Insectts Intestinal Organ for Symbiont Sorting

Insect’s intestinal organ for symbiont sorting PNAS PLUS

Tsubasa Ohbayashia, Kazutaka Takeshitaa,b, Wataru Kitagawaa,b, Naruo Nikohc, Ryuichi Kogad, Xian-Ying Mengd, Kanako Tagoe, Tomoyuki Horif, Masahito Hayatsue, Kozo Asanoa, Yoichi Kamagataa,b, Bok Luel Leeg, Takema Fukatsud, and Yoshitomo Kikuchia,b,1

aGraduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan; bBioproduction Research Institute, Hokkaido Center, National Institute of Advanced Industrial Science and Technology, Sapporo 062-8517, Japan; cDepartment of Liberal Arts, The Open University of Japan, Chiba 261-8586, Japan; dBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan; eEnvironmental Biofunction Division, National Institute for Agro-Environmental Sciences, Tsukuba 305-8604, Japan; fEnvironmental Management Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8569, Japan; and gGlobal Research Laboratory, College of Pharmacy, Pusan National University, Pusan 609-735, Korea

Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved August 11, 2015 (received for review June 11, 2015) Symbiosis has significantly contributed to organismal adaptation Those controlling mechanisms are of general importance for and diversification. For establishment and maintenance of such host– understanding symbiosis (6, 10). symbiont associations, host organisms must have evolved mecha- Stinkbugs, belonging to the insect order Hemiptera, consist of nisms for selective incorporation, accommodation, and maintenance over 40,000 described species in the world (15). The majority of of their specific microbial partners. Here we report the discovery of a the stinkbugs suck plant sap or tissues, and some of them are previously unrecognized type of animal organ for symbiont sorting. notorious as devastating agricultural pests (16). These plant- In the bean bug Riptortus pedestris, the posterior midgut is morpho- sucking stinkbugs possess a specialized symbiotic organ in their logically differentiated for harboring specific symbiotic bacteria of a alimentary tract: A posterior region of the midgut is morpho- beneficial nature. The sorting organ lies in the middle of the intestine logically differentiated with a number of sacs or tubular out- as a constricted region, which partitions the midgut into an anterior growths, called crypts or ceca, whose inner cavity hosts symbiotic nonsymbiotic region and a posterior symbiotic region. Oral adminis- bacteria (17–21). Usually, a single bacterial species dominates in tration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs the midgut crypts, and elimination of the symbiont causes re- showed that the symbionts pass through the constricted region and tarded growth and increased mortality of the host, which in- colonize the posterior midgut. However, administration of food col- dicates the specific and beneficial nature of the stinkbug gut orings revealed that food fluid enters neither the constricted region symbiosis (20–31). The initial symbiont infection is established by nor the posterior midgut, indicating selective symbiont passage at nymphal feeding, which may be either via vertical transmission the constricted region and functional isolation of the posterior mid- from symbiont-containing maternal secretion supplied upon gut for symbiosis. Coadministration of the GFP-labeled symbiont and oviposition (19–21) or via environmental acquisition from am- red fluorescent protein-labeled Escherichia coli unveiled selective pas- bient microbiota (21–23). What mechanisms underlie the selec- sage of the symbiont and blockage of E. coli at the constricted region, tive establishment of a specific bacterial symbiont in the midgut demonstrating the organ’s ability to discriminate the specific bacterial symbiotic organ despite the oral inoculum contaminated by symbiont from nonsymbiotic bacteria. Transposon mutagenesis and nonsymbiotic microbes has remained largely an enigma, al- screening revealed that symbiont mutants in flagella-related genes though recent studies have started to shed light on the sym- fail to pass through the constricted region, highlighting that both biotic mechanisms underlying the environmental acquisition host’s control and symbiont’s motility are involved in the sorting of specific Burkholderia symbionts in the bean bug Riptortus process. The blocking of food flow at the constricted region is con- pedestris (Hemiptera: Alydidae) (22, 32). Antimicrobial substances served among diverse stinkbug groups, suggesting the evolutionary produced by the midgut epithelia (33, 34) and some symbiont origin of the intestinal organ in their common ancestor. Significance stinkbug | gut symbiosis | partner choice | Burkholderia | flagellar motility In general, animals have a mouth for feeding, an anus for defe- iverse organisms are obligatorily associated with microbial cation, and a gut connecting them for digestion and absorption. Dsymbionts, which significantly contribute to their adaptation However, we discovered that the stinkbug’s gut is functionally and survival (1–3). In such symbiotic associations, the host or- disconnected in the middle by a previously unrecognized organ ganisms often develop specialized cells, tissues, or organs for for symbiont sorting, which blocks food fluid and nonsymbiotic harboring their specific microbial partners [for example, root bacteria but selectively allows passing of a specific bacterial nodules in the legume–Rhizobium symbiosis (4, 5), symbiotic symbiont. Though very tiny and inconspicuous, the organ light organs in the squid–Vibrio symbiosis (6, 7), and bacter- governs the configuration and specificity of stinkbug gut iocytes in the aphid–Buchnera symbiosis (8, 9)]. symbiosis, wherein the posterior gut region is devoid of food These microbial symbionts are either acquired by newborn flow, populated by a specific bacterial symbiont, and trans- hosts from the environment every generation as in the legume– formed into an isolated organ for symbiosis. Mutant analyses Rhizobium and the squid–Vibrio symbioses or transmitted verti- showed that the symbiont’s flagellar motility is needed for cally through host generations as in the aphid–Buchnera symbi- passing the host organ, highlighting intricate host–symbiont osis (10). In the environmentally acquired symbiotic associations, interactions underpinning the symbiont sorting process. it is essential for the host organisms to recognize and incorporate specific symbiotic bacteria while excluding a myriad of nonsym- Author contributions: T.O., T.F., and Y. Kikuchi designed research; T.O., K. Takeshita, W.K., N.N., R.K., X.-Y.M., K. Tago, T.H., M.H., K.A., Y. Kamagata, B.L.L., and Y. Kikuchi biotic environmental microbes (6, 11). In the vertically trans- performed research; T.O. and Y. Kikuchi analyzed data; and T.O., T.F., and Y. Kikuchi mitted symbiotic associations, it is important for the host wrote the paper. organisms to selectively transmit their own symbiotic bacteria The authors declare no conflict of interest. while excluding parasitic/cheating microbial contaminants (12– This article is a PNAS Direct Submission. 14). Hence, it is expected that the host organisms must have 1To whom correspondence should be addressed. Email: [email protected]. EVOLUTION evolved some mechanisms for selective incorporation, accom- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. modation, and maintenance of their specific microbial partners. 1073/pnas.1511454112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1511454112 PNAS | Published online August 31, 2015 | E5179–E5188 Downloaded by guest on September 30, 2021 factors, such as stress-responsive polyester accumulation, cell A wall synthesis, and purine biosynthesis (35–37) might be involved in the selective infection of the Burkholderia symbiont to the midgut crypts. Here we address this important symbiotic issue by the discovery of a previously unrecognized intestinal organ in the stinkbugs. Though very tiny and inconspicuous, the organ governs the configuration and specificity of the stinkbug gut symbiosis. Lying in the middle of the midgut, the organ blocks food flow and nonsymbiotic bacteria but selectively allows passing of specific symbiotic bacteria, whereby the stinkbug’s intestine is functionally partitioned into the anterior region specialized for digestion and absorption and the posterior region dedicated to symbiosis. The blocking of food flow by the organ is conserved B across diverse stinkbug families, suggesting the possibility that the organ evolved in their common ancestor and has played substantial roles in their symbiont-mediated adaptation and diversification. Results and Discussion Identification of Constricted Region in Stinkbug Midgut. As in diverse other stinkbugs (17–21), the midgut of the bean bug C D R. pedestris consists of the following morphologically distinct regions: the voluminous midgut first section (M1), the tubular midgut second section (M2), the ovoid midgut third section (M3), and the midgut fourth section (M4) with numerous crypts densely populated by a specific betaproteobacterial symbiont of the genus Burkholderia, which is orally acquired by nymphal insects from the environment every generation (22, 23, 32) (Fig. 1A). A swollen region adorally connected to the M4 is without crypts and called M4 bulb (M4B) (18, 34, 38) (Fig. 1A). Although biological roles of each section are not exactly known, it is Fig. 1. Midgut organization and the constricted region of R. pedestris. conjectured that the M1 serves for transient food storage and (A) Alimentary tract dissected from a third instar nymph. (B) Symbiont lo- digestion, the M2 and the M3 perform food digestion and ab- calization in the M4B and M4 regions of the dissected alimentary tract vi- sorption, and the M4 is specialized for harboring the symbiotic sualized using a GFP-labeled Burkholderia symbiont strain. Green and blue bacteria. Infection with a GFP-labeled strain of the Burkholderia signals indicate symbiont cells and host’s nuclear DNA, respectively. (C) En- symbiont (38) revealed symbiont localization to the M4B and the larged image of the constricted region. Note that the symbiont signal is M4 (Fig. 1B). Between the nonsymbiotic M3 and the symbiotic preferentially detected in M4B and not in M3. (D) Sectioned image of the constricted region of a second instar nymph, which was stained with periodic M4B a particularly narrow intestinal region was present, which – “ ” acid Schiff reagent (red) and hematoxylin (purple). (Inset) An enlarged im- we designated as constricted region in this study (Fig. 1C). age of the cavity of the constricted region. Note that the narrow lumen of Probably because of being very tiny and inconspicuous struc- the constricted region is filled with periodic acid–Schiff-positive material, turally, the constricted region has attracted little attention in indicating the presence of polysaccharide-rich mucous matrix. CR, con- previous anatomical studies on the alimentary tract of diverse stricted region; H, hindgut; M1, midgut first section; M2, midgut second stinkbugs (17–21). However, we discovered that the constricted section; M3, midgut third section; M4, midgut fourth section with crypts; region plays a pivotal role in the stinkbug gut symbiosis, as M4B, M4 bulb. Arrows indicate the constricted region. detailed below.

