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RESEARCH ARTICLE 

Characterization and localization of Rickettsia sp. in water beetles of 

genus Deronectes (Coleoptera: Dytiscidae) 

Stefan Martin Küchler, Siegfried Kehl & Konrad Dettner 

Department of Animal Ecology II, University of Bayreuth, Bayreuth, Germany 

Correspondence: Stefan Martin Küchler, 

Department of Animal Ecology II, University of 

Bayreuth, Universitätsstraße 30, 95440 

Bayreuth, Germany. Tel.: 10049 0921 55 

2733; fax: 10049 0921 55 2743; e-mail: 

stefan.kuechler@uni-bayreuth.de 

Received 22 September 2008; revised 3 

February 2009; accepted 3 February 2009. 

First published online 16 March 2009. 

DOI:10.1111/j.1574-6941.2009.00665.x 

Editor: Michael Wagner 

Keywords 

Rickettsia; Deronectes; Dytiscidae; citrate 

synthase; electron microscopy. 

Abstract 

In the present study, Rickettsia sp. was detected in four water beetles of the genus 

Deronectes (Dytiscidae) for the first time. Rickettsiae were found in 100% of 

examined specimens of Deronectes platynotus (45/45), 39.4% of Deronectes aubei 

(28/71), 40% of Deronectes delarouzei (2/5) and 33.3% of Deronectes semirufus 

(1/3). Analysis of 16S rRNA gene sequences revealed a phylogenetic relationship 

with rickettsial isolates of Limonia chorea (Diptera), tentatively classified as 

members of the basal ancestral group. Phylogenetic analysis of the gltA (citrate 

synthase) gene sequences showed that Deronectes symbionts were closest to 

bacterial symbionts from spiders. Ultrastructural examinations revealed typical 

morphological features and intracellular arrangements of rickettsiae. The distribution, 

transmission and localization of Rickettsia sp. in D. platynotus were studied 

using a diagnostic PCR assay and FISH. Eggs from infected females of D. platynotus 

were all Rickettsia-positive, indicative of a vertical transmission. 

Introduction 

Gram-negative bacteria of the genus Rickettsia belong to the 

family Rickettsiaceae in the subdivision of the monophyletic 

order Rickettsiales (Alphaproteobacteria). All members of the 

genus Rickettsia are obligate intracellular bacteria that reside 

free in the cytoplasm of eukaryotic cells. Mostly known 

as pathogenic endosymbionts of arthropods (ticks, mites, 

fleas and lice), rickettsiae cause human and animal diseases 

with a worldwide distribution (Eremeeva & Dasch, 2000). 

Some species are also capable of infecting and causing 

disease in plants (Davis et al., 1998). Rickettsiae are usually 

transmitted by arthropods to vertebrates via feces or salivary 

secretions. Because of their considerable medical importance, 

the genus Rickettsia was traditionally divided into two 

groups (Weiss & Moulder, 1984): the spotted fever group, 

including Rickettsia rickettsii and allied species, causative 

agents of spotted fever; and the typhus group, including 

Rickettsia prowazekii and Rickettsia typhi, the agents of 

murine and endemic typhus, respectively. In recent years, 

new rickettsiae have been discovered that do not belong to 

the previously described subgroups. A third group named 

‘ancestral group’, partially subdivided into the bellii, leech 

and limoniae groups (Perlman et al., 2006; Perotti et al., 

2006), was established (Stothard & Fuerst, 1995; Fournier & 

Raoult, 2007), and consists of Rickettsia bellii, Rickettsia 

canadensis and additional numerous rickettsial isolates that 

have been isolated from different terrestrial arthropods such 

as whiteflies, flies, aphids and beetles (Chen et al., 1996; 

Fukatsu et al., 2000; Campbell et al., 2004; Gottlieb et al., 

2006; Zchori-Fein et al., 2006). The ancestral group is still an 

initial theoretical classification that is not based on 

morphological criteria or biochemical features concerning 

basal standing rickettsial isolates in the genus. Rickettsiae of 

the ancestral group were not shown to be pathogens for 

humans or their animal hosts. However, insects are not the 

only vector of rickettsiae. Recently, rickettsiae have been 

found in spider mite Tetranychus urticae (Hoy & Jeyaprakash, 

2005), various spider species (Goodacre et al., 2006), 

amoeba Nuclearia pattersoni (Dykova et al., 2003), marine 

ciliate Diophrys appendiculata (Vannini et al., 2005) and 

several leeches from freshwater environments (Kikuchi et al., 

2002; Kikuchi & Fukatsu, 2005), which also cluster into the 

ancestral group. Analyses of 16S rRNA genes suggest that 

many of these rickettsial isolates could represent new genera 

within the order Rickettsiales. As already supposed (Yu & 

Walker, 2006), the family Rickettsiaceae could be more 

widespread than observed so far. 

