Article Figures & Data
Figures
- FIG. 1.
Midgut organization of R. clavatus and L. chinensis. (A) Dissected midgut of R. clavatus. (B) Dissected midgut of L. chinensis. (C) Tissue section of midgut crypts of R. clavatus, stained with hematoxylin and eosin. (D) Fluorescent in situ hybridization of a tissue section of midgut crypts of R. clavatus, targeting 16S rRNA of the Burkholderia symbiont. Green signals are due to the Burkholderia-specific probe Cy3-Alsym16S. Blue signals are nuclei of the host cells visualized by DAPI. The arrows indicate the Burkholderia signals in the main tract of the midgut and signals in the ducts connecting the crypts with the main tract. (A) Bar = 0.5 mm. (B) Bar = 0.2 mm. (C and D) Bars = 100 μm. Abbreviations: C, crypt: G, gut; M1, first midgut section; M2, second midgut section; M3, third midgut section; M4, fourth midgut section (symbiotic organ); H, hindgut.
- FIG. 2.
Phylogenetic relationship of the Burkholderia symbionts from R. clavatus and L. chinensis on the basis of 16S rRNA gene sequences. (A) Neighbor-joining tree. (B) Maximum-parsimony tree based on 50% majority-rule consensus. Pandoraea spp. were used as outgroup taxa for the genus Burkholderia. Bootstrap values greater than 50% are shown at the nodes. The numbers in brackets are nucleotide sequence accession numbers.
- FIG. 3.
Transmission electron microscopy of the midgut crypts of R. clavatus and L. chinensis. (A) Crypt of R. clavatus harboring many rod-shaped bacteria. Bar = 2 μm. (B) Enlarged image of the interface between the lumen and the epithelium of the crypt in R. clavatus. The bacteria are present only in the lumen. Bar = 1 μm. (C) Enlarged image of the rod-shaped bacteria in R. clavatus. A well-developed cell wall and a particle-like structure are present. Bar = 300 nm. (D) Crypt of L. chinensis harboring many rod-shaped bacteria. Bar = 2 μm. Abbreviations: B, symbiotic bacterium; M, mitochondrion; N, nucleus.
Tables
- TABLE 1.
Alydid stinkbug samples used in this study
Species Sample Locality Collection date Collector Riptortus clavatus a IB1 Mito, Ibaraki 30 June 2001 Y. Kikuchi IB2 Tomobe, Ibaraki 22 August 2002 M. Ishizaki IB3 Tsukuba, ibaraki 5 May 2001 Y. Kikuchi GN Numata, Gunma 15 July 2003 Y. Kikuchi NG Joetsu, Nigata 6 October 2003c,d H. Higuchi KY Kyoto, Kyoto 29 September 2003d C. Himuro NR Nara, Nara 16 October 2003d C. Himuro KC Nakamura, Kochi 26 September 2003 M. Takai SG Kanzaki, Saga 7 May 2002 T. Kashima KM Kikuchi, Kumamoto 3 October 2003d Y. Kikuchi OK Ishigaki, Okinawa 25 July 2002 K. Kohno Leptocorisa chinensis a FK Hanawa, Fukushima 14 August 2002 Y. Kikuchi IB1 Mito, Ibaraki 29 August 2002 Y. Kikuchi IB2 Tsukuba, Ibaraki 10 August 2002d Y. Kikuchi 10 October 2003c Y. Kikuchi GN Kiryu, Gunma 18 November 2003 E. Hara CB Maruyama, Chiba 8 September 2002 Y. Kikuchi KY Kyoto, Kyoto 29 September 2003d C. Himuro NR Nara, Nara 16 October 2003d C. Himuro SG Saga, Saga 3 June 2002 T. Kashima KM Kikuchi, Kumamoto 3 October 2003d Y. Kikuchi Riptortus linearis b Naha, Okinawa 20 June 2002 T. Fukatsu Leptocorisa acuta b Iriomote, Okinawa 26 May 2004 T. Yasuda Leptocorisa oratoria b Iriomote, Okinawa 26 May 2004 T. Yasuda Megalotomus costalis b Sapporo, Hokkaido 26 August 2002 K. Ito Daclera levana b Ishigaki, Okinawa 23 July 2002 K. Kohno Paraplesius unicolor b Monobe, Kochi 27 July 2002 M. Takai ↵ a One individual from each population was subjected to cloning and sequencing.
↵ b One individual was subjected to diagnostic PCR analysis.
↵ c Subjected to diagnostic PCR analysis to examine the localization in a body.
↵ d Subjected to diagnostic PCR analysis to survey the infection rate in a natural population.
- TABLE 2.