Burkholderia Symbiont Passing Through Constricted Region. Histo- tently observed not only with Congo red but also with other food – logical inspection revealed that, although very narrow, a lumen colorings of different color (Fig. 2 O T). Meanwhile, the colorings – was present in the constricted region, connecting the inner cav- appeared in the hindgut (Fig. 2 A and O T) and were excreted with ities of the M3 and the M4B (Fig. 1D). Our previous work feces (Fig. 2U). These results strongly suggested that (i) food fluid showed that when newly molted second instar nymphs were fed ingested by R. pedestris enters the M1, the M2, and the M3 but with GFP-labeled Burkholderia the symbiont cells aggregated at cannot pass the constricted region; (ii) the fluid is absorbed in the the entrance of the constricted region around 6 h after in- anterior regions of the midgut, excreted through Malpighian tu- oculation, subsequently migrated into the narrow lumen of the bules into the hindgut, and discarded with feces; (iii)asaresult,the constricted region, and finally reached the M4B and the M4 (38). M4B and the M4 do not contribute to the food flow; and (iv) These observations suggested that passing of the constricted therefore, the M4B and the M4 are, although structurally con- region comprises a rate-limiting step, or a bottleneck, for sym- nected to adoral and aboral regions of the intestine, functionally biont colonization to the midgut symbiotic regions M4B and M4. isolated and specialized for harboring the Burkholderia symbiont.

Oral Administration of Food Colorings Unveiled Restricted Food Flow Coadministration of GFP-Labeled Burkholderia Symbiont and Food at Constricted Region. When third instar nymphs that had been Coloring Revealed Selective Symbiont Passing at Constricted Region. inoculated with the Burkholderia symbiont at the second instar When newly molted second instar nymphs were fed with a mixture were fed with water supplemented with Congo red, the coloring of Congo red and the GFP-labeled Burkholderia cells, both the stained the M1, the M2, and the M3 but, strikingly, never appeared coloring and the bacteria were found in the M1, the M2, and the in the M4B and the M4 (Fig. 2 A and B). This phenomenon was M3. However, whereas the coloring did not enter the constricted found not only in third instar nymphs but also throughout all de- region (Fig. 3A), the Burkholderia cells appeared in the narrow velopmental stages including first, second, third, fourth, and fifth inner cavity of the constricted region (Fig. 3 B and C). These re- instars (Fig. 2 C–N). Furthermore, this phenomenon was consis- sults indicated that the constricted region is involved in selective

