Use of the Verrucomicrobia-Specific Probe EUB338-III and Fluorescent In Situ Hybridization for Detection of "Candidatus Xiphinematobacter" Cells in Nematode Hosts

Fluorescent in situ hybridization with a 16S rRNA probe specificfor Verrucomicrobia was used to (i) confirm the division-levelidentity of and (ii) study the behavior of the obligate intracellularverrucomicrobium "Candidatus Xiphinematobacter" in its nematodehosts. Endosymbionts in the egg move to the pole where the gutprimordium arises; hence, they populate the intestinal epitheliaof juvenile worms. During the host's molt to adult female, theendosymbionts concentrate around the developing ovaries to occupythe ovarian wall. Some bacteria are enclosed in the ripeningoocytes for vertical transmission. Verrucomicrobia in malesstay outside the testes because the tiny spermatozoids are notsuitable for transmission of cytoplasmic bacteria.

INTRODUCTION

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Introduction

Obligate intracellular symbioses of bacteria in Invertebrataare numerous (3, 4, 7, 8, 9, 10, 13, 21, 23) and constituteone of the fastest-growing fields in microbiology. This papersheds more light on an endocytobiosis of a special kind of Verrucomicrobiain nematodes, "Candidatus Xiphinematobacter spp." (24). Detailson the taxonomic status "Candidatus" for incompletely describedtaxa can be found in the work of Murray and Schleifer (17) andMurray and Stackebrandt (18).

Xiphinema spp. (Dorylaimida, Longidoridae) are ectoparasiticnematodes found on economically important plants like vinesand other fruit crops. The general morphology of a female isdrawn in Fig. 1. Xiphinema nematodes possess an oral stylet(400 to 450 nm in diameter) with which they penetrate root hairs.Adults are 1 to 7 mm long and reproduce either sexually or byfacultative thelytokous (mother-to-daughter) parthenogenesis.There are three or four juvenile stages (J1 through J4). Althoughtwo ovarian stem cells are present at J1, the ovaries do notdevelop before the molt from J3 or J4 to adulthood. Slightlylater, yolk material synthesized by the adjacent gut epitheliumis transferred to the ovarian epithelium, which then consistsof nurse cells. These cells inject yolk and other maternal constituentsinto the growing oocytes as they pass by on their way down tothe oviduct. The three incompletely described "Candidatus Xiphinematobacterspecies" are specifically associated with the predominantlyparthenogenetic hosts Xiphinema brevicollum, Xiphinema americanum,and Xiphinema rivesi (24); more such symbioses probably existwithin the so-called "X. americanum group." The obligate intracellularbacteria belong to the division Verrucomicrobia (11, 15, 25),where they occupy a unique branch within subdivision 2 (accordingto reference 12).

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FIG. 1. General morphology (A) and reproductive system (B) of an entire X. americanum female. G₁, anterior branch; G₂, posterior branch; ooc., oocyte; ova., ovary; ovi., oviduct; s., symbiotic bacteria; sph., sphincter; ut., uterus.

It has been suggested that the incomplete description, basedmainly on sequence characteristics, of new intracellular, inculturable,or uncultured "Candidatus species" be attended with in situhybridization data as a supplementary indication of identity.rRNA-targeted oligonucleotide probe-based fluorescent in situhybridization (FISH) allows for such cultivation-independentidentification (2, 22). FISH has been used by Daims et al. (6)and Petroni et al. (19) to detect and identify Verrucomicrobia.

The aim of this work is twofold. First, in order to prove thatthe Xiphinema intracellular symbionts are indeed Verrucomicrobia,we provide FISH results obtained with a probe specific for Verrucomicrobia,EUB338-III (6). This probe, a specifically modified versionof the older universal probe EUB338 (1), targets positions 338to 355 (Escherichia coli numbering) in the 16S rRNA and is 100%complementary only to members of verrucomicrobial subdivisions1 to 4. Second, we examined how the endosymbiont life cycleis tuned to that of the host at several crucial stages. We alsoreport for the first time on the behavior of the microbes inthe very rare Xiphinema males.

Nematodes.

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Nematodes.

X. brevicollum and X. americanum samples were collected in SouthAfrica and stored at 4°C in their natural medium beforebeing extracted from the soil as described by Vandekerckhoveet al. (24).

Preparation for FISH.

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Preparation for fish.

