Wolbachia infection in Drosophila simulans: does the female host bear a physiological cost?
In Drosophila simulans, Wolbachia cause cytoplasmic incompatibility (CI). CI is a significantly reduced egg hatch in crosses between infected males and uninfected females, whereas the reciprocal cross is normally fertile (unidirectional incompatibility). CI also occurs in both directions of crosses when the male and the female harbor two different Wolbachia (bidirectional incompatibility; Yen and Barr 1973; Breeuwer and Werren 1990; O'Neill and Karr 1990; Montchamp-Moreau et al. 1991; Mercot et al. 1995; Rousset and Solignac 1995). When both infected and uninfected individuals are present in a population, infected females have a selective advantage over uninfected ones: being compatible with all males, they have more offspring, to which they transmit the bacteria through the cytoplasm of the egg. Population models of Wolbachia infection dynamics have been developed (Caspari and Watson 1959; Fine 1978; Hoffmann et al. 1990; Turelli and Hoffmann 1991, 1995; Turelli 1994), outlining three parameters that would influence infection spread: (1) the level of CI; (2) the maternal transmission rate of Wolbachia; and (3) the possible deleterious effects on the host. These parameters have been estimated from field and laboratory data for one infection type in D. simulans (Hoffmann et al. 1990; Turelli and Hoffmann 1995). According to these models, a stable polymorphic equilibrium where both infected and uninfected individuals coexist in the same population is only possible if maternal transmission of the infection is not complete, leading to a residual amount of uninfected individuals being produced each generation. In these conditions, a second (and lower) equilibrium exists, but it is an unstable one. It corresponds to the critical threshold where CI just compensates for incomplete maternal transmission and for any deleterious effects on the female host. If the infection frequency is above the threshold, it will then rise to reach the higher stable polymorphic equilibrium. On the other hand, the infection will be lost if its frequency falls below the threshold. It follows that reaching the threshold when starting from the first infected female would require drift or a founder effect (Rousset and Raymond 1991), unless a neighboring infected population sends migrants (Turelli and Hoffmann 1991).
Different CI types caused by three Wolbachia variants that are mutually incompatible have been described in wild populations [TABULAR DATA FOR TABLE 1 OMITTED] of D. simulans (reviewed in Clancy and Hoffmann 1996). The variant wRi is found worldwide in continental areas. It has been associated with a deleterious effect on the infected female (Hoffmann and Turelli 1988; Hoffmann et al. 1990). In this work, we studied fitness traits in D. simulans strains infected by the Indo-Pacific Wolbachia variants wHa and wNo, found only on islands of the Seychelles archipelago and of the Pacific Ocean (Mercot et al. 1995). Because Wolbachia are only transmitted through the cytoplasm of the egg, we mainly concentrated our study on female fitness traits. Contrary to our expectations, we did not find any deleterious physiological effects on the female host.
MATERIAL AND METHODS
The strains used are listed in Table 1 and were reared at 25 [degrees] C on axenic medium (David 1962). Stocks were maintained as low-competition mass cultures (at least 200 reproductive couples of 4-5-day-old flies). Each strain generated two stocks as follows: infected females were left to lay eggs on standard medium to generate the infected stock (noted W). The same females were then transferred to lay eggs on medium treated with tetracycline, an antibiotic that eliminates Wolbachia. Tetracycline treatment was carried out for two generations according to Hoffmann et al. (1986). The stock obtained following tetracycline treatment is noted TC. The absence of Wolbachia in the TC stocks was checked by PCR three generations after treatment following the protocol described in Mercot et al. (1995). PCR amplification of bacterial 16S rDNA failed in DNA extracts from ovaries of 40 virgin females per TC stock, whereas it succeeded in ovaries extracts from infected controls. We have measured fitness traits in the three infected stocks (R1[A.sub.w], NH[a.sub.w], R3[A.sub.w]) and simultaneously in the corresponding cured stocks (R1[A.sub.TC], NH[a.sub.TC], R3[A.sub.TC]). Moreover, SimO (a naturally uninfected strain from the Tunisian population of Nasr'allah) was used as an uninfected control in productivity experiments, together with its tetracycline-treated stock [SimO.sub.TC].
Crossing Conditions in Fitness Experiments
In strains mono- or hi-infected by wHa and wNo Wolbachia, the fertility of females is slightly reduced when they are crossed with infected males, probably because some eggs are not infected enough for CI to be suppressed (Montchamp-Moreau et al. 1991; Mercot et al. 1995). To avoid any interference due to this phenomenon, the females used in all experiments were mated to uninfected males of their own TC stock.
