© 2002 by The Society for Integrative and Comparative Biology
Distribution of Wolbachia Within Drosophila Reproductive Tissue: Implications for the Expression of Cytoplasmic Incompatibility1
1 The University of Chicago, Department of Organismal Biology & Anatomy, 1027 East 57th Street, Chicago, Illinois 60637
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A PCR based quantitative assay was used to determine Wolbachia infection levels in three different Drosophila strains. In addition, confocal microscopy was used to confirm and calibrate these results. Wolbachia infection levels ranged from 2,600 to 18,500 per egg. Single ovaries and testes from each of the three strains were also assayed using the calibrated quantitative PCR assay. A general correlation was found between bacterial levels in eggs and those found in ovaries and testis. These infection levels were consistent with the expression of cytoplasmic incompatibility (CI). In two strains of D. simulans, although the overall bacterial numbers were not significantly different, they exhibited different levels of CI. A direct correlation between the number of infected developing sperm cysts in these strains and CI levels was observed. This calibrated assay should provide a useful baseline for future comparative work, particularly between laboratories.
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INTRODUCTION |
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Wolbachia are rickettsial-like bacteria that infect a wide variety of mainly arthropod hosts. Maternally inherited, Wolbachia have evolved a number of different strategies for manipulating host reproduction, which results in increased transmission in the population. These manipulations include feminization, male killing, parthenogenesis, and cytoplasmic incompatibility (CI), the inability of infected males to successfully fertilize eggs from uninfected females (for recent reviews see Bourtzis and O'Neill, 1998
; McGraw and O'Neill, 1999; Stouthamer et al., 1999). Recently, Wolbachia have also been found in non-arthropod hosts, most notably filarial parasitic nematodes (Sironi et al., 1995).
Repeated studies regarding host-Wolbachia interactions have highlighted the importance of bacterial numbers. Both Wolbachia levels and host factors seem to be at least in part responsible for the levels of effects on the host. In Drosophila, not only can different host strains infected with the same Wolbachia type, have different levels of infection, but different Wolbachia types can have different densities in the same host strain. Wolbachia numbers also seem to be correlated to levels of CI (Boyle et al., 1993; Bourtzis et al., 1996; Poinsot et al., 1998). Similarly, CI has also been shown to be correlated to bacteria numbers in Nasonia vitripennis (Breeuwer and Werren, 1993) and Aedes albopictus (Sinkins et al., 1995). In Muscidifurax uniraptor, when Wolbachia densities were experimentally manipulated via differential doses of antibiotics, the ability of Wolbachia to induce parthenogenesis was correlated with bacterial load (Zchori-Fein et al., 2000). The effects of Wolbachia numbers on general life history traits have been less clear. There have been inconsistent reports of costs and benefits of Wolbachia on fitness components. Understanding host factors controlling Wolbachia density and the effects of Wolbachia numbers on host fitness may give clues to the molecular and cellular mechanisms of CI. Further, deeper understanding of factors influencing bacterial density will most likely lead to deeper understanding of the population dynamics of Wolbachia infections. However, surprisingly little is known about the biology of Wolbachia, and essentially nothing is known about the factors affecting Wolbachia growth and maintenance in its host. One important tool needed for further progress is a reliable and convenient method to quantify bacteria in their hosts.
Wolbachia have been quantified using several different methods. Light microscopy based estimations, dot blots and PCR have been used to estimate relative infection status (Boyle et al., 1993; Stouthamer and Werren, 1993; Bourtzis et al., 1996; Sinkins et al., 1995). Each of these methods has drawbacks that have prevented them from being adopted by other researchers. Relative measures, having never been calibrated to actual infection levels, are of little use when comparing the work of different laboratories. Confocal microscopy techniques have been developed to accurately measure Wolbachia densities (Boyle et al., 1993; Kose and Karr, 1995, Zchori-Fein et al., 2000), but these techniques are time consuming and require resources and facilities not available to all Wolbachia researchers. With the recent focus on the importance of bacterial densities in Wolbachia infections, quantitative-PCR is currently being used to study Wolbachia in several laboratories. A simple, standard, calibrated Wolbachia quantification assay would be a useful tool to many in the Wolbachia research community.