Levels of similarity for the Burkholderia symbionts detected in local samples of R. clavatus and L. chinensis and other Burkholderia strains based on 16S rRNA gene sequencesa
Sample R. clavatus L. chinensis Burkholderia sp. B. kurkii B. glathei B. sordidicola B. capacia B. graminis IB1 IB2 IB3 GN KY NR KC SG KM OK type 1 OK type 2 FK IB1 IB2 GN CB KY NR SG KM type 1 KM type 2 S4.9 AK-5 WD206 NF100 R. clavatus IB1 1 0.98 0.983 0.974 0.98 0.978 0.977 0.974 0.984 0.971 0.969 0.991 0.981 0.978 0.992 0.983 0.983 0.983 0.98 0.985 0.983 0.978 0.979 0.981 0.996 0.972 0.971 0.972 0.968 0.954 IB2 1 0.974 0.981 0.985 0.983 0.991 0.976 0.982 0.982 0.977 0.98 0.973 0.981 0.98 0.977 0.974 0.974 0.976 0.974 0.977 0.983 0.974 0.973 0.981 0.969 0.969 0.965 0.959 0.957 IB3 1 0.982 0.98 0.983 0.977 0.985 0.991 0.974 0.977 0.987 0.998 0.983 0.988 0.988 1 0.992 0.977 0.991 0.987 0.984 0.992 0.998 0.985 0.969 0.974 0.971 0.961 0.952 GN 1 0.986 0.991 0.982 0.985 0.988 0.986 0.988 0.982 0.981 0.989 0.981 0.983 0.982 0.987 0.98 0.987 0.989 0.991 0.988 0.98 0.979 0.976 0.974 0.961 0.958 0.953 KY 1 0.993 0.99 0.98 0.985 0.985 0.979 0.981 0.979 0.994 0.98 0.98 0.98 0.98 0.976 0.98 0.983 0.995 0.98 0.977 0.984 0.976 0.979 0.968 0.962 0.96 NR 1 0.986 0.983 0.989 0.986 0.982 0.985 0.983 0.996 0.985 0.983 0.983 0.984 0.979 0.984 0.986 0.999 0.984 0.981 0.983 0.973 0.974 0.965 0.96 0.959 KC 1 0.976 0.981 0.986 0.977 0.976 0.974 0.986 0.977 0.976 0.977 0.974 0.971 0.974 0.976 0.987 0.974 0.975 0.977 0.971 0.971 0.961 0.956 0.957 SG 1 0.983 0.983 0.991 0.983 0.984 0.982 0.983 0.99 0.985 0.989 0.991 0.989 0.985 0.983 0.989 0.982 0.979 0.973 0.971 0.963 0.959 0.958 KM 1 0.98 0.98 0.988 0.989 0.988 0.988 0.987 0.991 0.988 0.976 0.991 0.994 0.99 0.988 0.988 0.986 0.974 0.977 0.967 0.965 0.953 OK type 1 1 0.985 0.976 0.973 0.988 0.975 0.977 0.974 0.981 0.979 0.981 0.983 0.988 0.978 0.973 0.971 0.971 0.966 0.955 0.949 0.953 OK type 2 1 0.978 0.977 0.98 0.977 0.983 0.977 0.982 0.987 0.982 0.983 0.983 0.982 0.976 0.974 0.973 0.97 0.959 0.955 0.957 L. chinensis FK 1 0.986 0.984 0.999 0.99 0.987 0.987 0.988 0.988 0.986 0.985 0.987 0.985 0.996 0.966 0.972 0.972 0.967 0.955 IB1 1 0.982 0.987 0.988 0.998 0.991 0.976 0.99 0.986 0.983 0.991 0.996 0.984 0.668 0.973 0.971 0.96 0.949 IB2 1 0.983 0.983 0.983 0.985 0.977 0.985 0.987 0.998 0.983 0.98 0.981 0.973 0.974 0.963 0.959 0.956 GN 1 0.991 0.988 0.986 0.988 0.988 0.985 0.985 0.986 0.985 0.995 0.967 0.971 0.971 0.966 0.955 CB 1 0.988 0.99 0.985 0.991 0.987 0.984 0.99 0.986 0.985 0.971 0.97 0.966 0.965 0.952 KY 1 0.992 0.977 0.991 0.987 0.984 0.992 0.998 0.985 0.969 0.974 0.971 0.961 0.952 NR 1 0.981 0.997 0.993 0.985 0.997 0.99 0.984 0.974 0.974 0.966 0.96 0.95 SG 1 0.981 0.979 0.979 0.981 0.974 0.984 0.967 0.965 0.966 0.959 0.956 KM type 1 1 0.996 0.985 0.994 0.988 0.987 0.975 0.973 0.965 0.963 0.95 KM type 2 1 0.987 0.991 0.985 0.985 0.977 0.975 0.965 0.964 0.951 Burkholderia sp. S4.9 1 0.985 0.982 0.983 0.974 0.975 0.965 0.961 0.959 AK-5 1 0.99 0.984 0.972 0.974 0.966 0.961 0.951 WD206 1 0.982 0.967 0.971 0.969 0.958 0.949 NF100 1 0.971 0.975 0.976 0.971 0.957 B. kirkii 1 0.968 0.955 0.959 0.952 B. glathei 1 0.972 0.96 0.956 B. sordidicola 1 0.967 0.952 B. cepacia 1 0.948 B. graminis 1 a The levels of similarity were calculated by using Kimura's two-parameter model.
- TABLE 3.
Diagnostic PCR detection of the Burkholderia symbionts in the tissues of R. clavatus and L. chinensis
Species Sex No. positivea Foreleg Proboscis Foregut Midgut section 1 Midgut sections 2 and 3 Midgut section 4 (crypts) Hindgut Testis or ovary R. clavatus Male 0 0 0 0 2 10 4 1 Female 0 0 0 2 2 10 1 0 L. chinensis Male 0 0 0 0 0 10 0 0 Female 0 0 1 0 1 10 1 0 ↵ a Ten individuals were examined for each species and each sex.
- TABLE 4.
Detection of the Burkholderia symbionts in midgut crypts in natural populations of R. clavatus and L. chinensis
Species Sample % Detection (no. positive/total no.) R. clavatus NG 100 (26/26) KY 100 (11/11) NR 100 (10/10) KM 95 (40/42) Total 98 (87/89) L. chinensis IB2 97 (70/72) KY 100 (12/12) NR 100 (11/11) KM 100 (21/21) Total 98 (114/116)