E5180 | www.pnas.org/cgi/doi/10.1073/pnas.1511454112 Ohbayashi et al. Downloaded by guest on September 30, 2021 3rd instar B C 1st instar D PNAS PLUS A RRed Red Red CR Red colonized by the Burkholderia symbiont during the second and M1 M3 CR CR H CR M3 M3 M4B M4B4B third instars, whereas the colonization efficiency drops significantly from the fourth instar and afterward, indicating that there M4B M1 M2 M3 M2 M1 M4 1.0 mmm M4 H 0.3 mm M4B 1.01 mmm M4 0.5 mm M2 exists a permissive time window for infection (39). Whether the

rd O SScarlet 3 instar Bluee P E Redd 2nd instar, 6 h after inoculation RRed F constricted region is involved in the establishment of the in- M1 M1 M2 M2 M3 CR M3 fection time window is of interest and deserves future studies. M3 H M3 H H M4B M2 M4B M1 M4B M4 M2 M CR M4 Fine Structure of Constricted Region. We observed the constricted 1.0 mmm CR 1.0 mm 1.01 mmm M B CR 444 0.5 mm regions dissected from second instar nymphs of R. pedestris using Q YYellow 3rd instar Purplee R G Redd 2nd instar, 24 h after inoculation RRed H M1 M2 M3 M1 M4B M4 CR M4B light microscopy (Figs. 1D and 4A) and transmission electron M4 M4 M4 microscopy (Fig. 4 B–E). The lumen of the M3 was filled with the H CR H H M4B CR M4B M2 symbiont cells, and the inner surface of the M3 was covered with M1 M3 H 1.0 mmm M2 M3 CR 1.0 mm 1.011 mmm 0.5 mm M3 a well-developed layer of microvilli (Fig. 4 C and D). The inner 3rd instar nd S Green M2 Brownn T I Redd 2 instar, 48 h after inoculation RedR J M1 M4B M4B4B MM1 M44 surface of the constricted region was also covered with a dense M1 M3 M4 layer of microvilli, which occupied most of the narrow inner CR H H M3 M2 M3 space (Fig. 4 B, C, and E). Only a very thin canal was left at the M2 M3 H M4B 1.0 mmm CR M4 1.0 mm 1.0 mmm CR M4B M4 0.5 mm CR

th U K Red M2 4 instar Red L CR M1 CR M3

M4B AD M4B M3 H 1.0 mm m M4 0.25 mm

M Red M33 5th instar Red N M2 M4B

M1 M4B CR 1.0 cm 1.0 mm M4 H 0.25 mm M3

Fig. 2. Dissected alimentary tracts of R. pedestris fed with water supplemented with food colorings. (A–N) Congo red. (O) New coccine. (P) Brilliant blue FCF. (Q) Gardenia yellow. (R) Purple sweet potato color. (S) Gardenia yellow and Gardenia blue. (T) Kaoliang color. The developmental stages of R. pedestris are at the first instar (C–D), the second instar (E–J), the third instar (A and B and O–T), the fourth instar (K and L) and the fifth instar (M BE and N). Abbreviations are as in Fig. 1. (U) Third instar nymphs of R. pedestris fed with soybean seeds and water supplemented with New coccine. Arrow- heads indicate feces of the insects, in which the red coloring is excreted.

passing of the Burkholderia symbiont while restricting passing of the food fluid.

Coadministration of GFP-Labeled Burkholderia Symbiont and Red Fluorescent Protein-Labeled Escherichia coli Uncovered Selective Symbiont Sorting at Constricted Region. When newly molted second instar nymphs were fed with a mixture of the GFP-labeled CF Burkholderia cells and red fluorescent protein (RFP)-labeled E. coli cells (104 cells per μL each), both the Burkholderia cells and the E. coli cells were found in the M1, the M2, and the M3 (Fig. 3 D and E). However, whereas the Burkholderia cells soon appeared in the constricted region, the M4B and the M4, the E. coli cells were not observed in these posterior midgut regions (infected insects/inoculated insects = 0/5) (Fig. 3 E and F). In addition to E. coli, orally administrated Pseudomonas putida and Bacillus subtilis did not colonize the M4B and the M4 as determined by plating and counting cfu of dissected midgut regions (infected insects/inoculated insects = 0/5, respectively). These results corroborated that the constricted Fig. 3. Symbiont sorting at the constricted region of R. pedestris.(A–C)Co- region is involved in selective passing of the Burkholderia symbiont inoculation of Congo red and a GFP-labeled Burkholderia symbiont strain. and suggested that the selection mechanism can discriminate the Second instar nymphs of R. pedestris were fed with water containing the coloring and the bacteria, and their alimentary tracts were dissected and Burkholderia symbiont from nonsymbiotic bacteria. observed 6 h after inoculation. (A) Differential interference contrast microscopic (DIC) image, wherein Congo red signal is observed in the M3 only. Constricted Region As a Host Organ for Selective Symbiont Sorting. (B) Fluorescence microscopic (FM) image, wherein green Burkholderia In this way, we identified a previously unknown type of intestinal symbiont signals are found in the constricted region. (C) Merged image. organ, the constricted region, whose function is to ensure se- (D–F) Coinoculation of the GFP-labeled Burkholderia symbiont strain and an lective passing of the Burkholderia symbiont to the posterior RFP-labeled E. coli strain. Second instar nymphs of R. pedestris were fed with symbiotic midgut. Considering that R. pedestris orally acquires water containing the bacterial mixture, and their alimentary tracts were dis- Burkholderia sected and observed 6 h after inoculation. Green and red signals indicate the symbiont from the microbe-rich environment the Burkholderia symbiont cells and the E. coli cells, respectively. (D)FMimage. every generation (22, 23), this organ must play a central role for

(E) Laser scanning microscopic (LSM) image. (F)EnlargedLSMimageofthe EVOLUTION establishment and maintenance of the specific Riptortus–Bur- constricted region, wherein red E. coli cells are indicated by arrowheads. Ab- kholderia symbiotic association. The M4 crypts are efficiently breviations are as in Fig. 1.

