Correspondence: Matthias Kador, Animal Ecology II, University of Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany. Tel.: +09203 729911; fax: +09203 729913; e-mail: firstname.lastname@example.org
Bacterial endosymbionts from female Paederus rove beetles are hitherto uncultured, phylogenetically related to Pseudomonas sp., and produce the polyketide pederin, which exhibits strong cytotoxic effects and antitumoral activities. The location of such endosymbionts inside beetles and on beetles' eggs is hypothesized based on indirect evidence rather than elucidated. Thus, an endosymbiont-specific and a competitor oligonucleotide probe (Cy3-labelled PAE444 and unlabelled cPAE444, respectively) were designed and utilized for FISH with semi-thin sections of Paederus riparius eggs. Cy3-PAE444-positive cells were densely packed and covered the whole eggshell. Hundred percent of EUB338-Mix-positive total bacterial cells were PAE444 positive, indicating a biofilm dominated by Paederus endosymbionts. Analysis of different egg deposition stadiums by electron microscopy and pks (polyketide synthase gene, a structural gene associated with pederin biosynthesis)-PCR supported results obtained by FISH and revealed that the endosymbiont-containing layer is applied to the eggshell inside the efferent duct. These findings suggest that P. riparius endosymbionts are located inside unknown structures of the female genitalia, which allow for a well-regulated release of endosymbionts during oviposition. The novel oligonucleotide probes developed in this study will facilitate (1) the identification of symbiont-containing structures within genitalia of their beetle hosts and (2) directed cultivation approaches in the future.
Kellner (1999, 2001a, b, 2002a) demonstrated that a Pseudomonas-like endosymbiont is associated with the transfer of pederin production capabilities to the female progeny of Paederus beetles via endosymbiont-harbouring eggs. Analysis of metagenomic DNA from Paederus fuscipes beetles revealed the existence of a mixed modular polyketide synthase (pks)-gene cluster that is responsible for pederin biosynthesis (Piel, 2002). Specific PCR primers were designed from conserved regions of single cluster modules and utilized for the amplification of pks-gene fragments from endosymbionts in beetle or egg specimens (Piel, 2002). However, direct evidence for the localization of Pseudomonas-like endosymbionts on eggs is lacking, and it is still unresolved, where such endosymbionts are located within Paederus beetles.
FISH is an appropriate tool for the in situ localization of specific phylogenetically defined groups of bacteria (Amann et al., 2001; Amann & Fuchs, 2008). Thus, the objectives were to (1) design and evaluate a specific 16S rRNA gene-targeted oligonucleotide probe for Pseudomonas-like Paederus riparius endosymbiont detection; (2) localize endosymbionts within serial egg thin-sections by FISH; and (3) determine where within the host symbionts are transferred to eggs by surface comparison of different egg stadiums using electron microscopy and pks-targeted PCR.
Materials and methods
Type strains of Pseudomonas aeruginosa (DSM50071) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany) and grown overnight in brain–heart infusion medium (Fluka, Germany) at 37 °C. Bacterial cultures were prepared for FISH according to Hugenholtz et al. (2001).
Extraction of P. riparius endosymbionts
Female P. riparius rove beetles were killed by freezing before dissection. The abdomen was cut with a scalpel behind the elytra, put onto a glass slide and covered with sterile PCR-H2O. Tergites were removed with two sterile tweezers (Dumont INOX. 5; tip diameter: 0.025 × 0.005 mm) and the entire abdominal intestinal tract was extracted using a specific pair of micro-spring-scissors (Fine Science Tools; tip diameters: 0.15 mm with straight blades and 0.1 mm with 90° angulated blades). Whole internal female genitalia were removed, homogenized and preserved in 100% ethanol.
Semi-thin section procedure of P. riparius eggs
Paederus riparius eggs were fixed in ice cold 4% paraformaldehyde solution at 4 °C for at least 2 days, rinsed twice with phosphate-buffered saline [130 mM NaCl, 10 mM sodium phosphate buffer (NaPi), pH 7.4], and dehydrated in ethanol (30%, 50%, 85%, 95%, 100%, 30 min each). Eggs were subsequently embedded in UNICRYL resin (British BioCell International) as specified by the manufacturer. Serial semi-thin sections of P. riparius eggs were produced with a rotary microtome (Leica Jung RM2035). Section thickness of eggs was 5 μm. Every section was placed on top of a water drop on the surface of a Teflon-coated adhesive slide (Roth, Germany), and slides were dried at 55 °C on a heat table. Slides were stored in Petri dishes at room temperature until analysis.
Probe design and FISH
Oligonucleotide probes targeting 16S rRNA gene of Pseudomonas-like Paederus endosymbionts and closely related nontarget organisms were designed with the probe design-tool of the software package arb (the arb project: http://www.arb-home.de; Ludwig et al., 2004). Specificity was checked with probe match implemented in arb and blastn (http://www.ncbi.nlm.nih.gov/BLAST/). Probes were labelled at the 5′-end with the sulphoindocyanine dye Cy3 when appropriate.
