Wolbachia injection from usual to naive host in Drosophila simulans (Diptera: Drosophilidae)

Wolbachiapipientis (Hertig) (Rickettsiaceae) is an endocellular bacterium infecting numerous species of arthropods. The bacterium is harboured by males and females but is only transmitted maternally because spermatocytes shed their Wolbachia during maturation. The presence of this endosymbiont can lead to feminisation of the host, parthenogenesis, male-killing or reproductive incompatibility called cytoplasmic incompatibility (Cl). Although Wolbachia transmission is exclusively maternal, phylogenetic evi­ dence indicates that very rare inter-species transmission events have taken place. Horizontal transmission is possible in the labora­ tory by transferring cytoplasm from infected to uninfected eggs. Using this technique, we have artificially infected lines of the fruit fly Drosophila simulans Sturtevant (Drosophilidae). Recipient lines came from two different D. simulans populations. One (“naive” host) is not infected in the wild. The other (“usual” host) is a population naturally carrying Wolbachia in the wild. In this second case, recipient flies used in the experiment came from a stock culture that had been cured off its infection beforehand by an antibi­ otic treatment. Infected D. simulans laboratory stocks were used as donors. We assessed the three following parameters: (i) trans­ infection success rate (ratio of infected over total female zygote having survived the injection), (ii) level of cytoplasmic incompatibility expressed by trans-infected males three generations post-trans-infection, and (iii) infection loss rate over time in trans-infected lines (percentage of lines having lost the infection after 20 to 40 generations). We observed that parameter (i) did not differ significantly whether the recipient line came from a “naive” or a “usual” host population. However, both (ii) and (iii) were sig­ nificantly higher in the “naive” trans-infected stock, which is in agreement with earlier theoretical considerations.


INTRODUCTION
Wolbachia are endocellular bacteria infecting arthro pods (Werren, 1997).They are found in germinal, as well as in somatic tissues, where they live in intra-cytoplasmic vacuoles, and it is estimated (Werren et al., 1995a) that more than 16% of insect species are infected (within a given species, a population can be entirely infected, poly morphic for the infection, or entirely uninfected).Although Wolbachia are harboured by male and female hosts, transmission only occurs maternally through the cytoplasm of the egg, because sperm cells shed their bac teria during maturation (Binnington & Hoffmann, 1989).
The most common phenomenon, cytoplasmic incom patibility (Cl), is an embryonic mortality induced by the presence of Wolbachia during the maturation of male reproductive cells.Cl occurs when the Wolbachia strain present in the male is absent in the egg.Then, fertilisation is apparently normal but subsequent mitoses are disrupted leading to the death of the zygote (Callaini et al., 1996).This occurs when the female is uninfected (Hoffmann et al., 1986) or when the Wolbachia strain harboured by the female is different from that of the male (Breeuwer & Werren, 1990;O'Neill & Karr, 1990).Since the eggs laid by uninfected females are not protected from CI, the fit ness of uninfected individuals is lowered, and the infec tion is expected to spread in an initially uninfected population.
The strict vertical transmission of Wolbachia does not hold when considering long evolutionary periods.Molecular data indicate that very closely related Wolba chia can be found in hosts as distant as insects and crusta cean isopods (Werren et al., 1995b;Zhou et al., 1998).The large and repeated discrepancies between the phylogenies of hosts and those of their Wolbachia demonstrate that inter-species transmission (and therefore horizontal transmission) has occurred repeatedly in the past.Moreo ver, it is possible to transfer Wolbachia from one species to another through micro-injection experiments in fertil ised eggs (Boyle et al., 1993;Braig et al., 1994;Clancy & Hoffmann, 1997;Poinsotetal., 1998).
We have used the same experimental approach to con duct intra-species transfer of Wolbachia using Drosophila simulans both as a donor and as a recipient host.The recipients were of two types.One type, the "naive host", came from a natural D. simulons population that is entirely uninfected in the wild.The other type, the "usual host", came from a natural population that is entirely infected.In this second case the eggs used as recipients came from a laboratory stock that had been artificially cured off its infection beforehand by an antibiotic treat ment.Two different Wolbachia variants were used in the experiments, and they were injected either separately (using mono-infected D. simulons donor stocks) or together (using a bi-infected donor stock).We assessed the three following parameters: (i) trans-infection success rate (ratio of infected over total female zygotes having survived the injection), (ii) level of cytoplasmic incom patibility expressed by trans-infected males three genera tions post-trans-infection, and (iii) infection loss rate in trans-infected lines (percentage of lines having spontane ously lost the infection after 18-20 months of routine laboratory rearing).