FEMS Microbiol Ecol 68 (2009) 201–211 

c 2009 Federation of European Microbiological Societies 

Published by Blackwell Publishing Ltd. All rights reserved

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) 


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. 





Screening for the presence of Rickettsia sp. 

Six Deronectes species and one subspecies were examined 

using Rickettsia-specific primers in a diagnostic PCR assay 

(Table 1). A total of 45 D. platynotus were screened. Rickettsia 

sp. could be detected in 100% of all tested D. platynotus. In 

contrast, only 39.4% of 71 examined individuals of D. aubei 

were infected. Both species were collected in different seasons 

and populations. Just two individuals of D. delarouzei and 

one individual of D. semirufus werefoundtobepositivefor 

the Rickettsia symbiont. For elaboration of infection rates in 

these two species, more specimens from different populations 

have to be examined for rickettsiae. No rickettsiae were 

detected in seven individuals of D. latus. In the same way, 

three surveys of D. aubei sanfilippoi indicated a negative signal 

for Rickettsia. Two examined individuals of D. moestus 

inconspectus also did not show any infection of Rickettsia. 

However, only a few individuals of D. latus, D. aubei 

sanfilippoi and D. moestus inconspectus could be examined 

for the presence of rickettsiae. All examined species of 

D. platynotus and D. aubei exhibited a well-balanced sex ratio. 

Furthermore, there was no evidence for male killing, parthenogenesis 

or other sex ratio distortion in numerically examined 

species of D. platynotus and D. aubei. Currentstudies 

indicated that further water beetles are associated with 

Rickettsia sp. too (data not shown). Examined species of 

Agabus melanarius, Agabus wasastjernae, Agabus guttatus, 

Hydroporus gyllenhalii, Hydroporus tristis, Hydroporus umbrosus 

and Hydroporus obscurus also exhibited Rickettsia infections. 

In situ hybridization of Rickettsia symbiont 

FISH revealed a remarkable affection of the Rickettsia 

symbiont (Fig. 1a). All tissues that are in contact with the 

hemolymph are affected by rickettsiae. The symbionts could 

be identified in all compartments of Deronectes, including 

the head (ommatidia) (Fig. 1b) and legs. Actually, internal 

parts of elytra with soft tissue exhibited bacteria. Especially, 

numerous rickettsiae could be detected in tissue of active 

metabolism, such as the fatbody and internal reproduction 

organs. A lower number of rickettsiae was found in muscles. 

In addition, Rickettsia sp. is more abundant in females than 

males. Accessory glands of males will be affected predominantly 

(Fig. 1c). The strongest signals of a Rickettsia-specific 

probe were found in females of D. platynotus. Minor signals 

of symbionts were detected in D. aubei, D. semirufus and 

D. delarouzei (data not shown). 

The distribution of the Rickettsia was also investigated in 

eggs and larvae of D. platynotus. The presence of large 

numbers of symbionts in oocytes and follicle cells (Fig. 1d), 

and the detection of Rickettsia in eggs (Fig. 1e) are indicative 

of the vertical transmission of symbionts. In the second and 

third larval stages of Deronectes, rickettsiae could be detected 

in the entire body. The number of symbionts increased from 

stage to stage. 

Fig. 1. FISH with specific probe Rick_B1 (Cy3) of 

square sections of water beetle Deronectes 

platynotus adults. (a) Rickettsia sp. infection of 

fatbody in a female abdomen. Scale bar = 40 mm. 