Ohbayashi et al. PNAS | Published online August 31, 2015 | E5181 Downloaded by guest on September 30, 2021 Transposon Mutagenesis and Screening for Motility-Deficient Mutants AB of Burkholderia Symbiont. We performed a random transposon- insertion mutagenesis of the Burkholderia symbiont using the min- iTn5 system (40), and resultant transconjugants were screened for motility on 0.4% semisolid nutrition agar. Of 6,212 transconjugants subjected to the assay, 7 were identified as motility-deficient. Furthermore, an additional 4,252 transconjugants were subjected to screening for biofilm formation, because previous studies had C reported that biofilm-deficient bacterial mutants also tend to be motility-deficient (41, 42). Of 25 biofilm-deficient mutants obtained, 7 were confirmed as motility-deficient by the semisolid agar assay. Southern hybridization analysis detected a single Tn5 insertion in 13 of the 14 motility mutants. In this way, we obtained 13 Tn5-inserted motility-deficient mutants of the Burkholderia symbiont (Fig. 5A). Of these, 9 mutants without visible motility in liquid medium under light microscopy were designated as no-motility mutants NM1- NM9, whereas the other 4 mutants that were motile in liquid medium but not in semisolid agar were designated as altered-motility mutants AM1–AM4 (Table S1). Most of the mutants contained aTn5 insertion associated with bacterial flagella-related genes: a flagellin gene (fliC) in NM1, an MS-ring gene (fliF) in NM2-NM6, a motorapparatusgene(fliM) in NM7, a flagellar export apparatus gene (fliR) in NM8, a rod regulation gene (flhA)inNM9,aregu- lation gene for hook length (fliK) in AM1, and a regulation gene for DE flagellar formation (flhF)inAM2(Fig.5B and C). In addition, a chemotaxis-related gene, cheA, was disrupted by the transposon in the mutant AM3, whereas a putative gene of unknown function contained a Tn5 insertion in the mutant AM4 (Fig. 5B). All these genes were single-copy in the Burkholderia symbiont genome (43). Flagella formation of the 13 motility-deficient mutants was determined by electron microscopy (Fig. 6). Whereas the wild- type strain RPE75 exhibited multiple polar flagella (Fig. 6N), – Fig. 4. Fine structure of the constricted region in second instar nymphs of most of the no-motility mutants had no or few flagella (Fig. 6 A I R. pedestris.(A) Semiultrathin section stained with toluidine blue and observed by light microscopy. (B–E) Ultrathin sections observed by transmission electron microscopy. (B) Longitudinal section of the constricted region, whose inner surface is lined with a dense layer of microvilli, leaving a very AC narrow canal at the center. (C) Posterior end region of the M3 in connection to the constricted region. The inner surface of the M3 and of the constricted region is lined with a dense layer of microvilli. The inner cavity of the M3 harbors numerous Burkholderia symbiont cells, whereas the inner canal of the constricted region is full of a mucous matrix. (D) Enlarged image of the M3 cavity. (E) Enlarged image of the constricted region. *, inner canal of constricted region; CR, constricted region; ML, microvilli layer; S, Burkholderia symbiont cell.

center of the constricted region, which was one or a few micro- meters in width and filled with a slightly electron-dense material B (Fig. 4 B, C, and E). Periodic acid–Schiff staining of semiultrathin sections of the constricted region identified the material in the canal as polysaccharide-rich, presumably a mucous matrix (Fig. 1D). These observations suggested that (i) the inner cavity of the constricted region is nearly filled with the highly developed microvilli layer, (ii) the very thin canal left at the center of the constricted region is filled with the mucous matrix, (iii) the microvilli and the mucous matrix plug the inner cavity of the constricted region, thereby hindering the passing of food colorings and E. coli cells; (iv) however, the Burkholderia symbiont can manage to pass through the constricted region despite these obstacles, (v) and, therefore, although speculative, it seems likely that the Burkholderia symbiont somehow interacts with Fig. 5. Motility-deficient mutants of the Burkholderia symbiont obtained the microvilli and/or the mucous matrix for passing through the by transposon mutagenesis and screening. (A) Growth of no-motility mu- – – constricted region. Because no ciliary layer was observed on the tants (NM1 NM9) and altered-motility mutants (AM1 AM4) on a semisolid YG agar plate. Note the limited diffusion of the mutant colonies in com- inner surface of the constricted region, host-driven movement of parison with the wild-type symbiont colony. (B) Transposon insertion sites the symbiont cells seemed unlikely. Hence, we suspected that for the motility-deficient mutants. Note that the majority of the disrupted motility of the Burkholderia symbiont may play a role in passing genes are flagella-related. (C) Structural presentation of the flagella-related through the constricted region. genes disrupted in the motility-deficient mutants.

E5182 | www.pnas.org/cgi/doi/10.1073/pnas.1511454112 Ohbayashi et al. Downloaded by guest on September 30, 2021 − PNAS PLUS and Table S1). Some cells of the mutant NM7 (fliM ) formed rates ranging from 50 to 100%, whereas 8 of 9 no-motility mutants morphologically intact flagella (Fig. 6G), but the mutant showed did not infect the symbiotic midgut regions (Table S1). The ex- no motility, probably because of its disrupted motor apparatus. ceptional no-motility mutant NM6, which carries a Tn5 insertion All of the altered-motility mutants AM1–AM4 were equipped near the 5′ end of fliF (Fig. 5B), exhibited a partial infection rate of − with flagella (Fig. 6 J–M). The mutant AM2 (flhF ) formed a 40%, which might be, although speculative, due to leaky or con- single flagellum on a lateral side of the cell (Fig. 6K) as reported ditional expression of the flagella-related gene from a promoter in flhF mutants of Pseudomonas aeruginosa (44) and Vibrio encoded within the Tn5 cassette. The essentiality of fliF for flagella alginolyticus (45). formation, motility, and colonization to the symbiotic midgut regions was confirmed by inspection of a fliF-deleted mutant, ΔfliF, Flagellar Motility Is Important for Passing Through Constricted Region. generated by homologous recombination (Fig. 7 H–J and Table Colonization ability of the 13 motility-deficient mutants to the S1). These results strongly suggested that the flagellar motility of symbiotic midgut regions M4B and M4 was investigated by oral the Burkholderia mutants is important for passing through the administration of the cultured bacteria to second instar nymphs of constricted region and infection to the symbiotic midgut regions R. pedestris. When the posterior midgut regions were dissected M4B and M4. from the inoculated insects at the third instar and subjected to To quantitatively evaluate the infection process of the motil- diagnostic PCR, all 4 altered-motility mutants exhibited infection ity-deficient mutants, second instar nymphs of R. pedestris were orally administrated with the cultured bacteria and dissected 48 h after inoculation. The dissected midgut sections (M1, M2, M3, M4B, and M4) and hindgut were homogenized and spread on AB C nutrition agar plates for counting cfu. The wild-type strain RPE75 was motile, flagellated, and detected in all of the midgut sections, being the most abundant in the M4 section at around − 105 cfu per tissue (Fig. 7 A, D, and G). By contrast, the fliC no- motility mutant NM1pBBR122 (NM1 transformed with the empty plasmid pBBR122) was not motile, without flagella, and not detected in the M4B and M4 sections at all, although a high level of infection was detected in the M3 section at around 106 cfu per − DE F tissue (Fig. 7 B, E, and G). The genetically complemented fliC mutant NM1fliC (NM1 transformed with a pBBR122 plasmid containing intact fliC gene) restored flagella formation, motility, and colonization ability to the M4B and M4 sections (Fig. 7 C, F, and G). Furthermore, the altered-motility mutants AM1 − − (fliK ) and AM2 (flhF ) were detected in the M4B and M4 sec- − tions, whereas the no-motility mutants NM2 and ΔfliF (both fliF ) GH I were scarcely detected in the symbiotic midgut regions (Fig. 7K). These results further corroborated the idea that the flagellar motility of the Burkholderia symbiont is important for passing through the constricted region and infection to the symbiotic midgut regions M4B and M4.

