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202 S.M. Küchler et al. The purpose of this study was to investigate and identify symbiotic bacteria in selected water beetles of the palaearctic genus Deronectes. The association of endosymbiotic bacteria with water insects, especially water beetles, was already presumed in previous studies (Maddison et al., 1999; Shull et al., 2001). Representatives of the genus Deronectes are predacious and colored uniformly black or brown. Deronectes species usually live among gravel in small, swift or strongly flowing streams with sparse vegetation. Most species prefer mountainous regions, some occurring at a high altitude. To date 55 species have been described (Fery & Brancucci, 1997; Fery et al., 2001). In the present molecular study, we report the first finding of a coccobacillus bacterium in Deronectes, which could be identified as a member of the genus Rickettsia. A total of 136 specimens from seven Deronectes species were examined for the existence of Rickettsia sp. using a diagnostic PCR assay. Phylogenetic analysis inferred by 16S rRNA gene and particularly the rickettsial gltA (citrate synthase) gene disclosed the phylogenetic position of beetle Rickettsia in the existing phylogenetic system of Rickettsia endosymbionts. FISH was used for the investigation of rickettsial distribution and vertical transmission rates. The morphological characteristics of the bacterium were analyzed by electron microscopic observations. Materials and methods Samples A total of 136 Deronectes specimens were collected between 2003 and 2008 (Table 1) by kick-sampling (Schwoerbel, 1994). Deronectes platynotus and Deronectes latus were obtained from Fichtelgebirge, a mountain range in northeastern Bavaria, Germany. Deronectes aubei was collected from Black Forest (Germany), Italy and French alps. Deronectes delarouzei, Deronectes semirufus, D. aubei sanfilippoi and Deronectes moestus inconspectus came from Spain, Italy and France. Beetles were brought to laboratory alive and embedded for histology/FISH or dissected for diagnostic PCR immediately. Histology Before all the beetles were fixed in 4% paraformaldehyde overnight, the elytra were carefully removed. The fixed beetles were washed in 1 phosphate-buffered saline and 96% ethanol (1 : 1), dehydrated serially in ethanol (70%, 90%, 2 100%) and embedded in UNICRYL TM (Plano GmbH, Germany). Serial sections (2 mm) were cut using a Leica Jung RM2035 rotary microtome (Leica Instruments GmbH, Germany), mounted on epoxy-coated glass slides and subjected to FISH. DNA extraction, cloning and sequencing DNA was extracted using a QUIAGEN DNeasy s Tissue Kit (QUIAGEN GmbH, Germany) following the protocol for animal tissue. The eubacterial 16S rRNA gene was PCR amplified using the primer set 07F (5 0 -AGAGTTT GATCMTGGCTCAG-3 0 ) and 1507R (5 0 -TACCTTGTTAC GACTTCAC-3 0 ) (Lane, 1991). All PCR reactions were performed in a Biometra thermal cycler with the following program: an initial denaturating step at 94 1C for 3 min, followed by 34 cycles of 94 1C for 30 s, 50 1C for 2 min and 72 1C for 1 min. A final extension step of 72 1C for 10 min was included. PCR products of the expected sizes were cloned using the TOPO TA Cloning s Kit (Invitrogen, CA). Suitable clones for sequencing were selected by restriction fragment length polymorphism (RFLP). Inserts were digested by restriction endonucleases HaeIII and TaqI. Plasmids containing the DNA inserts of the expected sizes were sequenced at the DNA analytics core facility of the Table 1. Infection rates of Rickettsia sp. in natural populations of Deronectes spp. Species Locality Date of collection No. of individuals % of infection (infected/total) D. platynotus Fichtelgebirge, Bavaria, Germany 2007/2008 45 100 (45/45) D. aubei Black Forest, Germany, Italy, France 2003–2008 71 39.4 (28/71) D. latus Fichtelgebirge, Bavaria, Germany 17.10.2007 7 0 (0/7) D. delarouzei Bonansa, El pont de Suert, Spain 05.10.2007 2 Llesp, El pont de Suert, Spain 10.08.2004 1 40 (2/5) 05.10.2007 1 Saldes, Spain 05.10.2007 1 D. semirufus Grotta all’Onda, Casoli, Lucca, Tuscany, Italy 09.10.2003 1 Sospel, Moulinet, Apuane-Maritimes, France 07.10.2003 1 33.3 (1/3) Fociomboli, Apuane Alps, Italy 07.09.2007 1 D. aubei sanfilippoi Vernet les Bains, Pyr. Orientales, France 07.10.2007 3 0 (0/3) D. moestus inconspectus Saldes, Spain 17.10.2007 2 0 (0/2) c 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved FEMS Microbiol Ecol 68 (2009) 201–211