Each preparation of nematodes was triplicated and comprisedabout 20 individuals covering most developmental stages (exceptmales, which are extremely rare). All of the following stepswere carried out in Eppendorf vessels with the simple replacementof fluids for each procedure. The specimens were surface sterilizedfor 1 min in 0.1% (wt/vol) benzalkonium chloride and rinsedtwice for 2 min in sterile water with a physiological makeup(0.85% [wt/vol] NaCl) appropriate for nematodes. They were fixedfor 10 min in a 1:1 blend of glacial acetic acid and ethanol,whereupon they were transferred into pure ethanol (twice for5 min). Next, they were put for 10 min into a 1:1 mixture ofmethanol and phosphate-buffered Tween 20 (PBT; 150 mM NaCl,10 mM Na₃PO₄, 0.1% [wt/vol] Tween 20 [pH 7.4]); they were thenput for 30 min in 1% (vol/vol) formaldehyde in PBT, whereuponthey were washed twice for 2 min each time in PBT.

FISH.

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Fish.

The 16S rRNA probes EUB338-III (5'-ACACCUACGGGUGGCAGC-3') andLax659 (5'-UGGUAGAGGGGGGUGGAA-3') were covalently linked attheir 5' ends to the sulfoindocyanine chromophore Cy5 (carboxymethylindocyanin-succinimidylester;Amersham Life Science, Inc.). Lax659 is complementary to a variable16S rRNA region of the chemoautotrophic, sulfur-oxidizing ectosymbioticbacteria that grow on a Laxus sp. (Nematoda, Desmodoridae) (20)and served as a control for nonspecific binding of probe RNAand its fluorescent moiety under both high- and low-stringencyconditions. Aliquotted stock solutions of 10 µM in TEbuffer (10 mM Tris-HCl, 1 mM EDTA [pH 7.4]) were made; 10 µlthereof was used for each 100 µl of final hybridizationmixture. The latter contained 20 mM Tris-HCl (pH 7.4), 0.02%(wt/vol) sodium dodecyl sulfate, 0.9 M NaCl, 5 mM EDTA, 60%(vol/vol) formamide, and a 1 µM concentration (±5ng · µl^-1) of the probe.

The hybridization conditions were largely adopted from the workof Manz et al. (16) and Daims et al. (6); high stringency wasapplied by keeping the closed vessels at 46°C for 3 h. Thiswas followed by two 30-min washes at 48°C in a hybridizationbuffer containing no formamide; instead, equally stringent conditionswere created by lowering the NaCl concentration to 0.008 M accordingto the formula of Lathe (14).

The images in Fig. 2A and 3 were taken under low-stringencyconditions, with hybridization at 42°C and 0% formamide;washing also occurred at 42°C. This gave a roughly threefold-more-intensesignal with better contrast on the confocal microscope.

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FIG. 2. FISH results after low-stringency hybridization with Lax659, a nonspecific probe (A), and high-stringency hybridization with EUB338-III, a verrucomicrobium-specific probe (B to F), to detect "Candidatus Xiphinematobacter brevicolli" (A, B, E, and F) and "Candidatus Xiphinematobacter americani" (C and D). (A) Newly molted female; (B) gravid female with endosymbionts at one egg pole; (C) newly molted female; (D) organism at a slightly later stage than that shown in panel C, with bacteria spreading downstream along the ovaries; (E) adult female showing endosymbionts and host cell nuclei after DAPI staining; (F) superimposed images at the same focal plane of the same female stained with DAPI and Cy5, with all endosymbionts being marked as Verrucomicrobia. ge, germinal zone of ovary; gr, growth zone of oocytes; i, intestine; m, body muscles; n, host cell nucleus; v, vulva. Single-headed arrows indicate directions of growth; ovarial apices are marked by asterisks.

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FIG. 3. Low-stringency FISH images of "Candidatus Xiphinematobacter americani" organisms. (A and B) Adult females, with an ovary (A) and intestine (B) in the focal plane; (C) adult male. n, nucleus of growing oocyte. Arrowheads in panels B and C point to serial pairs of endosymbionts.

Mounting, DAPI counterstaining, and microscopy.

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Mounting, dapi counterstaining,...

The specimens were pipetted out of the washing buffer onto microscopeslides. A droplet of a DAPI (4',6-diamidino-2-phenylindole)solution in distilled water (1 µg · ml^-1) was addedto stain the double-stranded DNA in any bacterium or host cellnucleus. The preparations were covered, sealed, and kept inthe dark at 4°C until they could be viewed. Images wererecorded with a Bio-Rad MRC-1024 confocal laser scanning systemequipped with a krypton-argon laser (excitation filter wavelength,647 nm; emission filter wavelength, 680 ± 32 nm) foruse with Cy5, an argon ion laser (excitation filter wavelength,357 ± 10 nm; emission filter wavelength, >460 nm)for use with DAPI, and a Nikon Eclipse TE300 microscope withapochromatic 60x or 40x oil immersion objectives (numericalaperture, 1.4 or 1.3, respectively). For image reconstruction,the software package Lasersharp (Bio-Rad) was used, as wellas Confocal Assistant 4.02 and Adobe Photoshop 4.0.

