertical symbiont transmission is generally observed in multi-cellular metazoans with sexual reproduction. Namely, the sym-bionts are transmitted vertically to the next host generation viainfection to eggs in the maternal body, but not via infection tosperms (8, 9). Exceptional reports of paternal symbiont trans-mission are venereal transmission cases of several symbioticbacteria (10, 11) and biparental transmission cases of some sym-biotic viruses (12). Oocytes accumulate a large quantity of cyto-plasm that provide a room for symbiont infection, whereassperms discard their cytoplasm (together with inhabiting symbi-otic bacteria) during spermatogenesis and transform into astreamlined shape with the small head consisting of condensednucleus and the slender tail made of microtubule bundles formotility (13, 14). Therefore, if such symbiotic bacterial cells canbe transmitted via sperm, a possible target may be the spermhead nucleus. Intranuclear bacterial symbionts, such as Hol-ospora and Caedibacter, have been relatively well-documentedfrom unicellular ciliates (6, 15), but reported only rarely frommulticellular metazoans (16, 17). In insects and other arthro-pods, intracellular Rickettsia and Orientia pathogens/symbiontsare sometimes observed to localize not only to the cytoplasm, butalso to the nucleus of the host cells (18–21). In bathymodiolinmussels inhabiting hydrothermal vents and cold seeps, intra-nuclear bacterial parasites “Candidatus Endonucleobacter bath-ymodioli” have been described (16). Thus far, no case of paternalsymbiont transmission via intrasperm passage has been reported.Considering the extremely streamlined sperm structure, bacterial

infection to the sperm head nucleus is expected to impair geneticmaterial and normal functioning of the sperm and, thus, intraspermvertical symbiont transmission may seem unlikely to occur. In thisstudy, however, we demonstrate that such a case exists in an insect.The green rice leafhopper Nephotettix cincticeps (Uhler)

(Hemiptera: Cicadellidae) (Fig. 1A), known as a notorious pestof rice in East Asia, is associated with two bacteriome-associatedobligate symbionts, Sulcia and Nasuia, and a facultative symbiontof the genus Rickettsia (22). The Rickettsia symbiont of N. cinc-ticeps represents a basal lineage of the genus Rickettsia (Fig. 1B)and exhibits high infection frequencies among N. cincticepsstrains established from natural populations in Japan (Table 1).Previous histological studies described that Rickettsia-like bac-terial cells are present not only in the cytoplasm but also inthe nucleus of various cells and tissues of several leafhopperspecies including N. cincticeps (23, 24). Here we report that, inN. cincticeps, the Rickettsia symbiont efficiently targets andinfects the host’s cell nuclei including sperm head nuclei, andvertically transmitted to the next host generation not only ma-ternally via ovarial passage but also paternally via intraspermpassage with high fidelity.

Results and DiscussionWhen we observed various cells and tissues of our N. cincticepsstocks by transmission electron microscopy, the Rickettsia sym-biont was consistently found not only in the cytoplasm, butalso in the nucleus of Malphigian tubule cells, midgut cells,and other types of host cells (Fig. 2 A and B). In an attemptto microbiologically characterize the Rickettsia symbiont, asep-tically dissected ovaries of N. cincticeps were subjected to culti-vation with insect cell lines, by which we established continuous

Significance

Diverse organisms are commonly associated with bacterial endo-symbionts, which often affect hosts’ biology and phenotypes ina variety of ways. The majority of these symbionts are generallypresent in the host cell cytoplasm and maternally transmittedthrough host generations. Here, however, this conventionalknowledge is countered by our discovery of intrasperm verticaltransmission of nuclear-targeting bacterial symbiont (Rickettsia)in an insect (leafhopper Nephotettix cincticeps), which poten-tially erodes the nuclear-cytoplasmic conflict that governs themajority of endosymbiotic associations. The molecular and cel-lular mechanisms underlying the sperm head infection withoutdisturbing sperm functioning are of not only basic but also ap-plied interest, whichmay provide insights into the developmentof sperm-mediated genetic transformation and/or material de-livery technologies.

Author contributions: K.W., T.F., and H.N. designed research; K.W., F.Y., Y.M., and H.N.performed research; Y.M. contributed new reagents/analytic tools; K.W. and H.N. ana-lyzed data; and T.F. and H.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1Present address: Graduate School of Environmental Science, Hokkaido University, Sapporo,Hokkaido 060-0810, Japan.