Wolbachia strains
The two Wolbachia strains studied are wHa and wNo (Rousset & Solignac, 1995).They are known only in popula tions from indo-pacific islands and are bidirectionally incom patible (Merçot et al., 1995).

Drosophila simulans strains
Donors.R1A, NHa and R3A derive from a naturally infected population (Nouméa, New Caledonia).In the wild, Nouméa individuals are either infected simultaneously by wHa and wNo, or mono-infected by wHa.The laboratory strain R1A only har bours bi-infected individuals.NHa is a Nouméa stock that was found in the laboratory to be infected by wHa only.R3A was obtained by substituting the Nouméa genome by that of SimO for 11 generations.It was then found to be infected by wNo only (Merçot et al., 1995).
Recipients.SimO is a naturally uninfected strain from Nar'allah, Tunisia (Montchamp-Moreau et al., 1991).It is used as a recipient in the trans-infection experiment into a naive host.Females from the standard SimO stock are also used in test crosses to detect the expression of CI in males.R1ATC is an uninfected stock derived from R1A by an antibiotic treatment (tetracycline) which cured its Wolbachia infection (Poinsot & Merçot, 1997).The asymbiotic status of R1ATC is regularly verified using PCR.R1ATC is used as an uninfected recipient strain in the trans-infection experiment into an usual host.

Experimental methods
Micro-injections.They were carried out as described in Santamaria (1987).Using a microcapillary needle (Femtotips, Boehringer) cytoplasm was drawn from infected embryos (R1A, NHA or R3A) and then injected into slightly dehydrated unin fected embryos (SimO or R1ATC).Isofemale lines were estab lished after crossing the emerging females with uninfected males bearing the same genetic background (i.e.SimO or R1ATC).
Rearing conditions.Initially, injected lines were maintained as low density mass cultures in bottles at 25°C, on axenic medium (David, 1962).After 10 generations, of this regime, all strains were transferred to vials at 18° and 25°C, with two repli cates per temperature.The lines were then maintained routinely by mass transfer for the rest of the experiment.
Pcr/relp.Total DNA was extracted from ovaries following the method of Kocher et al. (1989) or from whole individuals following the method of O'Neill et al. (1992).The Wolbachia 16S ribosomal subunit DNA sequence was amplified using spe cific primers 99F and 994R as previously described in O 'Neill et al. (1992).PCR amplification products were subjected to VspI digestion at 37°C for 3 h.The 16S rDNA sequence of wNo presents a VspI restriction site whereas the sequence of wHa does not, which allows discrimination of the two strains by RFLP.PCR products of bi-infected flies exhibit a three-band pattern (Poinsot et al., 2000).
Ci measurements.Tests were performed at 25°C.Fifteen 4 or 5-day-old virgin females were allowed to mate for 8 h with 25 virgin 3-day-old males in a bottle containing fresh axenic medium.Flies were then transferred to a box for oviposition.The aperture was fitted with a small Petri dish containing stan dard axenic medium darkened with vegetable charcoal powder.After 24 h, the adults were discarded and the eggs kept at 25°C for at least another 24 h duration before the hatching rate was estimated, generally from 200 eggs per line.
Level of cytoplasmic incompatibility.We used CIcorr, a cor rected index of Ci.The aim was to minimise the background noise due to the natural mortality of the cross (that is not related with Ci).This background mortality was estimated from the mortality induced by males from injected but uninfected isofe male lines (i.e.lines where the bacteria had failed to be trans ferred) in crosses with uninfected SimO females.Failure of the infection transfer was defined as the absence of a positive PCR signal in the isofemale line in G3.Since males from these lines are not infected, the cross with SimO females is compatible and any mortality is background mortality (BgM.).
Cicorr is then defined as the percentage of eggs that do not hatch among those that would have survived in the absence of CI.
where %Unhatched is the percentage of unhatched eggs observed in the incompatible cross.Cicorr was set at 0 in the very rare cases where %Unhatched was lower than %BgM.
Statistical analysis.The trans-infection rate was analysed as a function of the donor strain, the recipient strain and their inter action using the partition of % 2 test (Maxwell, 1961).This test provides, for discrete variables, a statistical analysis equivalent to an ANOVA (Winer, 1971).The same test was carried out to analyse the loss of the infection depending on the rearing tem perature and the recipient strain.The level of Ci induced by trans-infected lines was tested by an ANOVA [GLM procedure type iiiSS of SAS (1989)], after arcsine transformation, as a function of the infection status, the recipient strain and their interaction.

