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Hered 364 master.hered 364 . page254Heredity 81 (1998) 254–260
Received 6 August 1997, accepted 2 January 1998 Distribution and reproductive effects
of Wolbachia in stalk-eyed flies
AHMAD R. HARIRI†§, JOHN H. WERREN‡ & GERALD S. WILKINSON*† †Department of Biology, University of Maryland, College Park, MD 20742, USA and ‡Department of Biology, University of Rochester, Rochester, NY 14627, USA Wolbachia are cytoplasmically inherited bacteria capable of altering the reproductive biology oftheir hosts in a manner which increases their spread within a population. These microbes cancause cytoplasmic incompatibility, parthenogenesis and feminization of genetic males. BecauseWolbachia have been associated with female-biased sex ratio distortion, we used a PCR assayto examine 17 species of stalk-eyed flies (Diptera: Diopsidae), two of which exhibit female-biased sex ratios, for the presence of these microbes. Type A Wolbachia was detected in fourdiopsid species, three from the genus Sphyracephala, none of which exhibit biased progeny sexratios. The reproductive effects of the microbe were examined in one of those species, S. bec-carii, by conducting reciprocal crosses between infected and uninfected strains. In this species,Wolbachia do not cause detectable cytoplasmic incompatibility or reduce host fecundity. Incontrast, our results are consistent with an association between the microbes and enhancedmale fertility. Possible explanations for the pattern of distribution and effects on male fertilityinclude a predisposition for acquiring Type A Wolbachia by these flies and accommodation bythe host genome to bacterial presence.
Keywords: cytoplasmic bacteria, Diopsidae, Sphyracephala, stalk-eyed flies.
promotes the spread of the microorganisms. Theeffects of Wolbachia infection include unidirectional Wolbachia are a monophyletic group of proteobac- and bidirectional cytoplasmic incompatibility (CI), teria with two major divisions, Type A and B, which parthenogenesis, and feminization of genetic males have been associated with a wide range of repro- (see Werren, 1997 for review). Wolbachia can have ductive changes in arthropods (Werren et al., either positive effects on host fecundity (Girin & 1995a). They are primarily found within the cells of Bouletreau, 1995; Stolk & Stouthamer, 1996; the gonadal tissues of infected individuals (O’Neill, Poinsot & Mercot, 1997) and fertility (Wade & 1995) and are typically inherited by vertical trans- Chang, 1995) or negative effects on host fecundity mission through the maternal cytoplasm (Hoffmann (Hoffmann et al., 1990). The positive or negative & Turelli, 1988). Horizontal transmission, especially consequences of infection may be related to differ- of Type A Wolbachia, is believed to play an import- ences in the mode of microbe transmission and ant role in spreading the infection between species maintenance in different groups of organisms.
and maintaining phylogenetically similar strains in Models of symbiont transmission predict that nega- diverse Orders of arthropods (Werren et al., 1995b).
tive effects on host reproduction will be prevalent in Wolbachia have not been found extracellularly and systems where symbiont populations are maintained their existence and proliferation appear to be inti- by horizontal transmission, and positive effects will mately linked with that of their arthropod hosts be more common when vertical transmission domi- Wolbachia typically alter the reproductive biology Recently, female-biased sex ratio distortion has been of their hosts in a manner which ultimately reported in two species of stalk-eyed flies (Diptera:Diopsidae), Cyrtodiopsis dalmanni and C. whitei *Correspondence. E-mail: email@example.com§Present address: Brain Research Institute, UCLA, Los Angeles, (Burkhardt & De La Motte, 1983), and ascribed to X chromosome meiotic drive (Presgraves et al., 1997).
1998 The Genetical Society of Great Britain.
WOLBACHIA IN STALK-EYED FLIES 255
Because Wolbachia have been associated with was confirmed by measuring product length from an female-biased sex ratio distortion, either through agarose gel. All solutions were filter-sterilized parthenogenesis in haplodiploids (Stouthamer et al., (0.22 m pore diameter) to reduce potential 1993) or feminization in diploids (Rousset et al., bacterial contamination. Control DNA samples were 1992), we decided to determine whether or not these prepared from pupae of known infected and unin- microbes are present in C. dalmanni and C. whitei.
fected strains of Nasonia vitripennis and compared In order to determine the potential distribution of with amplified products from each stalk-eyed fly Wolbachia infection in stalk-eyed flies an additional species to determine the presence of Wolbachia.