2To whom correspondence may be addressed. E-mail: hnada@affrc.go.jp.

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Rickettsia cultures with the AeAl2 mosquito cell line and theaff3 silkworm cell line. In these heterospecific host cells,strikingly, the Rickettsia symbiont exhibited localizations notonly to the cytoplasm but also to the nucleus (Fig. 2 C andD). The consistent nuclear localization in the different host

species, which represent the distinct insect orders Hemiptera,Diptera, and Lepidoptera, strongly suggests that the Rickettsiasymbiont itself must have some mechanism for targeting insectcell nuclei.

Rickettsia symbiont of leafhopper Nephotettix cincticeps [AB702995]

65/57

100/99

97/80

99/80

92/64

61/*

88/6881/*

99/60

92/*

99/88

93/50

93/61

93/60

63/*

Rickettsia bellii [CP000087]

Rickettsia symbiont of aphid Acyrthosiphon pisum [AB196668]

Rickettsia rickettsii [CP000766]

Rickettsia symbiont of weevil Curculio sp. [AB604673]

Rickettsia symbiont of seed bug Nysius expressus [JQ726774]

Rickettsia symbiont of birch catkin bug Kleidocerys resedae[JQ726775]

belli

i

Rickettsia symbiont of psocid Liposcelis bostrychophila [DQ407744]

Rickettsia felis [NC_007109]

Rickettsia akari [CP000847]

Rickettsia conorii [NC_003103]

Rickettsia prowazekii [CP001584]

Rickettsia symbiont of springtail Onychiurus sinensis [AY712949]

Rickettsia symbiont of ladybird Adalia bipunctata [U04163]

Rickettsia canadensis [CP00409]

Rickettsia symbiont of bean beetle Kytorhinus sharpianus [AB021128]

Rickettsia symbiont of leech Torix tagoi [AB066351]

Rickettsia symbiont of psocid Cerobasis questfalica [DQ652596]

Rickettsia symbiont of ciliate Diophrys sp. [AJ630204]

Orientia tsutsugamushi [NC_010793]

Wolbachia wMel of fruit fly Drosophila melanogaster [AE017196]

Rickettsia typhi [AE017197]

spotted fever

onychiurus

adalia

rhizobius

tori

x

hydra

typhus

tran

sitio

nal

canadensis

B

A

1 mm

0.02

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 probabili-ties for the maximum likelihood phylogeny at 50% or higher are shown atthe nodes, whereas asterisks indicate support values lower than 50%. Se-quence accession numbers are shown in brackets. Major Rickettsia groups(40) are indicated on the right side.

Table 1. Rickettsia infection frequencies in laboratory strains ofN. cincticeps derived from different natural populations in Japan

Strain Origin Year Infection rate,* %

Tsukuba-A Yawara, Ibaraki 1988 100 (96/96)Tsukuba-B Tsukuba, Ibaraki 2006 100 (96/96)Jyouetsu Jyouetsu, Niigata 1993 100 (96/96)Kagoshima Kagoshima, Kagoshima 2001 100 (96/96)Total — — 100 (384/384)

*Diagnostic PCR was performed by using the primers NcRic_16S/f1 (5′-TGACGG TAC CTG ACC AAG A-3′) and NcRic_16S/r1 (5′-AAG GGA TAC ATC TCTGCT T-3′) as described (22).

A

n

n

n

n

2 m2 m

2 m 0.3 m

B

C D

2 m

F

0.3 m

H

sh

st

mv

*

1 m

G

E

0.5 m

Fig. 2. Transmission electron microscopy of nuclear localization of theRickettsia symbiont in tissues of N. cincticeps and cell lines of other insects.(A) Malphigian tubule cell of N. cincticeps. (B) Midgut cell of N. cincticeps.(C ) Cell line aff3 derived from the silkworm B. mori. (D) Cell line AeAl-2derived from the mosquito A. albopictus. (E ) Cross-section of sperm headsin testis of N. cincticeps. (F ) Longitudinal section of sperm heads in testis ofN. cincticeps. (G) Magnified image of the sperm heads. (H) Spermatheca ofRickettsia-uninfected female of N. cincticeps after mating with Rickettsia-infected male. mv, microvilli on the epithelium of spermatheca; n, nucleus;sh, sperm head; st, sperm tail. Asterisk in H highlights an intraspermRickettsia cell.