Possible Mechanisms of Selective Symbiont Sorting at Constricted Region. It should be noted that, although E. coli, P. putida, and JKL B. subtilis are motile with functional flagella, they were blocked at the sorting organ and unable to gain entry into the symbiotic midgut regions, which indicate that bacterial flagellar motility is indeed necessary but not sufficient for passing through the constricted region in R. pedestris. What mechanisms are involved in the selective passing of the Burkholderia symbiont through the constricted region is currently unknown and deserves future studies. It is conceivable, although speculative, that the Bur- M N kholderia symbiont may be capable of penetrating the constricted region by, for example, excreting specific enzymes that degrade the mucous matrix plugging the inner cavity of the constricted region. Alternatively, the constricted region may be producing some antimicrobials, such as antimicrobial peptides and lyso- zymes (33, 34), to which nonsymbiotic bacteria are sensitive but the Burkholderia symbiont is resistant. We have previously Fig. 6. Transmission electron microscopy of negatively stained bacterial identified several bacterial factors needed for stable symbiont cells of the motility-deficient mutants of the Burkholderia symbiont. colonization in the M4 crypts (35–37), which might also be in- (A) NM1 (fliC−) without flagella. (B) NM2 (fliF−) without flagella. (C) NM3 volved in the symbiont sorting process. Host’s control mecha- − − − (fliF ) without flagella. (D) NM4 (fliF ) without flagella. (E) NM5 (fliF ) ’ − − nisms over symbiont s population and localization have been without flagella. (F) NM6 (fliF ) without flagella. (G) NM7 (fliM ) with fla- demonstrated or suggested in aphids, weevils, and other insect– gella, though the majority of the bacterial cells are without flagella. (H) NM8 − − − microbe symbiotic associations (46–48). Transcriptomic analyses (fliF ) without flagella. (I) NM9 (flhA ) without flagella. (J) AM1 (fliK ) with flagella. (K)AM2(flhF−) with an abnormal lateral flagellum. (L)AM3(cheA−) of the constricted region will provide further insights into the with flagella. (M) AM4 (no homology gene−) with flagella. (N) RPE75 (wild molecular mechanisms operating at the symbiont sorting ma- chinery. Taken together, intricate host–symbiont interactions at

type) with flagella. Bacterial cells at a midexponential growth phase are EVOLUTION shown. Arrowheads indicate flagella. For details, also see Table S1. (Scale the constricted region, where both the symbiont motility and the bars, 0.5 μm.) host selection play important roles, must be involved in the

Ohbayashi et al. PNAS | Published online August 31, 2015 | E5183 Downloaded by guest on September 30, 2021 Wild-type fliC-/pBBR122 fliC-/fliC selective colonization and establishment of the Burkholderia (RPE75) (NM1 pBBR122) (NM1 fliC) symbiont in the midgut symbiotic organ of R. pedestris,ashas been established for the colonization of the symbiotic light ACB organinthesquid–Vibrio symbiosis (6, 7).

General Relevance of Constricted Region to Gut Symbiosis in Diverse Stinkbugs. The structural differentiation of the midgut into the morphologically distinct regions M1, M2, M3, M4B, and M4 1.0 cm 1.0 cm 1.0 cm (though M4B is not obvious in some species) has been observed across diverse plant-sucking heteropteran bugs (17–20, 49). In an D E F attempt to gain insight into the role of the constricted region in general, adult insects of the following stinkbug species, which represent different stinkbug families and harbor specific symbiotic bacteria within the M4 crypts, were subjected to feeding of water supplemented with Congo red and inspection of their al- 500 nm 500 nm 500 nm imentary tract: in addition to R. pedestris (the family Alydidae), Cletus punctiger and Acanthocoris sordidus (Coreidae), Togo #######"107 G Wild-type hemipterus and Paromius exiguus (Rhyparochromidae), Dolycoris !######"106 /pBBR122 * baccarum (Pentatomidae), Poecilocoris lewisi (Scutelleridae), /fliC Megymenum gracilicorne (Dinidridae), Adomerus triguttulus !#####"105 * (Cydnidae), Elasmucha putoni (Acanthosomatidae), and Mega- copta punctatissima (Plataspidae). In all of the stinkbug species !####"104 examined, strikingly, the coloring stained the M1, the M2, and the M3 but never appeared in the M4B and the M4 (Fig. 8), !###"103 suggesting that the sorting role of the constricted region is not restricted to R. pedestris but is commonly found across the di- !##"102 verse stinkbug groups. The stinkbugs of the superfamilies Cor- eoidea and Lygaeoidea, including R. pedestris, C. punctiger, CFU/midgut section 10!#" A. sordidus, T. hemipterus, and P. exiguus, are associated with betaproteobacterial gut symbionts of the genus Burkholderia (23, 0!" 32), which are orally acquired by nymphal insects mainly from M1 M2 M3 M4B M4 H the environment every generation (22, 23, 50). In these species, Midgut section the constricted region must play a pivotal role in selectively picking Wild-type fliF- fliF- up the Burkholderia symbiont from the diverse environmental (RPE75) ( flIF) ( flIF) microbiota. On the other hand, the stinkbugs of the superfamily Pentatomoidea, including D. baccarum, P. lewisi, M. gracilicorne, HIJ A. triguttulus, E. putoni, and M. punctatissima, are associated with gammaproteobacterial gut symbionts of the family Enter- obacteriaceae (24, 25, 30, 51–53), which are vertically transmitted through host generations by newborn’s feeding on either symbiont-containing excrements smeared on the egg surface (21, 1.0 cm 1.0 cm 500 nm 24, 26) or symbiont-encasing capsules deposited near the eggs #######"107 (25, 27, 54). In these species, although speculative, the con- K AM1 !"(fliK-) stricted region may play a role in excluding microbial contami- - !######"106 AM2 $"(flhF ) * nants for ensuring stable vertical transmission of the specific NM2 %"(fliF-) bacterial symbiont. Also, it should be noted that the constricted - !#####"105 fliF (&"fliF ) region functionally partitions the stinkbug’s alimentary tract into the anterior region for digestion and absorption and the !####"104 posterior region specialized for symbiosis. In A. sordidus and * M. punctatissima, strikingly, their constricted region was reduced !###"103 to a thread-like connective tissue, whereby the anterior region and the posterior region of their alimentary tract were struc- !##"102 turally completely disconnected (Fig. 8 D and T). Such discon-