Since its inception, PCR has spawned numerous applications, including the quantification of nucleic acids. Due to the sometimes-mercurial nature of PCR, strict internal controls are required when using PCR based quantification. The most commonly used form of quantitative PCR employs an internal competitor, usually a modified version of the target sequence, which contains the same primer sites. An unknown quantity of the target is coamplified with a known quantity of the competitor. The resulting products can be distinguished either electrophoretically or with the use of fluorogenic probes (for review see Zimmermann and Mannhalter, 1996; Orlando et al., 1998). Quantitative PCR assays have been successfully developed to determine infection status of viral (Liu et al., 1999), bacterial (Afghani et al., 1997) and filarial (Fischer, 1999) human pathogens. In this study, we describe a calibrated Quantitative-Competitive PCR (QC-PCR) based assay for use in determining Wolbachia infection levels.
Using these assays we have examined bacterial levels in three strains of Drosophila. Our results indicate the efficacy of the QC-PCR approach and led to new insights into the relationship between bacterial levels and CI expression.
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Drosophila strains
Two Drosophila simulans strains with different levels of Wolbachia infection were used: one containing high (DSR) infection levels (O'Neill and Karr, 1990
; Boyle et al., 1993), and one containing intermediate (C167) levels of infection (T. L. Karr, unpublished). A D. melanogaster strain, with a low level of infection (T. L. Karr, unpublished), yw (+) (yellow body and white eyes), used extensively by Drosophila researchers was also included. Flies were grown under standard laboratory conditions at 25°C on cornmeal/molasses media supplemented with yeast at a standard low larval density (50 larva/vial).
Construction of internal competitor
The assay described here is a modified and expanded version of that described by Sinkins et al. (1995). The internal competitor used in the QC-PCR assay was constructed by the method similar to that described in Zarlenga et al. (1995). An 849 bp fragment of the Wolbachia 16S rRNA gene was amplified using the Wolbachia specific primers 99F (5'-TTGTAGCCTGCTATGGTATAACT-3') and 994R (5'-GAATAGGTATGATTTTCATGT-3') from DSR genomic DNA. The product was then ligated into the pGEM®-T Easy Vector, propagated and purified. A deletion within the cloned portion of the 16S gene was created using two internal primers with 5' overhanging segments containing a HindIII restriction site (5'-TCTTTCGAATAATAGCAATAAATTTTTCCCCCG-3') and (5'-TCTTTCGAAAGGAGACTCCTACGGGAGGCAG-3'). These primers were used to amplify the entire plasmid and all but a 100 bp fragment of the 16S region. The amplification product was then digested with HindIII and the ends were ligated together and used in transformation. The control plasmid was purified using the Wizard® Plus miniprep DNA purification system following manufacturer's protocol and quantified using a spectrophotometer.
QC-PCR assay
Single eggs, ovaries and testis were placed in 50 µl STE (400 mM NaCl/10 mM Tris Cl, pH 8.0/1 mM EDTA, pH 8.0) with 2 µl Proteinase K (13.3 mg/ml), homogenized with an, acid-washed, sterile polypropylene Kontes pestle, and incubated at 37°C for 60 min, followed by 5 min at 95°C and then centrifuged briefly. For each egg, eight 20 µl PCR reactions were set up under standard conditions using the Wolbachia specific 16S rRNA gene primers (994F and 99R) with 2.5 mM MgCl2. To each reaction, 2 µl of egg extract was added as well 1 µl of serially diluted control plasmid. Each successive tube received one half the control plasmid concentration as the previous. Following 2 min denaturation at 95°C, forty cycles of amplification were performed with the following program: denaturation at 92°C for 30 sec, annealing at 50°C for 30 sec and extension for 30 sec at 72°C, followed by extended extension at 72°C for 5 min. The entire reaction was then run on a 1.5% agarose gel in 1x TAE, stained with ethidium bromide, and scanned for image analysis of band intensity.