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Our electron microscopic observations of the testis ofN. cincticeps revealed that, surprisingly, almost all sperm headnuclei contained bacterial cells (Fig. 2 E–G). Of 296 sperm headnuclei we inspected on electron microscopic images representinga single cross-section of elongate sperm heads, 181 (61.1%)exhibited one or two bacterial cells on the sectioned plane (Fig.2E). Longitudinal sections of the sperm heads revealed that thebacterial cells are arranged in a line along the long axis withineach sperm head: More than 10 bacterial cells were often ob-served in a sperm head nucleus (Fig. 2 F and G). Fluorescence insitu hybridization of the sperm heads using specific oligonucle-otide probes targeting 16S rRNA of the Rickettsia symbiontunequivocally demonstrated that the bacterial cells within thesperm head nuclei represent the Rickettsia symbiont (Fig. 3 A–C).Of 1,109 sperm heads inspected by in situ hybridization andfluorescence microscopy, 1,026 (92.5%) contained one or moreRickettsia cells, which were on average 5.33 ± 3.52 and rangingfrom 0 to 23 Rickettsia cells per sperm head (Fig. 4A).Using a selective symbiont curing technique by rifampicin ad-

ministration via rice seedlings (25), we established Rickettsia-infected (R+) and uninfected (R–) strains of N. cincticeps under thesame genetic background, with the obligate bacteriome symbiontsSulcia and Nasuia remaining intact. These R+ and R– insectstrains exhibited similar levels of fecundity, growth, and survival(Table 2), indicating no remarkable positive/negative effects ofthe symbiont infection on the host fitness at least under ourrearing condition, although the possibility cannot be excludedthat some effects at moderate levels would be detected withlarger sample sizes. Eggs produced by mating with R+ males

exhibited high rates of egg development almost comparable tothose with R– males (Table 3), indicating normal functioning ofthe Rickettsia-infected sperms. Meanwhile, statistical analysisshowed that the cross between R– females and R+ males pro-duced significantly less offspring than the other crosses (χ2 test;P < 0.001), suggesting slightly but significantly lower performanceof the Rickettsia-infected sperms. These patterns may look like alow level of cytoplasmic incompatibility, but more data should beaccumulated to test this hypothesis. Electron microscopy of dis-sected spermathecae confirmed transfer of intrasperm Rickettsiasymbiont cells from R+ males to R– females (Fig. 2H). When allpossible mating combinations within and between the R+ and R–

insect strains were examined, the Rickettsia symbiont exhibited100% maternal transmission and, notably, 61.8% (ranging from40.0 to 80.0%) paternal transmission (Fig. 4B). This result indicatesthat only a few Rickettsia cells residing in the sperm head nucleusare sufficient for establishing the paternal symbiont transmission.The paternally transmitted Rickettsia symbiont exhibited 100%vertical transmission in subsequent host generations.In conclusion, we discovered a previously unknown phenom-

enon: The intranuclear Rickettsia symbiont of N. cincticeps istransmitted efficiently through host generations via an intraspermpassage. In general, endosymbiotic bacteria are maternally trans-mitted and, thus, potentially incur an evolutionary conflict withtheir host organisms whose traits are biparentally inherited, whichunderlies such striking symbiont-mediated host phenotypes as cy-toplasmic incompatibility, male-killing, parthenogenesis induction,and feminization (26). The not only maternal, but also effi-cient, paternal symbiont transmission in N. cincticeps potentially

5 m

A B C

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 withinthe sperm heads, reflecting endonuclear localization of the Rickettsia symbiont cells. (C) Merged image. Note that a number of Rickettsia cells are arranged ina row within each sperm head.

0

20

40

60

80

100

120

140

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 < Number of Rickettsia cells per sperm head C

umul

ativ

e nu

mbe

r of s

perm

hea

ds

Mean ± SD = 5.33 ± 3.52

n = 1,109

[Female x Male]

Tran

smis

sion

rate

(%)

0

20

40

60

80

100

!" #" $" %"R+ x R+ R+ x R R x R+ R x R

9 pairs n = 107

13 pairs n = 187

8 pairs n = 107

7 pairs n = 94

A B

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 insitu hybridization images of 1,109 sperm heads obtained from seminal vesicles of four Rickettsia-infected adult males. (B) Vertical transmission rates ofRickettsia upon all mating combinations within and between the Rickettsia-infected and uninfected host strains. The numbers of parent pairs and those oftotal offspring are indicated above the columns.