Rickettsia endosymbiont in water beetles 203 University of Bayreuth with M13 forward and M13 reverse sequencing primers (Invitrogen). For diagnostic PCR, the 16S rRNA gene was amplified using the Rickettsia-specific primers Ri_170F (5 0 -GGGCTTGCTCTA AATTAGTTAGT-3 0 ) and Ri_1500R (5 0 -ACGTTAGCTCACC ACCTTCAGG-3 0 ). The citrate synthase gene (gltA) wasdetected using the primers CS5_F (5 0 -TCTTTATGGGGACC AGCC-3 0 )andCS4_R(5 0 -TTTCCATTGTGCCATCCAG-3 0 ). PCR primers were designed using the probe design tool of the ARB software package (Ludwig et al., 2004). Diagnostic PCR was performed under the temperature profile described above. FISH The following probes were used for FISH targeted to the 16S rRNA gene: eubacterial probe EUB338 [5 0 -(Cy5)-GCTGCCT CCCGTAGGAGT-3 0 ](Amannet al., 1995), EUB388 II [5 0 - (Cy3)-GCAGCCACCCGTAGGTGT-3 0 ], EUB338 III [5 0 -(Cy3)- GCTGCCACCCGTAGGTGT-3 0 ] (Daims et al., 1999) and Rickettsia-specific probe Rick_B1 [5 0 -(Cy3)-CCATCATCCC CTACTACA-3 0 ] (Perotti et al., 2006). Additionally, a nonsense probe complementary to EUB338, NON338 [5 0 -(Cy3)- ACTCCTACGGGAGGCAGC-3 0 ] (Manz et al., 1992) was used as a negative control of the hybridization protocol. Slides were hybridized in hybridization buffer [20 mM Tris- HCl (pH 8.0), 0.9 M NaCl, 0.01% sodium dodecyl sulfate (SDS), and 20% formamide] containing 10 pmol of fluorescent probes per milliliter, incubated at 46 1C for 90 min, rinsed in washing buffer [20 mM Tris-HCl (pH 8.0), 225 mM NaCl, 5 mM EDTA, and 0.01% SDS], mounted with antibleaching solution (VECTASHIELD s Mounting Medium; Vector Laboratories, UK) and viewed under a fluorescent microscope. Electron microscopy Male accessory glands of D. platynotus were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h, embedded in 2% agarose and fixed again in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) overnight. Specimens were washed in 0.1 M cacodylate three times for 20 min. Following postfixation in 2% osmium tetroxide for 2 h, the specimens were washed and stained en bloc in 2% uranyl acetate for 90 min. After fixation, specimens were dehydrated serially in ethanol (30%, 50%, 70%, 95% and 3 100%), transferred to propylene oxide and embedded in Epon. Ultrathin sections (70 nm) were cut with a diamond knife (MicroStar, Huntsville, TX) on a Leica Ultracut UCT microtome (Leica Microsystems, Vienna, Austria). Ultrathin sections were mounted on pioloform-coated copper grids, and stained with saturated uranyl acetate, followed by lead citrate. The sections were viewed with a Zeiss CEM 902 A transmission electron microscope (Carl Zeiss, Oberkochen, Germany) at 80 kV. Micrographs were taken using an SO-163 EM film (Eastman Kodak, Rochester, NY). Phylogenetic analysis High-quality sequences of the 16S rRNA gene and the gltA gene for citrate synthase were aligned with the CLUSTALW software in BIOEDIT (Hall, 1999) and transferred to the ARB software package and edited manually. Phylogenetic analyses using maximum parsimony were performed using PAUP version 4.0b10 (Swofford, 2000). The heuristic search included 10 000 random addition replicates and tree bissection–reconnection (TBR) branch swapping with the Multrees option. A 50% majority-rule consensus tree of the most parsimonious tree was constructed and exported to ARB program package, where branch lengths were calculated. Bootstrap analyses were performed with 500 replicates with TBR branch swapping, 25 random addition replicates and the Multrees option in PAUP . The gltA citrate synthase sequences were checked for chimeras using CCODE (Gonzalez et al., 2005). Sequence data Clone sequences of 16S rRNA gene and gltA sequences for the D. platynotus-, D. aubei-, D. semirufus- andD. delarouzeiassociated Rickettsia sp. were deposited in the DDBJ/EMBL/ GenBank nucleotide sequence databases with the accession numbers FM177868–FM177878 and FM955310–FM955315, respectively. Results Identification of bacterial symbiont A 1.5-kb segment of the eubacterial 16S rRNA gene was amplified by PCR and was subjected to cloning and RFLP typing. Overall, 19 clones were sequenced and compared with other sequences found in GenBank. The nucleotide sequences of clones exhibited 99% similarity to the sequence of Rickettsia limoniae (AF322442, AF322443) from cranefly L. chorea (Diptera, Limoniidae). The validity of the sequences was confirmed by FISH performed with probe Rick_B1. Furthermore, we sequenced a 416-bp fragment of the gltA gene (citrate synthase) from four Rickettsia-positive D. platynotus beetles as well as from D. aubei (404 bp), D. semirufus (367 bp) and D. delarouzei (401 bp). The gltA sequences from D. platynotus rickettsiae were 100% identical to rickettsial isolate (DQ 231491) from spider Pityophantes phrygianus (Goodacre et al., 2006). The gltA gene sequences from rickettsial symbionts of D. aubei and D. delarouzei exhibited 99% similarity to the sequence of P. phrygianus, whereas rickettsial gltA sequences from D. semirufus showed 98% similarity to Rickettsia endosymbionts of spider Meta menegi. FEMS Microbiol Ecol 68 (2009) 201–211 c 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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