Success rate of the trans-infection
The results, based on PCR + RFLPs carried out in G3, are presented in Table 1.The partition of % 2 reveals that only the type of donor effect is significant.This is due to a higher rate of trans-infection when the donor strain is bi-infected.indeed, the success rate is not significantly different between the two mono-infected donors (%2= 1.380, 1df, ns), at least when assessed in G3.However, the success rate obtained with the bi-infected donor strain is significantly superior to the average success rate of the two mono-infected donors (47.17% vs 24.24%; X2 = 6.842; 1df, P < 0.01).The absence of a recipient Table 1.Success rate of the transinfection as a function of the recipient and the donor strains, followed by a partition of x2 test.In the case of bi-infected donor strains, the type of infection found in the recipient isofemale lines after a successful transin fection is indicated (no individuals mono-infected by wNo were recovered).All PCR + RFLPs were carried out in G3 after trans infection.ns: not significant.effect shows that the infection is established as easily in the "naive" strain (SimO) as in its usual host (R1A genetic background).We also found that mono-infected as well as bi-infected isofemale trans-infected lines could be recovered when a bi-infected strain was used as a donor.In our experiments, all mono-infected lines recov ered in those conditions harboured wHa.The relative pro portion of mono and bi-infected lines recovered did not differ as a function of the recipient strain (x2 = 2.778, 1df, ns).

Cytoplasmic incompatibility
Before carrying out the injection experiment, we meas ured the CI level induced by the three donor strains.The percentage of unhatched eggs (estimated on 300 eggs per cross) were 71.3% (R1A); 57.7% (NHa) and 60.7% (R3A).Contrary to previous repeated observations (Merfot et al., 1995;Merfot & Poinsot, 1998), the wHa variant (NHa strain) did not induce a significantly higher CI than the wNo variant (R3A strain) (x2 = 0.581, 1df, ns).This might reflect a low level of infection of our NHa strain at the time of our injection experiment.Indeed, it has been found that, for a given Wolbachia, the level of CI is correlated with bacterial load in males (Breeuwer & Werren, 1990;Boyle et al., 1993;Bressac & Rousset, 1993;Solignac et al., 1994;Merfot et al., 1995).
Three generations after injection, we tested the level of CI induced by trans-infected lines.The results are shown in Table 2 and have been analysed using an ANOVA (Table 3).The recipient strain effect is significant (P < 0.05) as well as the infection status effect (P < 0.01).The interaction is not significant.The recipient strain effect is due to a higher CIcorr in trans-infected males of the "naive" SimO strain compared to males of the usual host (R1A genetic background) (67.2 ± 7.4% vs 37.3 ± 6.2%).Concerning the infection status, we compared the means of each infection status (LSMEANS/TDIFF statement in SAS 1989).This comparison shows that CIcorr was sig nificantly higher in bi-infected males than in males mono infected by wHa (77.5 ± 7.1% vs 43.4 ± 7.4%, T = 3.31, P < 0.01) or by wNo (77.5 ± 7.1% vs 35.9 ± 8.3%, T = 2.31, P < 0.05).There was no significant difference between wHa and wNo-infected males (T = 0.19, n.s).This latter result is in agreement with the absence of sig nificant difference in CI levels between NHa and R3A donor strains before the trans-infection experiment began.Nevertheless, we found the usual significant difference in favour of wHa when some of the trans-infected lines pre sented here were assessed again after a longer period (over 40 generations) in the laboratory.The percentage of unhatched eggs (estimated on 200 eggs per cross) were 88.5% (R1AwHa6 line) and 67.0%(R1AwNo17 line) (x2 = 26.72,1df, P < 0.001).