Probes (Table 1) were purchased from MWG-Eurofins (Ebersberg, Germany). FISH was performed using previously described protocols (Hugenholtz et al., 2001; Pernthaler et al., 2001). In brief, samples were incubated in 9 μL hybridization buffer (0.9 M NaCl; 20 mM Tris-HCl, pH 8.0; 0.01% sodium dodecyl sulphate; 0–80% formamide) plus 1–2 μL of probes (50 ng μL−1) at 46 °C for at least 2 h and then placed in washing buffer (20 mM Tris-HCl, pH 8.0; X mM NaCl; Y mM EDTA; H2Odest at 50 mL; 0.01% sodium dodecyl sulphate; X and Y were adjusted according to the formamide concentrations utilized during hybridization) at 48 °C for 15 min. Hybridized cells were quantified relative to total cell counts [as determined by staining with 4′,6-diamidino-2-phenylindole-hydrochloride (DAPI)]. Mounting was performed in Vectashield (Vector Laboratories). Fluorescence microscopy was performed with an Olympus C-35AD-4 fluorescence microscope equipped with filter-sets Cy3-HQ (for Cy3) and 02 (for DAPI). Cell suspensions of the P. riparius endosymbionts (target organism) were obtained from homogenized internal genitalia of female P. riparius. Probe specificities were evaluated with such cell suspensions of the Pseudomonas-like endosymbiont and the closely related P. aeruginosa (nontarget organism). A minimum of 300 DAPI-positive cells of randomly chosen areas on microscopic slides were evaluated.
Table 1. Sequence and target sites of 16S rRNA gene-directed oligonucleotide probes used for the FISH experiments
Scanning electron microscopy (SEM) studies of P. riparius eggs were carried out with a Philips FEI XL 30 ESEM. Subsequent to dehydration in ethanol (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 30 min each), specimens were coated with gold (Edwards S150B).
DNA extraction and PCR
DNA was extracted using Qiagen DNA extraction kits (Qiagen, Hilden, Germany) according to the manufacturer's protocol. A 157-base pair fragment of pks-gene encoding a ketosynthase involved in pederin biosynthesis was amplified with primers KS1F (5′-TGGCATCGT GGGGAAAGGCTG-3′) and KS1R (5′-GGCGCAGGTGCTGACACGC-3′) (pks-PCR; Piel, 2002). Primers were purchased from MWG-Eurofins (Ebersberg, Germany). PCR was performed in a total volume of 50 μL containing 24.8 μL PCR-H2O, 10.0 μL 5 × Q-Solution, 5.0 μL 10 × PCR buffer, 5.0 μL ddNTP Mix (2 mM), 2.5 μL of each primer (10 pmol μL−1), 0.2 μL Taq (5 U) (Qiagen). Thermal cycling was at 96 °C for 5 min followed by 35 cycles of denaturation at 96 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 1 min.
Design of endosymbiont-specific probes
Several potential target sequences for 16S rRNA gene-directed oligonucleotide probes were identified on the 16S rRNA gene sequence of the Pseudomonas-like bacterial endosymbiont of P. riparius (accession number: AJ316018; Kellner, 2002a). On the basis of different probe parameters (e.g. length of probe, hybridization temperature, GC content, Escherichia. coli position, etc.), the probe with target site 444–461 (E. coli numbering according to Brosius et al., 1981) was selected (Table 1) and checked with the probe match-tool (the arb project: http://www.arb-home.de) for specificity. The probe (PAE444) was complementary to the target sequence of the Paederus endosymbiont and displayed high probe accessibility within its target region according to a 16S rRNA gene secondary structure model of E. coli (Fuchs et al., 1998; Behrens et al., 2003). PAE444 exhibited only two mismatches to the closest related nontarget sequence (P. aeruginosa). Thus, a competitor (cPAE444) complementary to the P. aeruginosa sequence was designed in order to achieve full mismatch discrimination (Table 1; Manz et al., 1992).