15 diopsid species from six genera were also Additional amplifications were performed using examined. We also report on the reproductive Type A and Type B specific primers for ftsZ effects of infection in one of those species, Sphyr- acephala beccarii, which harbours Type A Wolbachia.
Materials and methods
Flies were maintained in modified mouse cages with a 12 L:12D photoperiodic cycle and 30 min simu- We collected flies from six of the 11 diopsid genera lated dawn and dusk periods (Lorch et al., 1993).
(Feijen, 1989): Teleopsis, Cyrtodiopsis, Diasemopsis, Flies were allowed to feed ad libitum on a standard Diopsis, Sphyracephala and Eurydiopsis. These six genera contain over 96% of the described species with a commercial mould inhibitor (Wilkinson, and occupy the entire geographical range of diopsids 1993). For breeding purposes, :35–40 mL of (Steyskal, 1972). All Teleopsis species (breviscopium, medium was presented in small plastic cups and rubicunda, quadriguttata) and Cyrtodiopsis species changed biweekly. Newly eclosed flies were isolated (dalmanni, whitei, quinqueguttata) were collected in by sex and maintained as virgins until they were January 1989 in peninsular Malaysia. All Diasemop- reproductively mature and then utilized in reciprocal sis (aethiopica, dubia, munroi, silvatica) and Diopsis (apicalis and fumipennis), as well as Sphyracephala In order to determine the optimum concentration munroi, were collected in December 1994 in the of antibiotic, which would maximize elimination of Natal Province, South Africa. Sphyracephala brevi- the infection and minimize the mortality caused by cornis were collected in October 1994 in Maryland the toxicity of the antibiotic, flies were bred on and S. detrahens and Eurydiopsis subnotata in media treated with 0.5 mg mL1, 1.0 mg mL1, or January 1996 in peninsular Malaysia.
2.0 mg mL1 concentrations of aqueous tetracycline, Sphyracephala beccarii were collected on 22 prepared by dissolving tetracycline hydrochloride November 1993 along a stream near Sudwala Caves, (Sigma T-3383) in water over low heat. Fecund 40 km west of Nelspruit, Transvaal Province, South females (3 weeks post eclosion) from these treat- Africa. Approximately 20 flies were used to establish ment classes were then tested for the presence of a laboratory population and were subsequently bred infection using the PCR assay described above.
in the laboratory for 10 months prior to the start of These analyses, in addition to the number of pupae this study. We estimate that the flies used in the first produced by five pairs of flies from each of the three antibiotic treatment experiment (see below) were at tetracycline concentrations, were used to identify the least three generations removed from those optimum tetracycline concentration for curing.
These results indicated that the optimum concen- tration of tetracycline was 1.0 mg mL1. At thisconcentration, the infection was eliminated and the average daily pupal production was 8.0<1.9 (SE) The presence of Wolbachia was determined for each per pair. At 0.5 mg mL1, average daily pupal species by extracting DNA from pooled ovaries of production was higher (10.0<1.5), but the infection two to four fecund females and then using the poly- was not eliminated. At 2.0 mg mL1, the infection merase chain reaction (PCR) to amplify a Wolba- was eliminated, but average daily pupal production chia-specific bacterial cell-cycle gene, ftsZ, sequence (Werren & Jaenike, 1995). All samples were Bacteria were eliminated by allowing 20 females screened with universal 28S primers as a positive and 20 males to mate and oviposit ad libitum for control for amplification ability (see Werren et al., 48 h on 1.0 mg mL1 tetracycline-treated medium.
1995a, for details). Bacterial ftsZ DNA amplification Twenty pairs of progeny were then bred to produce The Genetical Society of Great Britain, Heredity, 81, 254–260.