Loss of the infection
The lines where trans-infection had been successful (as judged by a positive PCR in G3) were assessed by PCR + RFLP a second time after 18-20 months of routine main tenance in vials in the laboratory.During that time, two different temperature regimes had been used (18°C, i.e. about 20 generations, or 25°C, i.e. about 40 generations) with two replicates per temperature.Due to losses during this long period of storage, only one of the original two replicates per temperature regime was available for sev eral lines at the time of our analysis.The proportion of cases did not differ significantly between SimO and R1ATC trans-infected lines both at 18°C (46.7% vs 58.8% respectively, x2 = 0.182, 1df, ns with Yates correc tion) and at 25°C (26.7% vs 15.0%, x2 = 0.470, 1df, ns).In order to keep as much information as possible, we cal culated the proportion of replicates that were still infected (instead of the proportion of lines).The results are pre sented in Table 4.The partition of x2 reveals a significant effect of temperature.The infection was nearly always lost in replicates maintained at 18°C (46 times out of 49) while it was lost in slightly more than half of the repli-

DISCUSSION
Day-to-day Wolbachia transmission is believed to be strictly vertical maternal transmission.Yet, phylogenetic evidence reveals that horizontal transmission has occurred several times in the past, even between very dis tant taxons (Werren et al., 1995b;Zhou et al., 1998), thus demonstrating that Wolbachia can colonise new host spe cies.The success of the establishment will then depend on the capability of the Wolbachia to grow in the new host, to induce Ci (if we consider only this phenotype) and to be efficiently transmitted maternally.Here, we have tried to mimic this colonisation process of an unin fected host within the species D. simulans, using a micro injection technique and two recipient strains.One strain, SimO, is uninfected in the wild while R1ATC is the usual host ofboth wHa and wNo Wolbachia variants.

Success rate of the trans-infection
We have been able to transfer in both recipient strains each of the three possible infection types, i.e. wHa mono infection, wNo mono-infection, as well as the wHa + wNo bi-infection.The rate of successful trans-infection among the isofemale lines obtained (41/119) was comparable to those reported in similar earlier experiments involving Drosophila species where the infection status of injected lines had been assessed in the very first generations after the injection (Rousset & de Stordeur, 1994: 6/12, Gior dano et al., 1995: 9/24, Poinsot et al., 1998: 17/33).The rate of success of the trans-infection was superior when using bi-infected donor eggs.This might result either from a greater infection of bi-infected females or from a synergistic effect between the wHa and wNo variants.On the other hand, the rate of successful transfer did not differ significantly between naive and usual host, sug gesting that the "naive" status did neither impede nor facilitate the initial establishment of the infection, at least in the conditions of our experiment.This finding is counter-intuitive (one might expect a more efficient transfer in the usual host).However, microinjection in embryos might not be representative of the (as yet unknown) routes followed by horizontal transmission in the wild.For example it has been demonstrated that Wol bachia can also be experimentally transmitted by injec tion in nymphs of Trichogramma (Trichogrammatidae) wasps (Grenier et al., 1998) or in the adult in terrestrial isopods (Juchault et al., 1994). in the case of the wood louse Armadillidium vulgare (Latr.),(Oniscidae), woundto-wound contact has been shown to be sufficient to induce transmission, since Wolbachia are present in hematocytes (Rigaud & Juchault, 1995).This suggests that inter-species natural transmission takes more indirect (and therefore more difficult) routes than injection into an egg.By injecting Wolbachia directly into the embryo at the most favourable stage (before pole cells formation), our protocol might have facilitated the installation of the bacteria to the point where potential differences between naive or usual hosts were not important for the success of the transfer.

Cytoplasmic incompatibility
The most interesting finding is that Ci was significantly higher in the naive host than in the usual host, regardless of the infection type (i.e.wHa, wNo or wHa + wNo). in a previous experiment, Rousset & de Stordeur (1994) trans infected a naturally uninfected D. simulans strain (Wat sonville, USA) with wHa and wNo.Seven generations post injection, they found that naive trans-infected males caused a weaker Ci than males from the infected donor strain.However, due to the absence of an injection con trol (injection into usual hosts from a strain cured from its Wolbachia beforehand) it even was not possible to unam biguously attribute the low Ci obtained to the naive genome (Watsonville) rather than to the injection proce dure itself.Other results involving inter-species transfers (Boyle et al., 1993;Braig et al., 1994) show that when the infection is in the first few generations following a trans infection, the level of Ci can be significantly weaker than what it becomes after several generations or rearing in good conditions.Our own present results suggest the same idea (Ci levels rising from 30-40% in G3 post infection to over 80% more than 40 generations later in our mono-infected trans-infected lines).