Analysis
Gel images were captured using an 8-bit Hamamatsu CCD camera and NIH Image 1.62. For clarity, images were inverted thus bands appear dark on a light background. The peak UV fluorescence intensity of each band was measured along a line transecting the center of the band. On 1% agarose gels with 1x TAE, only the two bands appeared. On 1.5% gels run for an adequate amount of time however, a heteroduplex molecule was observed in each QC-PCR assay. Heteroduplex formation and its consequences has been the subject of much discussion (Henley et al., 1996). An assay can be compromised if a heteroduplex is unknowingly segregating with one of the homoduplex PCR products. It is possible to reduce or eliminate the heteroduplex molecule by altering reaction conditions or by using denaturing gels (Chen et al., 1999), but the measures required often reduce the sensitivity of the PCR assay and/or increase the time and expense involved in doing the assay. Conversely, the heteroduplex molecule can be incorporated into the analysis and increase the precision of the assay. The latter strategy was taken and the inevitable heteroduplex band was similarly measured and used in the analysis as described by Serth et al. (1998) and Hoff et al. (1999). To determine the molar contents of the three bands, a correction factor of 1.134 (1.134 = length of 16S (849 bp)/length of competitor (749 bp)) was applied to both the competitor and the heteroduplex band. It was assumed that a heteroduplex molecule fluoresces at an equal intensity to the smaller, homoduplex molecule owing to their equal length of double stranded DNA to which EtBr binds. Therefore the ratio of 16S/16Scompetitor was calculated as:
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The point at which the 16S/16S competitor ratio was equal to one was calculated based on the slope between the two adjacent points. This was the point at which the initial 16S target sequence titer is equal to the control plasmid titer. In general, two crucial parameters are essential for the assay to yield reliable estimates: (1) the range of competitor needed to bracket the equivalency point, and (2) band intensities of the gel must be within the dynamic range of the camera used (see Fig. 1A for an example). The ranges of competitor needed to yield an equivalency point were easily determined by trial and error. Band intensities on the gel were also adjusted to the dynamic range of an 8-bit CCD camera empirically (Fig. 1A) by determining the amount of PCR reaction products loaded per lane that gave intensity values between 0 and 255. The competitor DNA and a sample of the Wolbachia DNA used in this study are available to researchers to serve as an internal standard for their experiments.
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Confocal imaging
Eggs and testes
In order to calibrate the PCR assay, confocal images of eggs from the three different strains were examined. Eggs were fixed following a procedure similar to that described in Kose and Karr (1995)
. Eggs were dechorionted in 50% bleach and fixed in 5 ml heptane, 5 ml methanol and 200 µl formaldehyde. Fixed eggs were incrementally rehydrated with TBST (50 mM TRIS, 150 mM NaCl, 0.1% Tween, 0.05% NaN3 pH 7.5) and RNased for 1 hr at 37°C and washed in TBST for at least 2 days at room temperature. Testes from 3 day old virgin males were harvested in TBST and fixed in 3.7% formaldehyde in TBST for 15 min, washed in TBST, RNased for 1 hr at 37°C and washed in TBST for at least 2 days at room temperature. Tissues were then stained with the SYTOX® Green nucleic acid stain (Molecular Probes). Wolbachia were then visualized using on a Zeiss LSM 510 confocal microscope with a Kr/Argon laser (488 nm) with 63x lens and a pinhole of 212 µm. Only embryos at or before nuclear cycle 4 were used for quantitation. At this stage, Wolbachia are evenly distributed throughout the cortex of the egg (Boyle et al., 1993) and are not undergoing growth (Kose and Karr, 1995). A Z-series comprised of 47 0.9 µm thick overlapping sections were taken in a 50 x 50 x 20 µm area starting at the surface at the center of the egg. Optical sections were projected onto a single two-dimensional image and Wolbachia numbers were tabulated.
Images of whole eggs were also captured using a 25x lens from which egg length and width were measured. Total egg volume as well as 20 µm thick egg cortex volume was calculated assuming nested prolate spheroid geometry according to the following:
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where r1 is the length of the major radius and r2 the minor.
Percent infected cysts were determined by scoring as either infected or uninfected, the 10 most proximal mature cysts per testis from DSR and C167.
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RESULTS |
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Eggs and ovaries
A comparison of Wolbachia densities estimated from confocal imaging analysis reveals a clear difference between three Drosophila strains (Fig. 2). Visual inspection of Wolbachia show lower numbers in yw(+) eggs with increasing numbers in C167 and DSR. This was confirmed by direct counts using confocal microscopy. Measured density using the confocal assay of C167 and DSR was 3.14 and 7.02 times respectively greater than yw(+). Comparable ratios were observed using the quantitative PCR assay (Fig. 3). Based on the quantified Wolbachia densities using both PCR and confocal microscopy, a standard curve was created and used to calibrate the PCR assay. (Fig. 4).