Development Time and Egg-to-Adult Viability
Development time was measured following Yonemura et al. (1991). Twenty eggs were transferred to a vial of axenic medium, with 20 vials per strain for each infection status. Adults were collected every six hours and sexed, until the end of the emergence period. Egg-to-adult viability was estimated by counting all the adults collected.
Forty virgin females less than one day old were mated to 50 males of the same age, in a vial containing fresh axenic medium. On day 3, the group was transferred for 24 hours in a plastic box with an aperture that was fitted with a laying dish. Upon removal, each laying dish was kept at 25 [degrees] C for 24 hours before hatch rate was estimated on 300 eggs.
Productivity is the number of offspring produced. This trait was measured following Boesiger (1961). Virgin females were collected during the day, then mated individually in a vial, during one night, to virgin males of the same age. The flies were then transferred to a fresh vial the next morning (the previous vial was discarded as no eggs were present). They were further transferred to a new vial every three days for 15 days. Dead males were replaced with mated males of the same age. The productivity of a female is the number of offspring that emerge from her series of vials. Females having died before day 15 were not considered in the analysis. To facilitate the statistical analysis using ANOVA and subsequent tests, all sample sizes were made equal by selecting 14 individuals at random among the 14 to 20 surviving females.
Fecundity is the number of eggs laid. Eighty virgin females aged 24 hours were mated with virgin males in groups of 10 females and 20 males, then aged with them in a vial over three days. Each group was then placed in a plastic box fitted with a darkened laying dish. In the next 48 hours, the laying dishes were replaced three times with fresh dishes and the eggs counted.
[TABULAR DATA FOR TABLE 2 OMITTED]
Number of Ovarioles
Fifty-two virgin females were aged 3-4 days on fresh medium to allow full oogenesis. After dissection, their ovaries were hardened at least 48 hours in a saturated solution of potassium dichromate, then functional ovarioles were counted following Thomas-Orillard (1984).
We used the statistical software SAS (SAS Institute 1989) for the following tests: ANOVA (procedure GLM), Scheffe (procedure GLM option Scheffe) and Student-Newman-Keuls (procedure GLM option SNK). Partitioning of [[Chi].sup.2] on egg hatch was performed as per Maxwell (1961) and Winer (1971).
In the mosquito Culex pipiens, Magnin and Pasteur (1987) report that females treated with tetracycline as larvae suffer a reduction in egg hatch. Our experiments were therefore carried out a minimum of three generations after the end of the tetracycline treatment, to avoid any direct effect of the antibiotic on physiology. Three generations after treatment, we measured development time, productivity and egg-to-adult viability. We made a second measurement of productivity five generations after treatment, together with a measurement of egg hatch. A third productivity experiment was carried out (on R3A and R3[A.sub.TC] only) 14 generations after treatment. Finally, we measured fecundity and the number of functional ovarioles 20 generations after treatment.
TABLE 3. Analysis of variance on development time; d = denominator of F-test: A = Vial, B = Sex by vial, C = Error. Mean Source of variation df square d F Infection 1 0.00007 A 0.05 Strain 2 0.12397 A 82.65(***) Sex 1 0.20600 B 429.17(***) Infection by strain 2 0.00096 A 0.64 Infection by sex 1 0.00100 B 2.08 Strain by sex 2 0.00732 B 15.25(***) Infection by strain by sex 2 0.00045 C 0.87 Vial 114 0.00150 C 2.85(***) Sex by vial 114 0.00048 C 0.91 Error 1612 0.00052 *** P [less than] 0.001. TABLE 4. Analysis of variance on egg-to-adult viability. Source of variation df Mean square F Infection 1 0.00005 0.00 Strain 2 2.1540 78.86(***) Infection by strain 2 0.0822 3.02 Error 114 0.0272 *** P [less than] 0.001.
Egg-to-Adult Development Time
The results (Table 2) were compared through a three-way ANOVA on log-transformed development times with vials nested within infection and strain (Table 3). There is no significant difference between infected and uninfected stocks. The sex factor is significant, because females develop faster than males in this species. The strain factor is also significant. A Scheffe grouping test shows that NHa develops faster than the two other strains (P [less than] 0.05).