Low-stringency hybridizations with the nonspecific Lax659 probegave only a very weak background signal, with not a single bacterialcell being detected (Fig. 2A). Moreover, this background wascompletely absent from high-stringency-hybridization specimens,indicating that EUB338-III probe binding was highly specific.A comparison of corresponding images (Fig. 2E and F) also revealedthat all endosymbiotic bacteria (DAPI stained) were Verrucomicrobia(Cy5 stained). On these grounds, the intracellular organismsfirst described and classified as three "Candidatus Xiphinematobacterspecies" (24) can be validly said to belong to the divisionVerrucomicrobia.

Endosymbionts were observed to heavily colonize the ovary wallepithelia of mature females (Fig. 2F and 3A). They were alsoreadily apparent within the gut epithelium (Fig. 3B and C).It sometimes happens—though very rarely—that fewsymbionts are found, but not a single female has been foundwith none. These conclusions are based on observations of hundredsof females and the majority of the known males for each Xiphinemaspecies.

"Candidatus Xiphinematobacter" organisms seem to have an amazingperception of their microhabitat. In the mononuclear intrauterineegg, these organisms migrate to one of the poles (Fig. 2B),and in the embryonated egg, they move further to surround oneparticular host nucleus: that of the intestinal stem cell (5).Logically, in juveniles, they occur only in the gut epithelialcells. But when a J4 organism molts into an adult female, thebacteria need to rearrange themselves in order to access thevertical transmission pathway: they must somehow invade thegrowing oocytes.

This decisive reshuffling can readily be visualized in newlymolted and in young-adult females (exemplified by Fig. 2C and D).In newly molted organisms, the vast majority of the microbesare situated in the germinal zones of both ovaries, i.e., wherethe oogonia multiply. In young-adult females, the growing oocytesare provided with yolk from the nurse cells. This implies thatthe bacteria have been taken up by the ovarian wall epitheliumprior to the provision of the yolk material. Yolk is primarilysynthesized by gut cells and is secreted into adjacent ovarianwall (or nurse) cells soon after the transfer of the symbionts.Clearly, indirect symbiont transfer to ripening oocytes vianurse cells is the best warrant for vertical transmission. Nevertheless,not all bacteria migrate to the nurse cells: some may stay insidethe gut cells (invisible on most confocal images because theyare in a different focal plane), and a minority invade the oocytesdirectly (5).

Once inside the nurse cells, the bacteria enter a phase of intenseproliferation (see, e.g., the difference in the occupation ofspace in Fig. 2C and D or 3A), but they never move farther thanthe growth zone, and the fluorescence can be seen to drop abruptlyalong the trajectory towards the oviduct (Fig. 3A, to the right).A secondary multiplication occurs inside the egg.

Interestingly, the endosymbionts also populate the male gutepithelium but not the ripe testes (Fig. 3C). Especially inmale X. americanum organisms, they are less numerous and morecoccus-like; they are more condensed than in females and areseldom found in serial pairs (compare Fig. 3B with C), as describedby Vandekerckhove et al. (24). Such pairs are evidence of activedivision and consist of a mother cell from which a daughtercell is budding. The rareness of actively dividing cells inadult males is not surprising in light of the impossibilityof vertical transmission: sperm cells do not offer enough spaceto harbor cytoplasmic symbionts. Instead, the symbionts in adultmales appear somewhat latent, and they cannot be triggered tomake the move to the testes, which are salient because of thecomplete absence of intracellular organisms.

All these findings point in the same direction: "CandidatusXiphinematobacter species," having coevolved with their nematodehosts for about 150 million years with no evidence of horizontaltransmission or survival in the external environment (24), aretotally reactive to and dependent on their female hosts—inalmost every stage of their life cycles—for the ultimatepurpose of each living organism: to maintain itself.

ACKNOWLEDGMENTS

We thank A. J. Meyer (University of Stellenbosch, Stellenbosch,South Africa) for the X. americanum and X. brevicollum specimensused in this study. P. Van Oostveldt (Laboratory for Biochemistryand Molecular Cytology, Faculty of Agricultural and AppliedBiological Sciences, Ghent University) is acknowledged for allowingus to use the confocal laser scanning microscope.

During the time this work was carried out, T. Vandekerckhovewas a Research Assistant of the Fund for Scientific Research(F.W.O.), Flanders, Belgium. A. Coomans and M. Gillis are indebtedto the F.W.O. for research and personnel grants. K. Corneliswas supported by a grant from the Interuniversity Poles of AttractionProgram (grant P4/15).

FOOTNOTES

* Corresponding author. Mailing address: Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Phone: 32 9 2648730. Fax: 32 9 2645092. E-mail: tvdkerck{at}yahoo.se.

REFERENCES

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