256 A. R. HARIRI ET AL.
a second generation of flies which had developed in could potentially influence the reproductive biology 1.0 mg mL1 tetracycline-treated media. Fecund females obtained after both one and two generations We explored the potential effects of these factors of tetracycline exposure were tested with the PCR assay to determine if there were detectable levels of (confirmed by PCR) in a subset of treated indi- Wolbachia. Progeny derived from both levels of viduals which were then used in reciprocal crosses tetracycline exposure were then bred on standard, with individuals that were untreated and known to tetracycline-free maize medium for three genera- be infected. A multiway contingency table analysis tions (20 males and 20 females in each generation) was then used to determine if there was an associa- in order to reduce the possibility of decreased fecun- tion between fertility, presence of Wolbachia and dity resulting from maternal exposure to tetra- infection status. If reproductive effects are the result of any of these factors, then reproductive perform-ance should not change in this second set of crosses.
If, however, the effects are associated with Wolba- chia, then the two sets of crosses should exhibit a After three generations of maintenance on tetra- cycline-free medium, uninfected individuals fromboth levels of tetracycline exposure were used in reciprocal crosses with infected individuals. Crosseswere conducted by pairing single males with single In fertile crosses between infected and uninfected females. Mating and oviposition on the medium was individuals, an effect of Wolbachia on egg produc- allowed to occur for 120 h. For each of the four tion or development would be indicated by inequal- possible crosses between infected and uninfected ities in mean daily pupal production between individuals, 20 replicates were established for flies reciprocal crosses. Analysis of variance was used to derived from one generation of tetracycline expo- determine if heterogeneity existed between crosses sure, and 15 replicates for flies derived from two in pupal production. Fisher’s Paired Least Signifi- cant Difference test (PLSD) was used to identify An effect of Wolbachia on fertility would be indi- significant differences between crosses in pupal cated by an association between the presence or absence of pupae and infection. Therefore, a multi-way contingency table analysis was conducted on the number of fertile and infertile pairs from each treat-ment using SYSTAT v.5.2 (Wilkinson, 1989). Fertility (pupae or no pupae) was used as the response vari- The results of the PCR screen for the Wolbachia able and the level of antibiotic exposure (one or two ftsZ gene are presented in Table 1. Four positive generations), female condition (infected or uninfec- amplifications were detected. Three species from the ted), and male condition (infected or uninfected) genus Sphyracephala and Eurydiopsis subnotata were were used as explanatory variables in a four-dimen- found to be positive for Type A Wolbachia, but sional analysis. The significance of each potential negative for Type B Wolbachia. Amplification interaction was determined by testing the difference products of the appropriate length, 1035–1047 2, calculated by finding the difference between like- bases, were present for all four species. Neither lihood-ratio 2 statistics of two models differing in Type A nor Type B Wolbachia was detected in C. dalmanni or C. whitei; thus, the microbes do not Reproductive performance between crosses may contribute to the observed female-biased sex ratio be influenced by several factors other than Wolba- chia. First, tetracycline-treated media yielded rela-tively few offspring. Therefore, inbreeding may have occurred for four generations prior to the reciprocalcrosses and could be responsible for fertility differ- Comparison of the fertility of flies from reciprocal ences between infected and uninfected individuals.
crosses between infected and uninfected strains Secondly, the two treated lines may differ genetically (Table 2) revealed a significant association between because of stochastic effects of small population size fertility and the presence of infection in males (diff.
following tetracycline exposure. Thirdly, antibiotic 21 = 11.53, P0.005), with 43% of 70 uninfected toxicity has an effect on the survival of eggs and males failing to produce pupae compared to only The Genetical Society of Great Britain, Heredity, 81, 254–260.
WOLBACHIA IN STALK-EYED FLIES 257
Table 1 PCR amplification of Wolbachia ftsZ gene in 17
17% of 70 infected males. No significant association was detected between fertility and presence of theinfection in females (diff. 21 = 0.55, P0.10) or fertility and number of generations of tetracyclineexposure (diff. 21 = 3.55, P0.05). Consequently, results from the two levels of tetracycline exposure were combined in subsequent analyses of fertility.
After recovery of Wolbachia in a subset of anti- biotic-treated individuals, the number of infertile males decreased from 43% to 18% (Table 3). A multiway contingency table analysis revealed a signi- ficant association between fertility and the presence of Wolbachia in males (diff. 21 = 6.66, P0.01), but not in females (diff. 21 = 4.09, P0.10). This analy- sis also indicated that the fertility effect observed in the first set of crosses was not present in the second set where there was recovery of Wolbachia (diff.