Loss of the infection
When checking the infection status of trans-infected lines after 18-20 months of routine maintenance at 25°C (i.e. about 40 generations) we found that infection loss had occurred in most naive trans-infected lines, but in only a third of usual host trans-infected lines, the differ ence being highly significant (P < 0.001).The loss of the infection can have two causes: (i) poor maternal transmis sion, (ii) lower viability of infected eggs (or individuals), especially in a situation of crowding.Our protocol does not allow to determine if only one or both factors played a role.We can however conclude that in temperature condi tions (25°C) which are optimal for the rearing of the host, Wolbachia maintenance was significantly easier in the usual host than in the naive host.This difference was not apparent in more hostile conditions, i.e. at 18°C, a tem perature found previously to be sub-optimal for the main tenance of the infection (Merfot & Poinsot, 1998).At this lower temperature, nearly all lines lost the infection regardless of the type of hosts (naive or usual), even though a period of 18-20 months at this temperature rep resents only about 20 generations of hosts (as compared to about 40 generations at 25°C).The rapid loss of a new infection has been found in other artificial transfer experi ments.Van Meer & Stouthamer (1999) injected the wRi Wolbachia variant from D. simulans to the microhymenopteran Muscidifurax uniraptor Kogan & Legner (Pteromalidae).The initial transfer was successful but maternal transmission was poor and the infection was lost in 6 generations despite optimal conditions of rearing for the host.In Isopods, Rigaud & Juchault (1995) found that in inter-species trans-injections, the initial transfer was often successful but that in most cases the bacteria were not transmitted to the offspring at all.Working with the same donor host (the woodlouse Armadillidium vulgare), Bouchon et al. (1998) found that in naive host species the infection could have lethal pathogenic effects, which was never the case in the usual host.On the other hand, Braig et al. (1994) obtained a stable infection after transferring the Wolbachia of the mosquito Aedes albopictus Skuse (Culicidae) into D. simulans, demonstrating experimen tally that Wolbachia can cross species barriers over rela tively large phylogenetic distances.

Conclusion
Our results show that when transferred into a new host, both wHa and wNo cause more CI if the host is naive than when it is not.We also found that these Wolbachia seem less able to maintain a durable association with the naive host.As previously stated, our protocol does not allow us to decide if this is due to poor transmission and drift alone or if a slightly deleterious effect linked to the infection played a role.In any case, our results are in full agree ment with the data and the ideas presented by Clancy & Hoffmann (1997), based in part on the model of host and Wolbachia coevolution developed by Turelli (1994).These authors conclude that CI will tend to be high in a new host (following horizontal transfer), when the host has not had time to evolve any resistance.Also, at this early stage, the bacteria are not yet fully adapted to the new host and are not expected to achieve a perfect maternal transmission, nor to be completely harmless for the female host, in particular regarding fecundity.Over time, the situation is expected to evolve on all fronts.First, any gene diminishing the effects of CI will be selected for in the host, and CI is expected to diminish with time.Indeed, CI is low and sometimes undetectable in Drosophila melanogaster Meigen (Drosophilidae), and totally absent in the wAu infection of D. simulans, both infections believed to be very ancient (Solignac et al., 1994;Clancy & Hoffmann, 1997).Second, Wolbachia obviously bears a strong selective pressure to improve its own transmission, and to a certain extent the female host will bear the same pressure (because infected eggs are protected from CI).Therefore, maternal transmission is expected to be highly efficient in old infections, espe cially if the CI capability has been lost and can not com pensate for a poor transmission any more.Finally, both the bacteria and the female host are selected to maximise female fecundity, so an old infection is not expected to reduce fecundity.Again, it seems exactly to be the case in the wAu infection of D. simulans, where transmission is probably nearly perfect in the field and where CI and deleterious effects on the female are not detectable (Clancy & Hoffmann, 1997).Such an old Wolbachia infection is however expected to slowly drift into extinc tion because, without CI, even a near perfect transmission is not perfect enough.Alternatively, this peaceful decline can be abruptly shortened by the sudden invasion of a new high-CI variant following horizontal transfer or migration.A case in point might be the spectacular spread of the wRi variant taking place right now in D. simulans populations, some of which probably harboured already the "harmless" (and apparently doomed) wAu variant (Turelli & Hoffmann, 1995).

Table 2 .
Cytoplasmic incompatibility in G3.See Materials and Methods for the calculation of the CIcorr index.se: stan dard error.

Table 4 .
infection loss in trans-infected isofemale lines after 18-20 months in the laboratory.