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erodes the nuclear-cytoplasmic conflict that governs the ma-jority of endosymbiotic associations (27, 28), thereby providinga unique empirical model that may shed light on the evolu-tionary aspects of symbiosis. For example, the biparental sym-biont transmission may entail occasional mixing of differentsymbiont lineages, which would potentially lead to the evolutionof some virulent phenotypes of the symbiont (29). Consideringthat some Rickettsia and allied pathogenic bacteria exhibit nu-clear infections in somatic cells of their hosts (18–21), it seemsplausible, although speculative, that the Rickettsia symbiont ofN. cincticeps has coopted the pathogenic nuclear infection mech-anism for establishing the sperm head infection and enablingthe efficient paternal transmission. The molecular and cellularmechanisms underlying the sperm head infection without dis-turbing sperm functioning are of not only basic but also appliedinterest, which would potentially provide insights into the de-velopment of sperm-mediated genetic transformation and/ormaterial delivery technologies that have long been anticipatedbut not yet realized (30, 31).

Materials and MethodsInsect Rearing. N. cincticeps was reared on rice seedlings at 25–26 °C undera 16-h light and 8-h dark cycle either in plastic boxes (30 cm × 28 cm × 24 cm) forstock 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)for individual rearing. The insect strain mainly used in this study was originallycollected from a rice field at Kagoshima, Japan, and harbor the bacteriomesymbionts Sulcia and Nasuia and the systemic symbiont Rickettsia (22).

Antibiotic Treatment. Rice seedlings were grown in glass test tubes with asmall cotton block at the bottom, to which water containing 200 μg/mL ri-fampicin was added. Newborn nymphs were introduced into the test tubesand reared to adulthood. Offspring of the antibiotic-treated insects weretransferred to new test tubes without the antibiotic, whereby isofemalelines were generated and maintained. After several generations, Rickettsia-negative isofemale lines were screened by diagnostic PCR (22), by which theRickettsia-uninfected strain of N. cincticeps was established.

Rickettsia Cultivation. The Rickettsia symbiont was cultivated with the AeAl-2cell line derived from the mosquito Aedes albopictus (32) by inoculating apart of dissected ovary of Rickettsia-infected N. cincticeps into a plastic dishcontaining the cells, according to the cultivation procedure for Wolbachia(33). The cultivated Rickettsia symbiont was further transferred to the aff3cell line from the silkworm Bombyx mori (34). The AeAl-2 and aff3 cells weregrown in medium IPL-41 (GIBCO BRL 11505) supplemented with 5% (vol/vol)FBS (Sigma) at 26 °C.

Electron Microscopy. The tissue and cell samples were prefixed with 0.8%glutaraldehyde and 1% paraformaldehyde in 0.06 M phosphate buffer for1–2 h on ice, postfixed with 2% (wt/vol) osmium tetroxide for 1 h at roomtemperature, and dehydrated through an ethanol series. The dehydratedsamples were embedded in Spurr resin, processed into ultrathin sections,stained with 2% (wt/vol) uranyl acetate and Sato’s lead solution, and ob-served under a transmission electron microscope (JEM-1010; JEOL).

Molecular Phylogenetic Analysis. A multiple alignment of the nucleotidesequences was generated by the program MAFFT version 7.127b (35). Thenucleotide substitution model, GTR + I + G, was selected by using the programjModelTest 2 (36, 37). The phylogenetic analyses were conducted by Bayesianand maximum-likelihood methods using the programsMrBayes v3.2.2 (38) andRAxML version 7.2.6 (39), respectively. Posterior probabilities were calculatedfor each node by statistical evaluation in Bayesian analysis, and bootstrapvalues were obtained with 1,000 replications in maximum-likelihood analysis.