CFU/midgut section nected alimentary tracts have been described from some 10!#" stinkbug species representing the Plataspidae, the Urostylididae, and other groups (19, 28, 49, 54). In these species, plausibly, after 0!" M1 M2 M3 M4B M4 H Midgut sections mutant NM1fliC to the midgut symbiotic regions of R. pedestris.(H) Normal motility of the wild-type symbiont RPE75 on semisolid agar. (I) No motility of Fig. 7. Colonization ability of the motility-deficient mutants of the Bur- the fliF− mutant ΔfliF on semisolid agar. (J) ΔfliF cell without flagella. (K) kholderia symbiont to the symbiotic midgut regions of R. pedestris.(A)Normal Colonization ability of the fliK− altered-motility mutant AM1, the flhF− al- motility of the wild-type symbiont RPE75 on semisolid agar. (B) No motility of tered-motility mutant AM2, the fliF− no-motility mutant NM2, and the fliF− − the fliC mutant NM1pBBR122 on semisolid agar. (C)Restoredmotilityofthe gene deletion mutant ΔfliF to the midgut symbiotic regions of R. pedestris. − complemented fliC mutant NM1fliC on semisolid agar. (D) RPE75 cell with Symbiont colonization was evaluated 48 h after oral administration to sec- normal flagella (arrowheads). (E)NM1pBBR122 cell without flagella. (F)NM1fliC ond instar nymphs. Means and SDs of cfu per dissected tissue (n = 3 or 4) are cell with restored flagella (arrowhead). (G) Colonization ability of the wild-type shown. Asterisks indicate statistically significant differences between the − − symbiont RPE75, the fliC mutant NM1pBBR122, and the complemented fliC strains (Kruskal–Wallis test; P < 0.05).

E5184 | www.pnas.org/cgi/doi/10.1073/pnas.1511454112 Ohbayashi et al. Downloaded by guest on September 30, 2021 ABCD PNAS PLUS

E FG H

I J K L

M N O P

Q R S T

Fig. 8. Dissected alimentary tracts of diverse stinkbugs fed with water supplemented with a food coloring. (A and B) C. punctiger (Coreidae). (C and D) A. sordidus (Coreidae). (E and F) T. hemipterus (Rhyparochromidae). (G and H) P. exiguus (Rhyparochromidae). (I and J) D. baccarum (Pentatomidae). (K and L) P. lewisi (Scutelleridae). (M and N) M. gracilicorne (Dinidridae). (O and P) A. triguttulus (Cydnidae). (Q and R) E. putoni (Acanthosomatidae). (S and T) M. punctatissima (Plataspidae). Adult insects were fed with water supplemented with Congo red for 3 d and then dissected. Arrows indicate the constricted region, of which red ones in D and T highlight reduction of the constricted region and consequent structural disconnection between the M3 and the M4B. Abbreviations are as in Fig. 1.

young nymphs have orally acquired the symbiotic bacteria and harbor crypt-associated symbiotic bacteria in the posterior mid- established infection in the posterior midgut, the constricted gut (19–21) (Fig. S1B). Considering that all predatory taxa such region is closed and degenerated during the subsequent nymphal as the Nepomorpha (water bugs) and the Gerromorpha (water development, thereby reinforcing the isolation of the posterior striders) constitute the basal lineages whereas all plant-sucking midgut for symbiosis. This unique configuration of the alimen- stinkbugs with the midgut symbiotic bacteria are restricted to the tary tract, which requires complete food absorption in the an- derived group Pentatomomorpha (58–60) (Fig. S1 B and C), it is terior region, must have been enabled by the sap-sucking lifestyle conceivable, although speculative, that the bacteriocyte-associ- of the stinkbugs. ated microbial symbiosis was lost in the predatory ancestor of the Heteroptera, and the gut-associated bacterial symbiosis evolved Evolutionary Origin of Constricted Region. Our finding that the in an ancestor of the Pentatomomorpha in association with the sorting function of the constricted region is conserved among evolutionary transition from predatory lifestyle to plant-sucking the diverse stinkbugs suggests that the novel intestinal organ lifestyle (61). Here we propose a hypothesis that the acquisition emerged in their common ancestor, which sheds new light on the of the novel intestinal organ, the constricted region, which evolutionary origin and relevance of the gut-associated bacterial functionally isolates the posterior midgut for symbiosis and en- symbiosis in the stinkbugs. Insects of the order Hemiptera, which sures colonization of specific bacteria therein, might be relevant consists of such higher taxa as the Sternorrhyncha (aphids, to the diversity and prosperity of the stinkbugs as a major group coccids, whiteflies, and psyllids), the Auchenorrhyncha (cica- of herbivorous insects. das, spittlebugs, leafhoppers, treehoppers, and planthoppers), the Coleorrhyncha (moss bugs), and the Heteroptera (water Conclusion and Perspective. In conclusion, we unveiled a unique bugs, water striders, bedbugs, and stinkbugs), are characterized configuration of the alimentary tract in the stinkbugs. The ali- by their needle-like mouthpart specialized for exploiting liquid mentary tract is, although structurally stretching from mouth to food sources (55) (Fig. S1A). Almost all members of the Ster- anus, divided in the middle by a tiny organ for symbiont sorting, at norrhyncha, the Auchenorrhyncha, and the Coleorrhyncha are which food materials and nonsymbiotic bacteria are blocked and plant sap feeders and obligatorily dependent on bacteriocyte- only symbiotic bacteria are selectively allowed to colonize the associated microbial symbionts (19) (Fig. S1A), which provide posterior midgut region. The sorting organ functionally partitions essential amino acids and other nutrients deficient in their host’s the stinkbug’s midgut into the anterior region for digestion and plant sap diet (8, 56, 57). In the Heteroptera, by contrast, absorption and the posterior region specialized for symbiosis. The whereas predatory species such as water bugs and water striders selective passing of the symbiotic bacteria and the functional iso- possess neither bacteriocytes nor microbial symbionts, the ma- lation of the posterior midgut probably ensure the establishment of EVOLUTION jority of plant-sucking species represented by diverse stinkbugs a specific bacterial association, the stable maintenance of a large