Experimental probe evaluation
PAE444 coverage alone was experimentally analysed by whole cell hybridization with cell suspensions of endosymbionts (extracted from P. riparius tissue, see Materials and methods) and pure cultures of P. aeruginosa. Probe dissociation curves were recorded to determine stringent hybridization and washing conditions. The nonhybridizing probe NON338 (Manz et al., 1992) and a 1 : 1 : 1 mixture of the bacterial domain level probes EUB338, EUB338-II and EUB338-III (Amann et al., 1990; Daims et al., 1999) were included (Table 1). Hybridization of P. riparius endosymbiont cells obtained from homogenized genitalia with Cy3-PAE444 resulted in intense fluorescent labelling of all cells evaluated over the whole range of formamide concentrations from 0% to 80% (Fig. 1a). However, PAE444 hybridized nonspecifically to nontarget sequences of P. aeruginosa cells from 0% to 60% formamide (Fig. 1b). Further increase of formamide concentration (stringency) resulted in a significant loss of cells' signal intensity. However, even the highest formamide concentration of 80% was not sufficient to cause dissociation of 50% of PAE444 from nontarget cells of P. aeruginosa. Hybridization of Cy3-cPAE444 to P. riparius endosymbiont cells (Fig. 1a) resulted in equal fluorescent labelling as shown for Cy3-PAE444 in case of P. aeruginosa cells (Fig. 1b). Hybridization of a 1 : 1-mixture of Cy3-cPAE444 and unlabelled PAE444 to endosymbiont 16S rRNA gene was observed for the lowest applied formamide concentrations of 0% and 5% only. With concentrations >10%, the weak fluorescence-signal completely disappeared, indicating specific discrimination of P. aeruginosa cells vs. endosymbiont cells. Hybridization of P. aeruginosa cells with Cy3-cPAE444 resulted in 100% fluorescence intensity over the whole range of formamide concentrations from 0% to 80%. Thus, the unlabelled oligonucleotide cPAE444 complementary to P. aeruginosa 16S rRNA gene was included as a competitor during hybridization to prevent the nonspecific hybridization of PAE444 with the nontarget sequence of P. aeruginosa. Hybridization of 1 : 1-mixed Cy3-PAE444 and unlabelled competitor probe cPAE444 to endosymbiont 16S rRNA gene resulted in intense fluorescent labelling of the cells hybridized with formamide concentrations from 0% to 80%, and nonspecific hybridization of Cy3-PAE444 to P. aeruginosa cells was not detectable at formamide concentrations >20%. Thus, formamide concentration in the hybridization buffer was adjusted to 30% in all following hybridizations, and the concentrations of NaCl (X) and EDTA (Y) in the washing buffer were 112 and 5 mM, respectively. The data indicate that the hybridization protocol allows for a specific detection of Pseudomonas-like Paederus endosymbionts.
Detection of endosymbionts on semi-thin sections of P. riparius eggs
The Pseudomonas-like Paederus endosymbionts were exclusively detected on a special layer entirely coating the egg shell in seven analysed section series of P. riparius eggs (Fig. 2a–d). Hundred percent of DAPI and Cy3-EUB-Mix-positive cells hybridized with Cy3-PAE444, indicating a ‘pure culture’ of endosymbionts (Fig. 2a–d). The interior of the investigated thin-sectioned eggs was always devoid of any bacteria as indicated by hybridization with Cy3-EUB-Mix (data not shown).
Analysis of P. riparius eggs with electron microscopy and pks-PCR
Surface investigation of P. riparius eggs by SEM identified a granular layer, indicating microbial cells completely covering the eggshell (Fig. 3a and b). Such findings underscore the data obtained by FISH that the eggshell is completely covered by the Pseudomonas-like P. riparius endosymbiont (Fig. 2a–d). The granular layer was found on all electron-microscopically investigated eggs (n=20), which had been oviposited by P. riparius. In order to check whether this granular layer is already applied to the eggshell in the ovaries during oogenesis or somewhere else in the internal female genitalia during egg passage, several eggs (n=9) were prepared out of the common oviduct and analysed by electron microscopy. These eggs always exhibited a strongly folded, smooth surface, indicating that a granular layer was absent (Fig. 3c and d).
In order to approve these findings molecularly, two eggs from the common oviduct (cf. Fig. 3e) and five already oviposited eggs from different female beetles (n=10) were analysed by pks PCR. pks gene fragments indicating Paederus endosymbionts were amplified from all oviposited eggs, but not from eggs originating from the common oviduct, indicating that the endosymbionts are applied to the egg shell inside the efferent duct (cf. Fig. 3e).
Many endosymbiotic bacteria are still unable to grow in vitro, potentially because of specific nutrients present exclusively within the source/host habitat and are not available in conventional culture media (Lewis, 2007; Davey, 2008). FISH allows the visualization of prokaryotic cells in their natural environment regardless of their culturability. The FISH method targets rRNA, which is essential to basic cellular metabolism and is thought to degrade soon after cell death (Nocker & Camper, 2009). Thus, this method is a very powerful tool for the detection and localization of unknown bacterial communities from a range of different habitats (Amann et al., 1995, 2001; Berchtold et al., 1999; Darby et al., 2005; Davidson & Stahl, 2006; Ferrari et al., 2006, 2008; Vartoukian et al., 2009), such as endosymbiotic bacteria that reside in invertebrates like insects within specific cells or symbiotic organs. Consequently FISH may facilitate isolation and potential cultivation of newly detected or previously uncultivable bacteria, as could be demonstrated recently (Vartoukian et al., 2010).