Analysis of variance failed to reveal any significantdifferences (F3,87 = 1.7, P = 0.17) in pupal production *Positive amplifications are Type A.
between fertile reciprocal crosses (Table 4).
However, there was a significant difference Table 2 Fertility of reciprocal crosses between strains of Sphyracephala beccarii
infected and uninfected with Wolbachia
Table 3 Comparison of the fertility of reciprocal crosses between strains of
Sphyracephala beccarii infected and uninfected with Wolbachia. Wolbachia (ǹ)
strains treated with tetracycline had subsequent recovery of Wolbachia
Wolbachia (), absent; (ǹ), present. I, infected (not treated with tetracycline);T, treated with tetracycline. Values are totals from Table 2.
The Genetical Society of Great Britain, Heredity, 81, 254–260.
258 A. R. HARIRI ET AL.
Table 4 Mean daily pupal production (<SE) per pair for all fertile reciprocal
crosses between strains of Sphyracephala beccarii infected and uninfected with
(F1,89 = 5.0, P = 0.03) in mean pupal production divergence in these species is needed to differentiate between lines receiving different levels of tetra- between these two potential routes of infection.
cycline exposure. Mean daily pupal production per Our results did not reveal any differences in pupal pair was 11.3<1.3 (SE) in lines with one generation production between fertile crosses. Specifically, of tetracycline exposure and 8.0<0.5 in lines with fertile crosses between uninfected females and two generations of exposure. There was no inter- infected males, which have significantly lower fecun- action between cross type and level of tetracycline dity in systems exhibiting CI, did not produce signifi- exposure for pupal production (F1,3 = 0.8, P = 0.47).
cantly different levels of pupae from all other PCR analysis of uninfected females used in all crosses. Thus, we can conclude that Wolbachia in crosses confirmed that these treated strains were this species do not cause CI or that the level of CI is sufficiently weak not to be detected by this assay.
The absence of CI in this system is not surprising.
For example, although some Wolbachia-infected Discussion
populations of Drosophila simulans exhibit CI, others The pattern of Wolbachia infection in the 17 species do not (Hoffmann et al., 1996).
of stalk-eyed flies screened, with three of the four The observed reduction in fecundity in lines positive amplifications occurring in the genus Sphyr- receiving two generations of tetracycline exposure in acephala, suggests the existence of a unique relation- comparison to those receiving only one, is most ship between the microbe and these species.
likely an artifact of the crosses being conducted at Assuming the overall probability of infection in two different times. Slight environmental differ- diopsids is 23% (4/17), the probability that at least ences, such as media quality or laboratory tempera- three of four Sphyracephala harbour the infection by ture, could translate into significant effects on both chance is only 0.04. There are two possible scenarios frequency of mating and survival of offspring.
which can explain this nonrandom pattern of infec- Interestingly, the results of our crosses are consist- tion. First, the Wolbachia may have been acquired ent with an association between the microbes and prior to the divergence of the Sphyracephala genus enhanced male fertility. Approximately 43% of unin- and then subsequently lost in S. detrahens. Secondly, fected males exhibit infertility compared to only given the high rate of horizontal transmission of 17% of infected males. This pattern is also evident Type A Wolbachia, the flies in this genus may have a in treated flies that did not lose Wolbachia compared predisposition for horizontal acquisition of the to those that did. Positive effects of Wolbachia on microbe and have been infected independently of host productivity have been reported for Trichog- each other. Although flies in the genus Sphyrace- ramma bourarachae (Girin & Bouletreau, 1995), phala can be found on several continents, all of the Tribolium confusum (Wade & Chang, 1995), Nasonia infected species are commonly found in dense aggre- vitripennis (Stolk & Stouthamer, 1996) and D. simu- gations near streams (Feijen, 1989). Possible sources lans (Poinsot & Mercot, 1997).
of infection include predatory mites (Johanowicz & The male fertility effect we found in S. beccarii is Hoy, 1995) and parasitoid wasps (Feijen & Schulten, unlikely to be caused by inbreeding depression, line 1981a,b). Determination of Wolbachia ftsZ sequence divergence or antibiotic toxicity. A multiway contin- The Genetical Society of Great Britain, Heredity, 81, 254–260.