In Situ Hybridization. By dissecting male seminal vesicles in PBS (137 mM NaCl,8.1 mMNa2HPO4, 2.7 mMKCl, and 1.5 mMKH2PO4), mature sperm suspensionwas prepared, smeared on MAS-coated glass slides (Matsunami Glass Ind.,Ltd.), and air-dried. The sperms were fixed with 4% (wt/vol) paraformaldehydein PBS for 60 min at room temperature. After rinsing twice with PBS, thesamples were treated with 0.1 mg/mL pepsin in 0.01 M HCl for 15 min at 37 °C,and washed with 100% ethanol twice and air-dried. Approximately 150 μL ofhybridization buffer [20 mM Tris·HCl at pH 8.0, 0.9 M NaCl, 0.01% SDS, and30% (wt/vol) formamide] containing 100 nM each of three oligonucleotideprobes specifically targeting 16S rRNA of Rickettsia spp., whose 5′ end waslabeled with AlexaFluor647 dye, namely Apis-Ric16R1 (5′-TCC ACG TCA CCGTCT TGC-3′), Ric-R1071 (5′-CTT ATA GTT CCC GGC ATT AC-3′), and Ric-R1405(5′-ACC CCA GTC GCT AAT TTT AC-3′), was applied onto the samples, coveredwith coverslip, and incubated in a humidified chamber at room temperatureovernight. For removing nonspecific probe binding, the samples were washedin the hybridization buffer without the probes for 30 min at 42 °C. Afterthorough washing, the samples were mounted in Slowfade antifade solution(Invitrogen) supplemented with 0.25 μM SYTOX Green (Invitrogen), and ob-served under a laser confocal microscope (Pascal 5; Carl Zeiss).

Fitness Measurement. Fecundity, nymphal growth, and survival were exam-ined for the Rickettsia-infected (R+) and uninfected (R–) strains of N. cincti-ceps. Fecundity was evaluated in terms of the number of offspring producedby 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 thenumber of newborn nymphs that emerged in each of the bottles (8 bottlesfor the infected strain and 9 bottles for the uninfected strain) was counted12 d later and divided by 6. Growth rate was evaluated in terms of days ofnymphal duration to adulthood. Each newborn nymph was reared in a testtube containing rice seedlings, and duration to adult emergence wasrecorded individually. Survival was evaluated in terms of percentage of adultemergence. Each newborn nymph was reared in a test tube containing riceseedlings until it became an adult or died. Number of the nymphs in-dividually reared in test tubes (#N) and number of the insects that reachedadulthood (#A) were recorded, and #A ÷ #N × 100 was calculated.

Crossing Experiments. For evaluating egg development rates, each of fourmating combinations of 30 females and 15 males (R+ × R+, R+ × R–, R– × R+,and R– × R–) was reared in each of four plastic boxes containing a rice plantwith four or five leaves. After 7 d, the plants were carefully dissected undera binocular microscope to isolate eggs, and the eggs were inspected for redeyespots as an indicator of embryonic development. For evaluating verticaltransmission rates of the Rickettsia symbiont, four types of mating combi-nations between a female and a male (R+ × R+, R+ × R–, R– × R+, and R– × R–;13, 9, 8, and 7 pairs for each type, respectively) were created in glass testtubes containing rice seedlings. From each of the test tubes in which nextgeneration nymphs emerged, 5–28 nymphs were picked up, and 94–187nymphs in total for each type were subjected to DNA extraction and di-agnostic PCR detection of the Rickettsia symbiont.

Table 2. Comparison of fitness parameters between Rickettsia-infected and uninfected strains of N. cincticeps

Fecundity* Growth rate† Survival,‡ %

Infectedstrain R+§

4.5 ± 2.8 (n = 8) 17.1 ± 0.7 (n = 42) 89.9% (89/99)

Uninfectedstrain R–§

6.6 ± 3.0 (n = 9) 17.0 ± 0.5 (n = 38) 91.5% (86/94)

P value{ P = 0.16 P = 0.75 P = 0.70

*Number of offspring per female in 2 d.†Nymphal duration in female (d).‡Adult emergence rate (%).§The uninfected strain R– was generated from the infected strain R+ by anantibiotic treatment (Materials and Methods).{P values were estimated by t test for fecundity and growth rate, and by χ2-test for survival.

Table 3. Egg development rates in crosses within and betweenRickettsia-infected and uninfected strains of N. cincticeps

Females ofinfected strain R+, %

Females ofuninfected strain R–, %

Males ofinfected strain R+

89.5* (196/219)† 75.9* (211/278)†

Males of uninfectedstrain R–

90.7* (107/118)† 94.4* (204/216)†

*Significantly different from expected values (χ2 test; P < 0.001).†Egg development rate (number of eggs with eyespots/total number of eggsinspected).

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ACKNOWLEDGMENTS. We thank Makoto Hattori and Masahiro Hirae forproviding N. cincticeps strains. This work was supported by Program

for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry.

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