A FISH approach using novel oligonucleotide probes was developed and demonstrated that essentially a ‘pure culture’ of the Pseudomonas-like pederin-producing endosymbionts of P. riparius covers the whole surface of P. riparius eggs, which extends previous reports suggesting that the endosymbiont is transmitted to the offspring via the egg (Kellner, 2001a, b, 2002a, b, 2003; Piel, 2002, 2004, 2005). Most bacteria appear to form biofilms, including P. aeruginosa, and such a multicellular mode of growth likely predominates in nature as a protective mechanism against hostile environmental conditions (Costerton et al., 1995; Costerton & Stewart, 2000). Consequently, this ability could also be existent for the Paederus endosymbiont because of its close relationship to P. aeruginosa (Kellner, 2002a; Piel, 2002; Piel et al., 2004). Indeed, SEM and FISH indicated a biofilm-like structure consisting of the Pseudomonas-like endosymbiont (Figs 2 and 3). Such a biofilm formation of Paederus endosymbionts might defend the egg and the symbiotic bacteria from adverse environmental conditions after oviposition like foreign colonization with moulds or pathogenic soil bacteria (O'Tool & Kolter, 1998). A biofilm might protect the oviposited eggs against the impact of rainwater and prevent that symbiotic cells are flushed away. However, the endosymbiont containing matrix might likewise represent a gelatinous secretion cyst that is released together with a definite amount of symbiotic bacteria from specific, not yet discovered, transmission organs and smeared on the egg surface during egg deposition (Meixner, 1932; Howard & Kistner, 1978; De Marzo, 1986; Hanley, 1996).
As smearing of the egg surface with endosymbiotic bacteria has been known as a transmission route for extracellular symbionts for a long time (Steinhaus, 1946), the explanation given above appears to be the most likely for the observed biofilm-like structure of an essentially ‘pure culture’ of endosymbionts on P. riparius eggs.
Generally, symbiont transmission from female host insects to their eggs is easily conceived, if endosymbionts are located within the insects' intestinal epithelium or intestinal lumen (Dettner, 2003). Symbionts mixed with faeces are deposited on the eggshell and can be orally ingested by the ‘sterile’ larvae (Dettner, 2003). However, more complicated methods of symbiont transmission occur for P. riparius endosymbionts that are located within the abdominal cavity outside the gut. The endosymbiotic bacteria of P. riparius are most probably located in not yet identified compartments within the female internal genitalia and are applied to the eggshell inside the efferent duct, into which the bacteria are released (Fig. 3). In case of certain stink bugs (Acanthosomatidae: Hemiptera), symbionts are located in a pair of transmission organs. Necessarily, only a well-dosed amount of these bacteria is squeezed into the genital opening by the passing eggs and thus gets onto the egg shell (Buchner, 1965). Such ‘smear-organs’ generally become essential where symbiotic bacteria are not harboured within the intestinal lumen. Mostly, special reservoirs that are lined with chitin are closely connected with the ovipositor, as in the case of certain weevils (Curculionidae: Coleoptera). These beetles harbour their endosymbionts within evaginations at the beginning of the midgut and exhibit two clubbed symbiont-containing organs with a narrow passageway leading to the egg depositor. These symbiont organs are endowed with well-developed longitudinal muscles that enable a dosed release of symbionts to the egg surface (Buchner, 1960, 1969).
Stereomicroscopic observation of several eggs with currently hatching P. riparius larvae during this study revealed that the eggshell bursts during eclosion and thus inevitably comes into contact with the sterile embryonal fluid (data not shown). According to Buchner (1960), many hatching larvae and nymphs (e.g. weevils, stink bugs) own biting mouthparts with which they feed parts of the eggshell during its burst and are thus infected with the bacteria. Larval infection of P. riparius with endosymbionts most probably takes place in the same manner.
However, where exactly the endosymbiotic bacteria of P. riparius are located inside the beetles was not resolved in this work and requires further FISH investigations with the novel oligonucleotide probes developed in this study.
We thank J. Piel (Kekulé Institute, University of Bonn) for the supply with the ketosynthase-specific primer pair KS1F/KS1R, H. Rödel (Institute of Animal Physiology, University of Bayreuth) for the kind introduction in sigmaplot 9.0, R. Grotjahn (Institute of Electron Microscopy, University of Bayreuth) for numerous electron-microscopical exposures, E. Helldörfer (Institute of Animal Ecology II, University of Bayreuth) for the creation of scientific figures of Paederus beetles' anatomy, W. Nowak and I. Nowak for the provision of several P. riparius specimens and Harold L. Drake for provision of a LINUX-based network for arb. Financial support by the Deutsche Forschungsgemeinschaft (DFG) is gratefully acknowledged (GRAKO 678).