WOLBACHIA IN STALK-EYED FLIES 259
gency analysis revealed a significant association References
between fertility and the presence of Wolbachia in BURKHARDT, D. AND MOTTE, I. DE LA 1983. How stalk-eyed males, suggesting that the bacteria, rather than these flies eye stalk-eyed flies: observations and measure- other factors, are responsible for the observed ments of the eyes of Cyrtodiopsis whitei (Diptera, fertility effect. Further evidence against inbreeding Diopsidae). J. Comp. Physiol. A, 151, 407–421.
depression as the cause of the 26% reduction in FEIJEN, H. 1989. Diopsidae. In: Griffiths, G. C. D. (ed.) male fertility comes from a study of artificial selec- Flies of the Nearctic Region, pp. 1–122. E. Schweizer- tion in another stalk-eyed fly, Cyrtodiopsis dalmanni bart’sche Verlagsbuchhandlung, Stuttgart.
(Wilkinson et al., 1998). After 22 generations of FEIJEN, H. AND SCHULTEN, G. G. M. 1981a. Egg parasitoids selection with an effective population size of 29 (Hymenoptera: Trichogrammatidae) of Diopsis macro-
phthalma (Diptera: Diopsidae). Neth. J. Zool., 31,
(coefficient of inbreeding estimated to be 53%), male fertility declined 10% in comparison to an FEIJEN, H. AND SCHULTEN, G. G. M. 1981b. Egg parasitoids outbred stock population (Wilkinson, unpubl. data).
of rice pests in Malawi, East Africa. Int. Rice Res. In contrast, a comparable estimate of the inbreeding Newsl., 6, 17–18.
coefficient for the S. beccarii used in the reciprocal FIENBERG, S. E. 1981. The Analysis of Cross-Classified Cate- crosses reported here was 5%. Nevertheless, it gorical Data. MIT Press, Cambridge, MA.
remains possible that other genetic changes in the FRANK, S. A. 1996. Host-symbiont conflict over the mixing flies could have caused a paternal fertility effect.
of symbiotic lineages. Proc. R. Soc. B, 263, 339–344.
Introgression of the uninfected genotype into the infected background is needed to address this possi- associated variation in host infestation efficiency in aparasitoid wasp, Trichogramma bourarachae (Hymenop- tera: Trichogrammatidae). Experientia, 51, 398–401.
One possible explanation for the male fertility HOFFMANN, A. A. AND TURELLI, M. 1988. Unidirectional effect in S. beccarii is that the bacteria were origin- incompatibility in Drosophila simulans: inheritance, ally CI producers which spread to near fixation, after geographic variation and fitness effects. Genetics, 119,
which the host genome accommodated to their presence by adjusting spermatogenesis to reduce the HOFFMANN, A. A., CLANCY, D. J. AND DUNCAN, J. 1996.
negative effects of CI in males. By eliminating the Naturally-occurring Wolbachia infection in Drosophila microbes with antibiotics, spermatogenesis may have simulans that does not cause cytoplasmic incompati- been altered causing reduced fertility. Turelli (1994) bility. Heredity, 76, 1–8.
has suggested that the host genome can be selected HOFFMANN, A. A., TURELLI, M. AND HARSHMAN, L. 1990.
Factors affecting the distribution of cytoplasmic incom- to suppress microbial effects in males, whereas patibility in Drosophila simulans. Genetics, 126,
suppression in females is generally not favoured. In addition, recent molecular studies have illustrated that Wolbachia interact with centrosomal microtu- evidence for A-Wolbachia endosymbiont in the preda- bules and host proteins, both important in game- tory mite Metaseiulus occidentalis. J. Cell. Biochem., 21,
togenesis (Kose & Karr, 1995). These interactions may provide a potential mechanism by which Wolba- KOSE, H. AND KARR, T. L. 1995. Organization of Wolbachia chia alter the reproductive biology of their hosts.
pipientis in the Drosophila fertilized egg and embryo Studies designed to determine how the bacteria revealed by an anti-Wolbachia monoclonal antibody.