(b) Square section of head; numerous Rickettsia 

signals from ommatidia (arrows). Rickettsia sp. 

clustered around the cerebral ganglia. Scale 

bar = 40 mm. (c) Male accessory glands infected 

by intracytosolic Rickettsia sp. Bacteria are 

located near the connective tissue (arrow). Scale 

bar = 20 mm. (d) Ovariole infected by Rickettsia 

sp. symbiont. Scale bar = 20 mm. (e) Bacterial 

symbionts are also present within the egg 

(arrow). Surrounding tissue of ovarioles habouring 

Rickettsia. Scale bar = 50 mm. FB, fat body; 

B, brain; E, egg; AGT, accessory glandular tissue; 

M, muscle; O, ovariole; OM, ommatidia. (a), (b) 

and (c) were taken by confocal microscope. 

(d) and (e) were taken by a conventional 

fluorescence microscope. 





205 

Fig. 2. FISH double hybridization of the 

Rickettsia symbiont on tissue (fatbody) crosssection 

from Deronectes platynotus (a–c, scale 

bar = 40 mm) and Deronectes aubei (d–f, scale 

bar = 20 mm). The left panels (a, d) show FISH 

with the probe Cy3-Rick_B1, which visualizes the 

Rickettsia symbiont, while middle panels (b, e) 

show FISH with the probe Cy5-EUB338, which 

recognizes diverse eubacteria universally. The 

overlay (c, f) does not show other bacteria. 

Double hybridization performed with Rick_B1, together 

with the universal probe EUB338, showed the absence of any 

other kind of endosymbiotic bacteria (Fig. 2), with the 

exception of gut bacteria inside adult and larval specimens 

of Deronectes. FISH accomplished with EUB388 II and 

EUB338 III did not show any positive signals (data not 

shown). 

Electron microscopy of the Rickettsia symbiont 

Ultrastructural examinations of male accessory glands of 

D. platynotus revealed coccobacillary rickettsiae that were 

observed free in the cytoplasm (Fig. 3a). Most rickettsiae 

ranged from 0.35 mm in width to 0.65 mm in length. Rickettsiae 

were delineated by an inner periplasmatic membrane, 

a periplasmatic space and a trilaminar cell wall with a thicker 

inner leaflet, characteristic of the genus Rickettsia. ARickettsia-typical 

slime layer or an electron-translucent zone, up 

to 60 nm thick, surrounded the cell wall and separated the 

bacterium from the cytoplasm of the host cell (Fig. 3b). 

Isolated bacteria were also found in the musculature, 

enclosing the male accessory gland (Fig. 3c). Some of the 

symbionts were much longer and had a ‘filamentous’ 

appearance. They could be as long as 2.74 mm (Fig. 3d). 

Phylogenetic analysis of the Rickettsia symbiont 

Phylogenetic analyses on the basis of the 16S rRNA gene 

sequence revealed that Rickettsia sp. from Deronectes belong 

neither to the ‘spotted fever group’ nor the ‘typhus group,’ 

but was placed within the ancestral group (100% bootstrap 

support) (Fig. 4). Clones of the endosymbiotic bacteria of 

D. platynotus, D. aubei and D. delarouzei were placed in the 

clade with R. limoniae, Rickettsia endosymbionts of Cerobasis 

guestifalica (Psocoptera, Trogiidae) and Lutzomyia apache 

(Diptera, Psychodinae), whereas Rickettsia sp. of D. semirufus 

cluster basal with rickettsiae from leeches. By phylogenetic 

analysis based on the gltA gene (Fig. 5), the Rickettsia strain 

from Deronectes clustered with Rickettsia endosymbionts of 

L. apache and different spider species in a terminal group 

(99% bootstrap support) that is distinctly separated from 

other Rickettsia species of the spotted fever, typhus and bellii 

groups. The phylogenetic position of Deronectes rickettsiae 

was the same in the 16S rRNA gene and gltA trees when the 

maximum-likelihood, parsimony and distance methods 

were used (data not shown). Only the position of the 

Rickettsia endosymbiont of D. semirufus varied with different 

methods (MrBayes, data not shown), and was sometimes 

added to the leech group or to the other rickettsial 

endosymbionts of the Deronectes species of the limoniae 

group. This explains the low bootstrap support values 

in the ancestral group in the phylogenetic tree of 16S rRNA 

gene. 