Ohbayashi et al. PNAS | Published online August 31, 2015 | E5185 Downloaded by guest on September 30, 2021 amount of beneficial bacteria, and the proper control over the inoculation), their symbiotic organs were dissected and examined for infection bacterial population in the stinkbug gut symbiosis. Symbiont mu- with the Burkholderia symbiont by diagnostic PCR as described (22, 32). tants of flagella-related genes tend to be rejected at the sorting Burkholderia organ, highlighting that not only the host’s control but also the Inoculation of Food Colorings, GFP-Labeled Symbiont, and RFP- E. coli symbiont’s phenotype is involved in the sorting process at the host– Labeled . To visualize the food passage in the alimentary tract, the nymphs were fed with DWA supplemented with the food colorings symbiont interface. (0.05% wt/vol) listed in Table S3 for 3 d and dissected for visual inspection In diverse animals including insects, fish, mammals, and oth- under a dissection microscope. To elucidate the relationship between food ers, specific microbial consortia within their alimentary tract, passage and symbiont colonization, second instar nymphs were fed with especially those in the posterior region such as the large intestine DWA supplemented with 0.05% (wt/vol) Congo red and 107 cfu/mL of the of humans, are generally involved in a variety of biologically GFP-labeled Burkholderia symbiont. Six hours after inoculation, the nymphs important traits including growth, health, immunity, and so on were dissected and their alimentary tracts were examined under a light and (62–66). In the stinkbugs, the peculiarity resides in the conspic- fluorescence microscope (Axiophot; Carl Zeiss). Coinoculation of the GFP- uous morphological specialization of the posterior symbiotic labeled Burkholderia symbiont and the RFP-labeled E. coli (Table S2) was midgut as well as the highly specific symbiotic microbiota usually conducted to evaluate symbiont selection in the stinkbug–Burkholderia symbiosis. The respective bacterial strains were grown to an early log phase, consisting of a single dominant bacterial species (20, 21). On the 7 grounds that microbial nutritional provisioning is generally im- harvested, diluted, and mixed to be 10 cfu/mL each in DWA. Six hours after inoculation, the nymphs were dissected and their alimentary tracts were portant for plant sap-feeding insects (8, 56, 57) and that the observed under the fluorescence microscope and a confocal laser scanning ancestral heteropterans were predatory and devoid of microbial microscope (LSCM Pascal5; Carl Zeiss). symbionts (58, 61), it is conceivable, although speculative, that the constricted region for symbiont sorting and midgut parti- Transmission Electron Microscopy. From second instar nymphs, the constricted

