Localization and visualization of a coxiella-type symbiont within the lone star tick, Amblyomma americanum

Abstract

A Coxiella-type microbe occurs at 100% frequency in all Amblyomma americanum ticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified the Amblyomma-associated Coxiella microbe in several types of tissue and at various stages of the life cycle of A. americanum by 16S rRNA gene sequencing and diagnostic PCR. We visualized Amblyomma-associated Coxiella through the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence of Amblyomma-associated Coxiella within oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of the Amblyomma-associated Coxiella symbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens.

FIG. 1.

Detection of Amblyomma-associated Coxiella symbiont in whole adult tick sections. (A) PCR detection of Amblyomma-associated Coxiella endosymbionts in DNA from A. americanum adults, larvae, and eggs. PCR amplifications used AAC-1f and AAC-2r1 primers. Lanes: −, negative control using an equivalent DNA concentration of an extract from D. variabilis; +, positive control using a plasmid carrying the Amblyomma-associated Coxiella amplicon; 1, single male tick; 2, single female tick; 3, single larva; 4, pool of 10 eggs. Filled arrows indicate the 835-bp Amblyomma-associated Coxiella-specific amplicon and the 600-bp size standard in the ladder. Sodium borohydrate-buffered 1% agarose gel stained with ethidium bromide was used. See Materials and Methods for details. (B) FISH of 2-μm section of the whole adult A. americanum stained with the specific Coxiella AAC-1r-Cy3 probe. (C) Negative control hybridization with non-AAC-1r-Cy3 probe. (D) RNase treatment control hybridized with AAC-1r-Cy3. Salivary gland from the whole tick body section stained with AAC-1r-Cy3 probe (E) and with DAPI (F) is shown at higher magnification. Arrows in panels E and F indicate fluorescent foci. ms, muscles; sg, salivary glands; sd, salivary duct.

FIG. 2.

Double FISH analysis with Coxiella-specific and eubacterial probes. Confocal microscopy of FISH with double staining of sections from adult female A. americanum. (A) Confocal micrograph of AAC-1r-Cy3-specific staining. (B) Confocal micrograph of Eub338 fluorescein staining. (C) Overlay image of panels A and B. cu, cuticle; ms, muscles; sg, salivary glands.

FIG. 3.

Confocal FISH microscopy of A. americanum larvae. Sections hybridized with AAC-1r-Cy3 probe. (A) A 2-μm section of a whole larva. (B) Higher magnification of the boxed section shown in panel A. (C) Epidermal tissue. Arrows in panels B and C show fluorescent foci. cu, cuticle; ms, muscles; sg, salivary glands; mp, Malpighian tubules.

FIG. 4.

Examination of dissected A. americanum salivary tissues. Salivary glands dissected from an adult unfed female A. americanum tick are shown. (A) Dissected salivary glands from a single tick and PCR with specific Amblyomma-associated Coxiella primers (lanes 1 and 2) show Amblyomma-associated Coxiella microbes present in salivary glands from two different ticks (identical conditions and controls as described in the legend of Fig. 1). Filled arrows mark Amblyomma-associated Coxiella amplicon and size standard as described in the legend of Fig. 1. (B) Epifluorescence microscopy of FISH of salivary glands stained with specific AAC-1r-Cy5 probe. (C) Higher magnification of the boxed section shown in panel B. Arrows show fluorescent foci. (D) Gimenez-stained smear preparations of dissected salivary glands observed by bright-field microscopy. Arrows mark presumptive bacteria.

FIG. 5.

Examination of dissected A. americanum reproductive tissues. Reproductive tissues dissected from adult unfed female A. americanum ticks and identically prepared D. variabilis ticks. (A) Ovary dissected from an engorged A. americanum female. PCR of DNA extracted from ovarian tissue (lane 1), oocyte dissected from oviduct (lane 2), and eggs at stage 0 h (lane 3) 20 h (lane 4), 48 h (lane 5), and 72 h (lane 6). Filled arrows mark Amblyomma-associated Coxiella amplicon and size standard as described in the legend of Fig. 1, and white arrows correlate samples to regions of dissected ovarian tissue. (B) Epifluorescence FISH of an A. americanum oocyte at high magnification. (C) Negative control of D. variabilis oocytes stained with AAC-1r-Cy5 probe. (D) Gimenez-stained section from a dissected A. americanum ovary. (E) FISH microscopy of tissue section from a dissected ovary of A. americanum shows oocytes stained with AAC-1r-Cy5 probe. (F) Negative control of tissue section in which no probe was added. Arrows show fluorescent foci and presumptive bacteria. yp, yolk plates; pm, plasma membrane.

FIG. 6.

TEM analysis of dissected salivary glands and ovaries from A. americanum. (A) TEM image of ovarian tissue from an A. americanum engorged female with endosymbiotic bacteria. Arrowheads indicate bacterium-type structures. (B) Higher magnification of the boxed section shown in panel A. TEM showing detailed structure of endosymbionts in ovary of engorged adult female (C) and in salivary glands of unfed adult female (D). mt, mitochondria; v, cytoplasmic membrane-limited vacuole; om, outer cell membrane; im, inner cell membrane.