Discussion 

The present study reports the first molecular identification 

of Rickettsia in four water beetle species of the genus 

Deronectes and generally for adephagan beetles. Evaluable 

results have been obtained of D. platynotus and D. aubei. 

Rickettsia infection was detected in 100% (45/45) of 

D. platynotus and 39.4% (28/71) of D. aubei. The frequencies 

of Rickettsia infection were maintained constant in 

different seasons. Individuals of D. delarouzei and D. semirufus 

were also tested positive for rickettsiae, whereas 

individuals of D. latus, D. aubei sanfilippoi and D. moestus 

inconspectus did not show any signal for symbiotic bacteria. 

Remarkably, Rickettsia-positive species of Deronectes do not 

cluster in a single group within the phylogenetic tree of 

Deronectes. In addition, the current analysis indicated six 

more species of Agabus and Hydroporus (Dytiscidae) that are 





Fig. 3. Electron photomicrographs of male 

accessory gland tissue from water beetle 

Deronectes platynotus infected by intracytosolic 

Rickettsia sp. (a) Coccobacillary rickettsiae 

resided free in the cytoplasm of accessory gland 

tissue (asterisk), located near connective tissue 

(arrow), which close about the glandular tissue. 

Scale bar = 1.1 mm. (b) High magnification of 

Rickettsia sp. showing the typical rickettsial 

morphology, with a periplasmic membrane 

(white arrowhead), an electron-lucent 

periplasmic space (black arrowhead), the 

trilaminar cell wall (black arrow) and with an 

electron-lucent ‘halozone’ (asterisk). Scale 

bar = 0.09 mm. (c) Rickettsia within accessory 

gland musculature. Scale bar = 0.6 mm. 

(d) A filamentous Rickettsia (asterisk), possessing 

a length of 2.74 mm, before extending out of the 

plane of section (arrow). Scale bar = 0.4 mm. 

associated with Rickettsia sp. too. To our knowledge, only six 

verifications of Rickettsia have been documented in Coleoptera 

so far (Coccinellidae: Werren et al., 1994; von der 

Schulenburg et al., 2001; Bruchidae: Fukatsu et al., 2000; 

Buprestidae: Lawson et al., 2001; Curculionidae: Zchori-Fein 

et al., 2006; and Mordellidae: Duron et al., 2008). All 

rickettsial isolates from beetles, with the exception of 

Mordellistena sp., Adalia bipunctata and Adalia decempunctata, 

have been allocated to the rickettsial ancestral group, 

which includes all members of a clade that are most basal in 

the genus Rickettsia. Interestingly, the ancestral group comprised 

various symbionts from aquatic hosts, such as leeches 

(Kikuchi et al., 2002; Kikuchi & Fukatsu, 2005), amphizoic 

amoeba N. pattersoni (Dykova et al., 2003) and marine 

ciliate D. appendiculata (Vannini et al., 2005). Even insects 

that spend most of their development in water, i.e. the 

cranefly L. chorea (Diptera, Limoniidae; AF322443), mothfly 

L. apache (Diptera, Psychodidae; EU223247) and biting 

midge Culicoides sonorensis (Diptera, Ceratopogonidae) 

(Campbell et al., 2004), have been shown to be infected with 

rickettsiae. The exact classification of these rickettsial isolates 

that are allocated to the ancestral group is still unclear. 

Only 16S rRNA gene sequences are available for most 

members of the ancestral group. However, for detailed 

phylogenetic analysis within the genus Rickettsia, 16S rRNA 

gene sequences are not convenient because of their conservatism 

(Roux et al., 1997). Nevertheless, rickettsial 16S 

rRNA clone sequences from D. platynotus are different 

in two to six positions from each other, mainly in two 

variable regions. In all sequenced clones, the sequence 

similarity ranges from 99.1% to 100%. Possible reasons 

for the microdiversity of rickettsial 16S rRNA gene clones 

in D. platynotus may be the presence of multiple different 

copies of the 16S rRNA gene in one genome, which has 

been demonstrated for numerous species of bacteria (Cilia 

et al., 1996). 