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Wolbachia infection in ovaries estimated using the calibrated QC-PCR assay follows the same trend as that found in single eggs, with ovaries from the DSR strain having the highest bacterial numbers followed by C167 and yw(+) (Fig. 5). Based on QC-PCR measurements of single ovaries, yw(+), C167 and DSR had 16.7, 7.9 and 7.7 fold, respectively, greater numbers of Wolbachia than in a single egg from the same strain.
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Testes
Wolbachia levels in testes estimated using the calibrated QC-PCR assay follows the same trend as that found in eggs and ovaries. Testes from the DSR strain have the highest bacterial numbers followed by C167 and yw(+) (Fig. 6). Based on measurements of single testes, yw(+), C167 and DSR had 4.9, 5.8 and 3.6 fold, respectively, greater numbers of Wolbachia than found in an egg from the same strain.
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Confocal images of Wolbachia in testes did not reveal the expected lower density suggested by the PCR assay results (Fig. 7). Comparing the two D. simulans strains, DSR and C167, the number of bacteria in each infected cyst was not obviously different (compare 7D to 7E). Instead, the difference in overall bacteria load was due to the number of infected cysts (Table 1). Interestingly, the percent infected cysts correlated closely to the percent CI elicited by each of these strains (Table 1).
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Interestingly, the D. melanogaster strain, yw(+) exhibited entirely different Wolbachia distribution within the testis (Fig. 7A). Bacteria in these testes were rarely found in the cysts (estimated to be <10%, data not shown), and the rare infected cysts in this strain were present at much lower levels than seen in DSR and C167. Most of the Wolbachia observed appeared to be associated with somatic sheath cells of the testes (Fig. 7A).
A summary of our data is shown in Figure 8, which diagrammatically illustrates the quantitative differences between cells and tissues of the three strains used in this study. Variation in the level of infection occurs both between strains and within tissue types of the same strain. For example, although bacterial levels in the testes of C167 and DSR are approximate equals, the ovaries of the DSR strain contained significantly higher levels.
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DISCUSSION |
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Repeated QC-PCR sampling of Wolbachia infected eggs from strains with different bacterial densities indicated that the assay is accurate and reproducible. Comparing estimated total Wolbachia/egg using QC-PCR and confocal microscopy revealed a close correlation suggesting both were accurate indicators of bacterial levels. Due to its ease of use and accessibility, QC-PCR should be most useful in the quantitation of Wolbachia in tissues and organs, such as ovaries and testes, not easily observed in the microscope.
QC-PCR is a straightforward technique for quantifying Wolbachia infections. The reagents and equipment required are relatively inexpensive and available to most researchers. The techniques employed are simple, standard and easily adaptable to different tissues or host species. The use of a system that is calibrated to actual Wolbachia counts, has the advantage that results can be compared between studies and researchers. This should be particularly advantageous for field studies that must rely on PCR-based assays for obtaining results of infection status. In this regard we are working out the conditions for the preservation of field specimens, subsequent DNA extractions and PCR conditions necessary for reproducible results.
The Wolbachia densities recorded here were comparable to those reported in (Hadfield and Axton, 1999). The regions measured by confocal microscopy ranged from 0.97 ± 0.10 x 106 Wolbachia per mm3 in the yw(+) strain, 2.91 ± 0.26 x 106 Wolbachia per mm3 in C167 and 5.40 ± 0.42 x 106 Wolbachia per mm3 in DSR. Hadfield and Axton (1999) report 3.04 ± 1.04 x 106 Wolbachia per mm3 in the D. melanogaster Canton S strain. Conversely, our bacterial loads are much lower than that reported by Poinsot et al. (1996). They estimate that DSR eggs have about 5 times as many Wolbachia as our measures. This may truly reflect strains with different bacterial densities or it may result from inherit difficulties in calibrating a dot blot assay.
Previous studies of Wolbachia levels have often found relationships between bacterial load and cytoplasmic incompatibility. In all instances however, bacterial numbers were either quantified in embryos (Breeuwer and Werren, 1993; Boyle et al., 1993; Poinsot et al., 1998; Zchori-Fein et al., 2000) or whole individuals (Sinkins et al., 1995; Bourtzis et al., 1996; Poinsot et al., 1998). No studies have previously examined bacterial numbers in either ovaries or testes. This was no doubt due to the general difficulty encountered in obtaining accurate images of bacteria in the testes and other thicker tissues and organs. These previous studies implicitly assume that there is a direct correlation between the bacteria load in an embryo or adult and the number of Wolbachia in an adult testis. A combined approach using confocal microscopy and PCR based assay however, such tests are feasible, and establish a relationship between the number of Wolbachia in a single egg and that found in ovaries and testes.