The results (Table 2) were compared through a two-way ANOVA following arcsin [square root of p] transformation (Table 4). There is no significant difference between the infected and the uninfected stocks. The strain factor is significant. A Scheffe grouping test shows that R3A has a lower egg-to-adult viability than the two other strains (P [less than] 0.05).
Results (Table 2), were analyzed through a partition of [[Chi].sub.2] (Table 5). The infection factor is significant, with infected stocks having a higher egg hatch than uninfected stocks (mean hatch rate of 90.6% vs. 87.2%). Nevertheless, the calculation of intrastrain [[Chi].sub.2] shows that only in R1A did the infected stock have a significantly higher productivity than the treated stock ([[[Chi].sub.2].sub.1df] = 4.87; P [less than] 0.05).
The number of offspring produced was measured first three generations after tetracycline treatment. We observed an important difference between infected and uninfected stocks (Table 6), which was contrary to that expected (cured stocks produced less than infected ones). We then decided to carry out a second measurement five generations after treatment (Table 6). All results were analyzed through a three-way ANOVA (Table 7). The infection, strain and generation factors and their interactions are all significant. A Student-Newman-Keuls test allowed us to compare and group the 12 mean productivities in three groups of means not significantly different one from another [ILLUSTRATION FOR FIGURE 1 OMITTED]. Among these groups, one corresponds to the lowest productivities observed (R1[A.sub.TC], NH[a.sub.TC], R3[A.sub.TC] in generation 3, and R3[A.sub.TC] in generation 5) and does not overlap any of the other two groups. Thus, three generations after treatment, all cured stocks had a significantly lower productivity than the corresponding infected stocks. The productivity decrease ranged from -57% to -90%. However, the same tetracycline treatment applied to the uninfected strain SimO did not result in any significant productivity decrease in two experiments carried out five months apart (Exp. 1: SimO = 241.4 offspring per female in 15 days vs. [SimO.sub.TC] = 213.7; n = 14 and 19, respectively; Exp. 2: SimO = 203.4 vs. [SimO.sub.TC] = 203.8; n = 26 and 27, respectively. Experiment effect: [[F.sup.1].sub.82] = 2.08, ns; Treatment effect: [[F.sup.1].sub.82] = 0.67, ns; Interaction: [[F.sup.1].sub.82] = 0.71, ns). This makes the possibility of a deleterious effect of the antibiotic unlikely.
TABLE 5. Analysis of sources of variation on fertility by partition of [[Chi].sup.2] test. Source of variation df [[Chi].sup.2] Infection 1 5.11(*) Strain 2 97.76(***) Infection by strain 2 2.83 Total 5 105.70(***) * P [less than] 0.05; *** P [less than] 0.001.
[TABULAR DATA FOR TABLE 6 OMITTED]
As only R3[A.sub.TC] still exhibited a significantly lower productivity than the corresponding infected stock five generations after treatment (130.8 vs. 252.4 offspring for R3[A.sub.w]), we carried out a last measurement on these two stocks 14 generations after treatment. It revealed that their productivity difference was no longer significant (R3[A.sub.TC] = 186.6 offspring per female in 15 days vs. R3[A.sub.w] = 224.3; n = 25 and 22, respectively; t = 1.30; ns). Therefore, all cured stocks experienced an important productivity drop following Wolbachia loss, but this was apparently a temporary phenomenon.
TABLE 7. Analysis of variance on productivity. Source of variation df Mean square F Infection 1 264656.10 38.48(***) Strain 2 253272.86 7.75(***) Generation (after treatment) 1 181897.52 26.45(***) Infection by strain 2 47340.72 6.88(**) Strain by generation 2 83016.90 12.07(***) Infection by generation 1 33886.88 4.93(*) Infection by strain by generation 2 23731.11 3.45(*) Error 156 6878.21 * P [less than] 0.05; ** P [less than] 0.01; *** P [less than] 0.001.
Fecundity and Number of Functional Ovarioles
Productivity depends on three factors: fecundity, egg-hatch, and larvo-nymphal viability. From our measurements of egg hatch and viability, we deduced that the main factor explaining the large productivity drop observed could only be fecundity. Yet this is only an indirect conclusion, as we did not measure fecundity early in this work. We however measured fecundity and one of its components (the number of functional ovarioles) 20 generations after treatment. Our hypothesis was that if the productivity drop observed was temporary, we should not be able to find any difference in fecundity between infected and TC stocks at this generation time. The TC stocks were checked again by PCR and found to be still uninfected. The results (Table 6) were compared through a two-way ANOVA (Table 8). As expected, there was no significant difference between infected and uninfected stocks. Only the strain factor is significant and a Scheffe grouping test reveals that R3A has a lower fecundity than the other strains (P [less than] 0.05). The absence of difference between infected and TC stocks is confirmed by our results on the number of functional ovarioles (Table 6). These results were compared through a two-way ANOVA (Table 8) following Blom's rank transformation (Blom 1958). No significant difference was observed between infected and uninfected stocks. The only significant difference observed is between strains, a Scheffe grouping test showing that the R1A strain has more functional ovarioles than the other strains (P [less than] 0.05).