207 

Fig. 4. Phylogenetic position of different clones of the endosymbiotic bacteria of water beetles Deronectes platynotus, Deronectes aubei, 

Deronectes semirufus and Deronectes delarouzei (bold) based on 55 16S rRNA gene sequences (1350 bp, maximum parsimony heuristic search, 50% 

majority-rule consensus tree of 840 most parsimonious trees, tree length = 497, CI = 0.71, RI = 0.86). The tree has been rooted with Orientia 

tsutsugamushi as an outgroup. Only bootstrap values of at least 50% are shown at nodes (n = 500). 





Fig. 5. Phylogenetic position of the endosymbiotic bacteria of water beetles Deronectes platynotus, Deronectes aubei, Deronectes semirufus and 

Deronectes delarouzei (bold) based on 46 gltA gene sequences (275 bp, maximum parsimony heuristic search, 50% majority-rule consensus tree of 30 

most parsimonious trees, tree length = 459, CI = 0.55, RI = 0.88). The tree has been rooted with Rhodopirellula baltica as an outgroup. Only bootstrap 

values of at least 50% are shown at nodes (n = 500). 





209 

Referred to rickettsiae of the ancestral group, the citrate 

synthase-coding gltA gene sequences, also used for phylogenetic 

analysis, are only available for Rickettsia sp. of L. apache 

(EU368001) and rickettsial isolates from spiders (Goodacre 

et al., 2006). Sequences of further protein-coding genes, 

mostly those belonging to the surface cell antigen (sca) 

family, which have been used for the classification of 

rickettsial isolates over the past few years (Fournier et al., 

1998; Roux & Raoult, 2000; Sekeyova et al., 2001; Ngwamidiba 

et al., 2006), are not available for rickettsiae of the 

ancestral group (Fournier et al., 2003). 

Phylogenetic analysis based on the 16S rRNA gene 

showed that Rickettsia sp. from Deronectes cluster into the 

‘limoniae group’, the basal subgroup of the ancestral group. 

Furthermore, we succeeded in partial sequencing of the gltA 

gene of these rickettsiae. Sequences from Deronectes symbionts 

resulted in a high concordance with rickettsial 

isolates from spiders P. phrygianus and M. mengei (Goodacre 

et al., 2006). However, 16S rRNA gene sequences are not 

available for spider rickettsiae in GenBank. It is interesting 

that sequences of the gltA gene of Rickettsia sp. isolated from 

water beetles are quite divergent from all previous valid 

Rickettsia strains from the spotted fever, typhus and bellii 

groups. Furthermore, gltA sequences of Rickettsia sp. show a 

high similarity to the related genus Bartonella, whose 

members are known as emerging human pathogens (Minnick 

& Anderson, 2006), but do not cluster with Bartonella 

sequences. Goodacre et al. (2006) presumed that spiderspecific 

Rickettsia form an isolated, terminal group that 

contains no symbionts of other arthropod hosts. They 

further assumed that frequent horizontal transmission of 

rickettsiae between spiders and their insect prey is unlikely 

because this would produce a heterogeneous assortment of 

non-spider-specific Rickettsia in spiders. In contrast, we were 

able to connect molecular data of the 16S rRNA gene 

sequences from basal rickettsiae strains and spider-specific 

Rickettsia by sequencing of 16S rRNA and gltA genes of the 

Deronectes symbiont. We assume that other Rickettsia strains 

of the ancestral group could exhibit comparable gltA 

sequences. Based on this assumption, Rickettsia from water 

beetles and spiders could belong to a new taxon, which might 

represent a near relative of the genus Rickettsia. Itcanbe 

speculated that sequences of the ompA, ompB genes and gene 

D will show appreciable deviations like gltA, if these genes are 

present in rickettsiae of the ancestral group in general. 

However, ultrastructural observations revealed typical 

morphological features and intracellular arrangements of 

rickettsiae, such as a thickened inner leaflet or electronlucent 

‘halos’ (Silverman, 1991). Additionally, some rickettsiae 

had a ‘filamentous’ appearance, similar to R. bellii 

from Amblyomma cooperi ticks (Labruna et al., 2004). The 

actual length of these bacteria cells could not be revealed by 

transmission electron microscopical sections. 