tioning may represent an evolutionary innovation with which the regions were dissected in 0.1 M phosphate buffer [25 mM NAH2PO4 and stinkbugs are currently prosperous as a major plant-sucking in- 75 mM Na2HPO4 (pH 7.2)] containing 2.5% (wt/vol) glutaraldehyde using sect group embracing many agricultural pests (16). What mo- fine forceps. The dissected tissues were prefixed in the fixative at 4 °C lecular, cellular, and physiological mechanisms underlie the overnight and postfixed in 2% (wt/vol) osmium tetroxide at 4 °C for 60 min. selective symbiont sorting at the constricted region is still an After dehydration through an ethanol series, the tissues were embedded in open question and deserves future studies. an epoxy resin (Epon 812; Okenshoji Co., Ltd). Ultrathin sections were made Most animals (namely, large-sized metazoans, except for those on an ultramicrotome (EM UC7; Leica), mounted on copper meshes, stained with a highly parasitic lifestyle) have a mouth for feeding, an with uranyl acetate and lead citrate, and observed under a transmission electron microscope (H-7600; Hitachi). For inspection of the flagellar ar- anus for defecation, and a gut connecting them for digestion and rangement and structure of the symbiont mutants, exponentially growing absorption. However, some animals intimately associated with bacterial cells were negatively stained with 1% (wt/vol) uranyl acetate on microbial symbionts represent striking deviations from this gen- copper meshes and observed under the transmission electron microscope. eral rule. For example, tubeworms gathering around hydrother- mal vents at the oceanic floor lack mouth, anus, and gut. By Histology for Light Microscopy. The Epon-embedded constricted regions were extending fan-like gills, the animals deliver hydrogen sulfide processed into semiultrathin sections at 0.3 μm on the ultramicrotome, from surrounding spring water to huge amounts of chemoauto- mounted on glass slides, and stained with toluidine blue. The dissected trophic symbiotic bacteria harbored in their body, thereby constricted regions were also fixed in sterilized PBS [137 mM NaCl, 8.1 mM acquiring energy and nutrition without feeding (67). Juvenile Na2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4 (pH 7.5)] containing 4% form- kleptoplastic mollusks such as Elysia chlorotica, so-called so- aldehyde for 1 h at room temperature. After washing with PBS several times, lar-powered sea slugs, accumulate functional chloroplasts from the fixed tissues were then dehydrated through an ethanol series, embedded in a hydroxyethylmethacrylate resin (Technovit 7100; Kulzer), sectioned food algae within the gut lining cells, and adult animals survive at 2 μm on a microtome (RM2165; Leica), mounted on glass slides, and without feeding for as long as 10 mo, solely depending on photo- – “ ” stained with hematoxylin and periodic acid Schiff reagent. These stained synthetic products of the cyanobacterium-derived stolen organ- tissue sections were observed under a light microscope. elles (68). Although not so drastic as these amazing cases, the stinkbugs commonly found in our backyard also exhibit an ex- Random Insertion Mutagenesis of Burkholderia Symbiont. For random Tn5 traordinary symbiosis-associated modification of their alimentary mutagenesis, we used pRL27, a plasmid carrying a hyperactive Tn5 trans- tract, which highlights the general relevance of animal–microbe posase gene adjacent to an insertion cassette comprising two inverted Tn5 symbiosis to organismal adaptation and diversification. termini bracketing a kanamycin-resistant gene kan and a R6K pir-dependent origin (69). The E. coli donor strain WM3064, which is diaminopimelic acid Materials and Methods auxotroph and carries pRL27 (70), was grown on LB medium [1% (wt/vol) Insects, Bacterial Strains, and Plasmids. The R. pedestris strain used in this tryptone, 0.5% (wt/vol) yeast extract, and 1% (wt/vol) NaCl] containing study was originally collected from a soybean field in Tsukuba, Ibaraki, Ja- 0.3 mM diaminopimelic acid, and the plasmid was transferred to the pan and maintained in the laboratory. The insects were reared in Petri dishes Burkholderia symbiont strain RPE75 by conjugation. Burkholderia trans- (90 mm in diameter, 20 mm high) at 25 °C under a long-day regimen (16 h conjugants containing the integrated Tn5 cassette were selected on YG agar μ light, 8 h dark) and fed with soybean seeds and distilled water containing plates containing 30 g/mL kanamycin. 0.05% ascorbic acid (DWA). Bacterial strains and plasmids used in this study are listed in Table S2. Screening for Motility Mutants of Burkholderia Symbiont. The transconjugants from the primary selection plates were individually stabbed into semisolid YG Inoculation of Burkholderia Symbiont. The Burkholderia symbiont strains agar [0.4% (wt/vol) agar and 10 μg/mL kanamycin in YG medium] in 96-well were grown at 30 °C to an early log phase in yeast–glucose (YG) medium microtiter dishes and incubated at 27 °C for 24–48 h. The transconjugants [0.5% (wt/vol) yeast extract, 0.4% (wt/vol) glucose, and 0.1% (wt/vol) NaCl] that grew but did not form halo-shaped colonies were presumed as motility supplemented with an adequate antibiotic on a gyratory shaker at 150 rpm. mutants. Absence of motility of transconjugants resulting from the first Colony-forming unit values were estimated by plating the culture media on screen was confirmed by a second semisolid agar assay. Biofilm-deficient YG agar plates (1.5% agar in YG medium) containing an adequate antibiotic. mutants were screened by a crystal violet staining assay as described (42) and The cultured symbiont cells were harvested by centrifugation, suspended in subsequently subjected to the semisolid agar assay. The transconjugants that DWA,andadjustedto107 cfu/mL. Newly molted second instar nymphs were formed no halo in semisoft agar and exhibited no visible motility in liquid deprived of DWA overnight, which made the insects thirsty and willing to in- medium under a phase-contrast microscope were categorized as “nonmotile gest the symbiont-containing DWA. The thirsty nymphs were fed with the mutants.” Meanwhile, the transconjugants that formed no halo in semisoft symbiont-containing DWA for 24 h, which was subsequently replaced by nor- agar but showed visible motility in liquid medium were designated as mal sterile DWA. Two days after the third instar molt (approximately 5 d after “altered-motility mutants.”

E5186 | www.pnas.org/cgi/doi/10.1073/pnas.1511454112 Ohbayashi et al. Downloaded by guest on September 30, 2021 Inoculation Assay of Motility-Deficient Mutants. Second instar nymphs were Identification of Transposon Insertion Site. Because the origin of replication PNAS PLUS fed with DWA containing each of the motility-deficient mutants, and 2 d after (R6K ori) is within the Tn5 cassette, transposon insertions along with the the third instar molt, the symbiotic midgut sections M4B and M4 were dis- flanking genomic DNA can be cloned in pir+ E. coli and sequenced to de- sected, subjected to DNA extraction, and analyzed by diagnostic PCR as termine the insertion site in the genome of the Burkholderia symbiont. Total described (23). To quantitatively evaluate the infection process of the mo- DNA was prepared from 1 mL of YG liquid culture grown overnight as de- μ tility-deficient mutants, the alimentary tracts were dissected from the sec- scribed (32). Then, 2.5 g of genomic DNA was digested with NcoI, self-ligated, and transformed into One ShotPIR1 Chemically Competent E. coli cells (Invi- ond instar nymphs 48 h after inoculation and carefully cut into the sections trogen) with selection on LB agar plates containing 100 μg/mL kanamycin. Se- M1, M2, M3, M4B, M4, and hindgut in PBS. After rinsing in sterilized PBS, lected colonies were picked and cultured overnight in kanamycin-containing LB each of the alimentary sections was homogenized and spread on YG agar liquid medium, from which plasmid DNA was extracted using QIAprep Spin plates containing 30 μg/mL kanamycin for cfu counting. As a control, the Miniprep Kit (Qiagen). The insert DNA in the plasmid was sequenced with the wild-type symbiont strain RPE75 was inoculated. primers tpnRL17-1 (5′-AAC AAG CCA GGG ATG TAA CG-3′)andtpnRL13-2(5′- CAG CAA CAC CTT CTT CAC GA-3′) (69). The sequence was then compared with Detection of Transposon Insertion. Whether a single Tn5 cassette was inserted the protein sequence databases using the BlastX search. in the genome of the symbiont mutants was examined by Southern blot hybridization. Bacterial genomic DNA samples (around 3 μg) were digested ACKNOWLEDGMENTS. We thank E. V. Stabb and D. K. Newman for with NcoI, electrophoresed on 0.67% (wt/vol) agarose gels, and blotted onto providing plasmids and P. Mergaert for comments on the manuscript. This Hybond N+ membranes (GE Healthcare) using the BacuGene XL Vacuum study was supported by the Institute for Fermentation, Osaka (Y. Kamagata); Blotting system (GE Healthcare). Hybridization was performed using an the Global Research Laboratory of the National Research Foundation of Korea Grant 2011-0021535 (to B.L.L. and T.F.); Ministry of Education, Culture, Sports, AlkPhos Direct Labeling and Detection System (GE Healthcare). A 1,761-bp Science and Technology Grant-in-Aid for Scientific Research 26117732 (to hybridization probe was prepared from pRL27 by digestion with SalI, which Y. Kikuchi); and the Scientific Technique Research Promotion Program for was hybridized to the blot at 55 °C overnight. Agriculture, Forestry, Fisheries and Food Industry (Y. Kikuchi).

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