Our main finding is that in long-established laboratory populations, the Indo-Pacific Wolbachia wHa and wNo do not seem to have a deleterious impact on their female host. This result was not expected, as similar experiments made in Californian populations of D. simulans on the wRi infection had revealed that the wRi Wolbachia do reduce fitness in laboratory conditions (Hoffmann et al. 1990). A second unexpected result is that D. simulans females apparently suffer from a temporary drop of productivity during a few generations, after being cured from the Indo-Pacific Wolbachia infection. We will discuss first this secondary result, for which we do not have any conclusive explanation. We will then discuss the possible reasons why Indo-Pacific Wolbachia would not have any negative impact on their host.
Temporary Drop of Productivity Following Tetracycline Treatment
The temporary nature of the productivity drop in treated stocks indicates that Wolbachia do not enhance productivity in infected stocks. This temporary effect can probably not be explained either by tetracycline toxicity or by selection of a less productive genotype by the antibiotic. Indeed, the treatment did not have any effect on the productivity of an uninfected strain (SimO) carrying the same nuclear genome than one of our infected strains (R3A). Drift and inbreeding are also unlikely to explain our results as our stocks were maintained with at least 200 reproductive couples per generation. We are then led to associate the temporary productivity drop to Wolbachia loss in treated stocks and not to a positive effect of Wolbachia in untreated stocks. The effect we observe might be explained in several ways. First, massive killing of Wolbachia by the antibiotic might have put an extra pressure on host physiology, either through the release of toxins or simply by the effort necessary to process dead bacteria/host cells. If important enough, these effects might have debilitated the host for a few generations. The productivity drop could also be explained if Wolbachia took part in the physiological processes of oogenesis. Curing a strain from its Wolbachia might then require an adaptation of the host to the symbiont-free status, which could take several generations. Regardless of its cause, if such a phenomenon took place in the wild, it would advantage the infection by diminishing the fitness of females having just lost their Wolbachia. However, Wolbachia loss in the wild is unlikely to be as drastic as in our heavy antibiotic treatment. The productivity drop we observe might then be an artificial laboratory by-product. As our results do not allow us to conclude on the nature or even the precise duration of the productivity drop we observed, further experiments would be necessary to address these issues.
A drop of fitness following the loss of Wolbachia is found in two other cases. In one strain of the hymenopteran Nasonia vitripennis, a significant drop of productivity is found in females [TABULAR DATA FOR TABLE 8 OMITTED] whose mothers have been cured from a heavy CI-Wolbachia infection by an antibiotic treatment. In an other strain where Wolbachia densities are lower, the treatment does not have a detectable effect (Stolk and Stouthamer 1996). It is not known yet if the effect is temporary or permanent in this species. In another parasitoid wasp, Trichogramma bourarachae, Girin and Bouletreau (1995) found a drop of productivity in females cured from Wolbachia, while the same treatment did not have any effect in an uninfected strain. The key difference compared to our findings is that in T. bourarachae the productivity in the treated stock remains low permanently, suggesting a positive effect of Wolbachia on fitness in this species (C. Girin, pets. comm.).
Absence of Deleterious Effects on the Female Host
A paradigm presented by some authors (May and Anderson 1983; Ewald 1987; Smith and Dunn 1991) is that exclusive maternal transmission of a parasite should favor an evolution toward mutualism, because the parasite and the host share the same route (the egg) to the next generation. Because Wolbachia are only transmitted through the egg, the absence of a negative impact on female physiology should not be surprising. Yet, CI could allow Wolbachia to spread regardless of a deleterious impact on the host, as long as the impact is not too high. Therefore, the fact that they are transmitted maternally is insufficient to predict the absence of deleterious effect of Wolbachia on the female host. Indeed, deleterious effects linked to CI-Wolbachia infection have been reported. In Tribolium confusum, Wade and Chang (1995) found the productivity of infected females to be lower than that of uninfected females. In D. simulans, Hoffmann et al. (1990) found in laboratory stocks that a negative effect on fecundity (a reduction of 8% to 18% in the number of eggs laid over 48 h) could be attributed to the presence of the Wolbachia variant wRi. In the beginning of our experiments, we were in fact expecting that curing our stocks of their Wolbachia would increase their fitness, which was clearly not the case.