Mech. Dev., 51, 275–288.
influence spermatogenesis are needed to confirm LORCH, P., WILKINSON, G. AND REILLO, P. 1993. Copulation duration and sperm precedence in the stalk-eyed fly
Cyrtodiopsis whitei (Diptera: Diopsidae). Behav. Ecol.
Sociobiol., 32, 303–311.
O’NEILL, S. L. 1995. Wolbachia pipientis: symbiont or para- We would like to thank Marion Kotrba, Daven site? Parasitol. Today, 11, 168.
Presgraves and Paul Reillo for collection of flies; POINSOT, D. AND MERCOT, H. 1997. Wolbachia infection in Sharvari Bhatt and Lili Crymes for maintenance of Drosophila simulans: does the female host bear a
physiological cost? Evolution, 51, 180–186.
laboratory populations and Renee Goodwin for PRESGRAVES, D. C., SEVERANCE, E. AND WILKINSON, G. S.
PCR assays; Daven Presgraves and two anonymous 1997. Sex chromosome meiotic drive in stalk-eyed flies.
reviewers for helpful comments on earlier versions Genetics, 147, 1169–1180.
of this manuscript. This research was supported by ROUSSET, F., BOUCHON, D., PINTUREAU, B., JUCHAULT, P.
grants from the National Science Foundation to responsible for various alterations of sexuality in arthro- The Genetical Society of Great Britain, Heredity, 81, 254–260.
260 A. R. HARIRI ET AL.
pods. Proc. R. Soc. B, 250, 91–98.
WERREN, J. AND JAENIKE, J. 1995. Wolbachia cytoplasmic STEYSKAL, G. 1972. A catalogue of species and key to incompatibility in mycophagous Drosophila and their genera of the family Diopsidae. Stuttg. Beitr. Naturkd., relatives. Heredity, 74, 320–326.
WERREN, J., GUO, L. AND WINDSOR, D. W. 1995b. Distribu- STOLK, C. AND STOUTHAMER, R. 1996. Influence of a cyto- tion of Wolbachia in neotropical arthropods. Proc. R. plasmic incompatibility-inducing Wolbachia Soc. B, 262, 147–204.
fitness of the parasitoid wasp Nasonia vitripennis. Proc. WERREN, J., ZHANG, W. AND GUO, L. 1995a. Evolution and Exp. Appl. Ent., 7, 33–37.
phylogeny of Wolbachia: reproductive parasites of STOUTHAMER, R., BREEUWER, J., LUCK, R. AND WERREN, J.
Proc. R. Soc. B, 261, 55–71.
1993. Molecular identification of microorganisms assoc- WILKINSON, G. S. 1993. Artificial selection alters allometry iated with parthenogenesis. Nature, 361, 66–68.
in the stalk-eyed fly Cyrtodiopsis whitei (Diptera: TURELLI, M. 1994. Evolution of incompatibility-inducing Genet. Res., 62, 213–222.
microbes and their hosts. Evolution, 48, 1500–1513.
WILKINSON, G. S., PRESGRAVES, D. C. AND CRYMES, L. 1998.
WADE, M. AND CHANG, N. 1995. Increased male fertility in Male eye span in stalk-eyed flies indicates genetic Tribolium confusum beetles after infection with the quality by meiotic drive suppression. Nature, 391,
intracellular parasite Wolbachia. Nature, 373, 72–74.
WERREN, J. 1997. Biology of Wolbachia. Ann. Rev. Ent., 42,
WILKINSON, L. 1989. SYSTAT: the system for statistics. Systat, The Genetical Society of Great Britain, Heredity, 81, 254–260.
Allard, R., Marshall, M., Plante, M-C. (1992). Intensive follow-up does not decrease the risk of repeat suicide attempts. Suicide and Life threatening behaviour, 22(3), 303-314. Andersen, U.A, Andersen, M., Rosholm, J.U., & Gram, L.F. (2001). Psychopharmacological treatment and psychiatric morbidity in 390 cases of suicide with special focus on affective disorders. Acta Psychiatrica Scand