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R E S E A R C H A R T I C L E

Characterizationand localizationofRickettsia sp. inwater beetles ofgenusDeronectes (Coleoptera:Dytiscidae)Stefan Martin Kuchler, Siegfried Kehl & Konrad Dettner

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

Correspondence: Stefan Martin Kuchler,

Department of Animal Ecology II, University of

Bayreuth, Universitatsstraße 30, 95440

Bayreuth, Germany. Tel.: 10049 0921 55

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

[email protected]

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 distribu-

tion, 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 impor-

tance, 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 & Jeyapra-

kash, 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 SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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. Dero-

nectes 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 endosym-

bionts. FISH was used for the investigation of rickettsial

distribution and vertical transmission rates. The morpholo-

gical 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 north-

eastern Bavaria, Germany. Deronectes aubei was collected

from Black Forest (Germany), Italy and French alps. Dero-

nectes 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 UNICRYLTM (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 DNeasys Tissue Kit

(QUIAGEN GmbH, Germany) following the protocol for

animal tissue. The eubacterial 16S rRNA gene was PCR

amplified using the primer set 07F (50-AGAGTTT

GATCMTGGCTCAG-30) and 1507R (50-TACCTTGTTAC

GACTTCAC-30) (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 Clonings Kit (Invitrogen, CA).

Suitable clones for sequencing were selected by restriction

fragment length polymorphism (RFLP). Inserts were di-

gested by restriction endonucleases HaeIII and TaqI. Plas-

mids 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)

FEMS Microbiol Ecol 68 (2009) 201–211c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

202 S.M. Kuchler et al.

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 (50-GGGCTTGCTCTA

AATTAGTTAGT-30) and Ri_1500R (50-ACGTTAGCTCACC

ACCTTCAGG-30). The citrate synthase gene (gltA) was de-

tected using the primers CS5_F (50-TCTTTATGGGGACC

AGCC-30) and CS4_R (50-TTTCCATTGTGCCATCCAG-30).

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 [50-(Cy5)-GCTGCCT

CCCGTAGGAGT-30] (Amann et al., 1995), EUB388 II [50-

(Cy3)-GCAGCCACCCGTAGGTGT-30], EUB338 III [50-(Cy3)-

GCTGCCACCCGTAGGTGT-30] (Daims et al., 1999) and

Rickettsia-specific probe Rick_B1 [50-(Cy3)-CCATCATCCC

CTACTACA-30] (Perotti et al., 2006). Additionally, a nonsense

probe complementary to EUB338, NON338 [50-(Cy3)-

ACTCCTACGGGAGGCAGC-30] (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 fluor-

escent 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 (VECTASHIELDs 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% glutar-

aldehyde 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). Ultra-

thin 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, Oberko-

chen, 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 ana-

lyses 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 repli-

cates 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- and D. delarouzei-

associated 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 Rick-

ettsia limoniae (AF322442, AF322443) from cranefly L. chorea

(Diptera, Limoniidae). The validity of the sequences was

confirmed by FISH performed with probe Rick_B1. Further-

more, 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.


203Rickettsia endosymbiont in water beetles

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 were found to be positive for

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, parthe-

nogenesis or other sex ratio distortion in numerically exam-

ined species of D. platynotus and D. aubei. Current studies

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 umbro-

sus and Hydroporus obscurus also exhibited Rickettsia infec-

tions.

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 predomi-

nantly (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 = 40mm.

(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 habour-

ing Rickettsia. Scale bar = 50mm. 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.



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. Rick-

ettsiae 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. A Rick-

ettsia-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 Ceroba-

sis 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 differ-

ent methods (MrBayes, data not shown), and was some-

times 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 frequen-

cies of Rickettsia infection were maintained constant in

different seasons. Individuals of D. delarouzei and D. semi-

rufus 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. 2. FISH double hybridization of the

Rickettsia symbiont on tissue (fatbody) cross-

section 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.



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

verifications of Rickettsia have been documented in Coleop-

tera 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 decempunc-

tata, 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 com-

prised 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), moth-

fly 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 iso-

lates 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 conser-

vatism (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).

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 2.74mm, before extending out of the

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



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).



Referred to rickettsiae of the ancestral group, the citrate

synthase-coding gltA gene sequences, also used for phyloge-

netic 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; Ngwami-

diba 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 sym-

bionts 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 (Min-

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

sequences. Goodacre et al. (2006) presumed that spider-

specific 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. It can be

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 electron-

lucent ‘halos’ (Silverman, 1991). Additionally, some rick-

ettsiae 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. Pre-

sumably, symbionts have been transported by the hemo-

lymph, 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 mycetome-

like 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 sym-

bionts. 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 fluctuat-

ing 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 possi-

ble. 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 indica-

tions 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 Dero-

nectes 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. Repro-

ductive 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. Persoh for

providing equipment and assistance in cloning, sequencing

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

gratefully acknowledged for assistance in electron micro-

scopy 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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