Furthermore, Rickettsia could be detected in all tissues of 

all investigated Rickettsia-positive Deronectes species. Presumably, 

symbionts have been transported by the hemolymph, 

comparable to observations in aphids (Chen et al., 

1996). This would support their appearance in the eyes and 

legs. However, there is no evidence of harboring of Rickettsia 

in specific sheath cells, secondary mycetocytes or mycetomelike 

structures in aphids or booklice (Sakurai et al., 2005; 

Perotti et al., 2006). Likewise, Rickettsia could not be 

detected in high densities close to midgut epithelia or 

malpighian tubules (Perotti et al., 2006). In contrast, the 

internal genitals and fatbody exhibited numerous symbionts. 

Rickettsiae depend on precursors of amino acids 

from their host. Equally, the de novo synthesis of purines as 

well as pyrimidine is not achieved by rickettsiae (Renesto 

et al., 2005). Hence, tissue of high metabolic activity, such as 

the fatbody, could be preferably infested with Rickettsia. The 

presence of bacteria within ovariols and eggs also indicates 

strong evidence for transovarial transmission, which must 

contribute to the maintenance of Rickettsia sp. infection in 

Deronectes populations (Whitworth et al., 2003). This is 

supported by the existence of Rickettsia sp. in the second and 

third larval stages of Deronectes, whereas the quantity of 

symbionts increased gradually in each stage. 

Thus, the amount of bacteria seems to be determined by 

several factors: (1) fidelity of vertical transmission, (2) 

frequency of horizontal transmission (Fukatsu et al., 2000), 

(3) fitness effect on the host and (4) selfish manipulation of 

the host reproduction (Tsuchida et al., 2002). Moreover, 

interactions between the endosymbionts and their hosts are 

complicated due to subpopulation structures and fluctuating 

environmental conditions. The resulting bottleneck 

during vertical transmission could be a reason for the 

fluctuated rate of rickettsial affection in D. aubei. More 

individuals of D. delarouzei and D. semirufus have to be 

investigated to verify the infection rates. Because of the 

predatory nature of Deronectes (Dettner et al., 1986), a 

horizontal transmission of Rickettsia sp. seems to be possible. 

Current research should demonstrate whether aquatic 

prey of Deronectes is also associated with Rickettsia sp. 

On the basis of the present results, there are no indications 

that Rickettsia sp. affection has an effect on the fitness 

of Deronectes. Neither reduced body weight and fecundity 

like in Rickettsia-infected host aphids (Sakurai et al., 2005) 

nor remarkable increases in host size as observed in leeches 

(Kikuchi & Fukatsu, 2005) could be observed. However, the 

research of fitness effects is quite difficult, because Deronectes 

cannot be bred under laboratory conditions. 

Parasitic living bacteria, such as Rickettsia, Spiroplasma, 

Cardinium and especially Wolbachia, have a tendency to 

manipulate host reproduction for their own benefit. Reproductive 

phenomena such as parthenogenesis, cytoplasmatic 

incompatibility, feminization and male killing have been 





documented (O’Neill et al., 1997). Male killing caused by 

Rickettsia was described until now in ladybird beetles A. 

bipunctata and A. decempunctata (Werren et al., 1994; von 

der Schulenburg et al., 2001) and buprestid beetle Brachys 

tessellates (Lawson et al., 2001). Probably, rickettsiae also 

influence oogenesis in bark beetle Coccotrypes dactyliperda 

(Zchori-Fein et al., 2006). Rickettsia-induced parthenogenesis 

was documented from eulophid parasitoid Neochrysocharis 

formosa (Hagimori et al., 2006). In contrast, all investigated 

species of Deronectes indicated a balanced sex ratio. 

In this context, further research is required to investigate 

in detail more about the influence of Rickettsia on Deronectes 

species, especially at the biochemical level. Furthermore, 

transmission and maintenance of Rickettsia in different 

populations of Deronectes species must be examined. Future 

studies will show, whether other Dytiscidae as well as their 

aquatic prey are also infected by Rickettsia sp. symbiont. 

Acknowledgements 

We thank M. Meyering-Vos, G. Rambold and D. Peršoh for 

providing equipment and assistance in cloning, sequencing 

and phylogenetic analysis. S. Geimer and R. Grotjahn are 

gratefully acknowledged for assistance in electron microscopy 

analysis, as well as S. Heidmann for the opportunity 

to use the confocal microscope, and for providing help. We 

also thank A. Kirpal for technical assistance. 

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