The behavior of Indo-Pacific Wolbachia might be explained when considering long-term Wolbachia evolution. Turelli (1994) showed by modeling that in a situation where all Wolbachia clones are compatible, the one to invade the population will be the one with the highest "effective relative fecundity" defined as F x (1 - [mu]), where F is the relative fecundity of the infected female (with F - 1 for uninfected females) and (1 - [mu]) is the maternal transmission rate (when [1 - [mu]] = 1, transmission is perfect). Turelli points out that, for a given clone, high Wolbachia densities in the host probably diminish F, but increase (1 - [mu]). The optimal effective relative fecundity for a given Wolbachia might then result from a trade-off (based on bacteria density) between host fecundity and transmission efficiency.
A key feature of this model is that F x (1 - [mu]), and not CI, is the main target of natural selection. Yet, a high CI will favor the initial invasion in an uninfected population, and high levels of CI have been correlated with a high density of Wolbachia in the host (Breeuwer and Werren 1990; Boyle et al. 1993; Bressac and Rousset 1993; Solignac et al. 1994; Mercot et al. 1995). Then, during the invasion phase, a high CI (linked to high Wolbachia densities and therefore to potential deleterious effects on the host) could be selected for. This might be the case for example in the wRi infection, which swept very recently through Californian D. simulans population (Hoffmann et al. 1986, 1990; Hoffmann and Turelli 1988; Turelli and Hoffmann 1995). But once the invasion is complete, a high CI ceases to be critical. From what is known about Indo-Pacific populations of D. simulans, the infection by wHa and wNo Wolbachia predates the divergence between D. simulans and the sibling species D. sechellia, which took place at least 0.5 M.Y.B.P. (Rousset and Solignac 1995). Because the populations of Indo-Pacific islands are probably small, the infection by wHa and wNo could have reached near fixation a very long time ago. On the other hand, the wRi infection is restricted to D. simulans continental populations harboring a precise mitochondrial subvariant, suggesting a more recent infection of wRi in this species (Baba-Aissa et al. 1988; Hale and Hoffmann 1990; Turelli et al. 1992; Rousset and Solignac 1995). Our hypothesis is that lowered densities of Wolbachia associated with the absence of deleterious effect have had time to be selected in Indo-Pacific populations. Indeed, when different infections have been compared, wHa Wolbachia density was found to be at least three times lower than that of wRi Wolbachia in the egg (Giordano et al. 1995; Sinkins et al. 1995) or in the adult male (Bourtzis et al. 1996). It is also clear that wHa and wNo transmission in the laboratory is not as efficient as wRi transmission (Hoffmann et al. 1990; Mercot et al. 1995; unpubl. data).
Hoffmann et al. (1996) found in Australian populations of D. simulans another Wolbachia without any deleterious impact on its female host, but they also found that its transmission was apparently perfect, even in field-captured females. This example shows that the relation between fitness effects and transmission efficiency is not straightforward. Interestingly, this Wolbachia did not exhibit any detectable amount of CI. Other Wolbachia not causing any detectable CI have been found in other D. simulans populations and also in D. mauritiana (Giordano et al. 1995; Rousset and Solignac 1995; Turelli and Hoffmann 1995). It remains to be seen whether such Wolbachia are maintained through a combination of perfect maternal transmission and the absence of deleterious fitness effect, or if they compensate a very small transmission leakage by a limited amount of CI. The example of Trichogramma bourarachae discussed earlier also suggests the possibility of positive rather than deleterious effects of the infection. Some Wolbachia might have gone all the way from endoparasites to endosymbionts.
We would like to thank A. Atlan, D. Higuet, S. Netter, H. Quesneville, and two anonymous reviewers for very useful comments on an earlier version of this manuscript; C. Girin and R. Stouthamer for sharing unpublished information; and M. Thomas-Orillard for advice on functional ovarioles observation techniques. We also thank E. Aspect and V. Delmarre for technical assistance. DP is supported by a grant from the French Ministere de l'enseignement superieur et de la recherche.
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|Author:||Poinsot, Denis; Mercot, Herve|
|Date:||Feb 1, 1997|
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