However, the correlation does not seem direct and care should be taken when using bacterial load in one tissue or cell type to estimate bacterial load in another. Direct measurement and PCR estimates of total bacterial numbers was especially illuminating in the comparison between DSR and C167 D. simulans strains (Figs. 6 and 7). The combined results using both assays clearly showed that, although overall DSR harbored more bacteria than C167, individual cysts within C167 contained similar numbers of Wolbachia (Fig. 7D and E). These results suggest the intriguing possibility that variation in CI levels are the result of the number of infected cysts, not the total number of Wolbachia in the testis of these two strains and further implies two types of sperm, "imprinted" (Wolbachia present) and "non-imprinted" (Wolbachia absent).
It is unclear what host factors are responsible for the lower number of infected cysts in the C167 strain. Perhaps a lower Wolbachia density in the egg decreases the probability of incorporation into pole cells and subsequent infection. Another possibility is host factors that influence intercellular interactions between host and symbiont, thus lowering Wolbachia number in spermatocysts in the C167 strain.
Our results also are consistent with previous observations in other studies. Bressac and Rousset (1993) observed an ‘all-or-none’ phenomenon concerning cyst infection. Infected cysts of old males seemed to have comparable levels of bacteria as infected cysts of young males, although the number of infected cysts decreased with age. Poinsot et al. (1998) also observed a greater number of infected cysts in a high-CI transinfected line as compared with the low-CI donor strain. Our results provide a quantitative underpinning to these studies and further define the parameters of the dynamics of Wolbachia infection in Drosophila.
Our microscopic analysis of Wolbachia in the testes of the yw(+) D. melanogaster strain suggested that the majority of bacteria are localized to non-cyst cells. These testes lack the localization of bacteria in the cysts as has been described previously in strains of D. simulans (Binnington and Hoffmann, 1989; Bressac and Rousset, 1993; Snook et al., 2000). Further examination of numerous infected strains revealed that Wolbachia are also present in non-cyst cells of a variety of other infected strains, including DSR and an infected strain of Canton S (data not shown). However, Wolbachia were rarely if ever observed in cysts in the yw(+) strain. It is unclear what costs, if any, these extra-spermatocyst Wolbachia have on the host or if they contribute to cytoplasmic incompatability. However, it further suggests that host factors play an important role in the localization and growth of bacteria.
There are three general mechanisms to explain the absence of Wolbachia in yw(+) cysts: either they were not present in sperm progenitor cells in the mature testis, were actively excluded from cysts in subsequent stages of spermatogenesis, or the intra-cyst environment does not support bacterial viability. Because Wolbachia exist in both germ line and somatic cells in Drosophila adults (Dobson et al., 1999; our unpublished results), Wolbachia can originate from two cell types: 1. those derived from somatic cells of the embryo that give rise to the imaginal disks (all cells are infected after cellularization of the blastoderm [Kose and Karr, 1995]), and 2. those cells that are the descendant of the pole cells that formed during the syncytial stages. Because gametes arise directly from pole-cell progenitors, the absence of Wolbachia in yw(+) sperm cysts may be due to the early stages of pole cell formation where Wolbachia are first included into the germ line (Karr and O'Neill, 1990). The very low relative number of Wolbachia in yw(+) eggs suggests the intriguing possibility that too few bacteria are incorporated into pole cells initially and are therefore incapable of survival to the adult stage. If true, this line of reasoning further suggests that a critical number of Wolbachia per cell are needed that below which the infection cannot be maintained. We are currently examining these various possibilities in yw(+) and other, strains.
In conclusion, the QC-PCR technique when coupled with direct microscopic examination has proven useful for the analysis of the dynamics of Wolbachia in its host, Drosophila. Our approach should be useful to other infected species and animal groups.
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ACKNOWLEDGMENTS |
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We are grateful for the careful reading and editorial suggestions made by Yuval Gottlieb and Cort Anderson. This work was supported by a grant from the National Science Foundation IBN-9604287.
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FOOTNOTES |
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1 From the Symposium Living Together: The Dynamics of Symbiotic Interactions presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 3–7 January 2001, at Chicago, Illinois.
2 E-mail: tkarr{at}midway.uchicago.edu
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