Student Short review

Fly 4:1, 80-87; January/February/March 2010; © 2010 Landes Bioscience

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Spiroplasma are one of only two heritable bacterial endosymbi-onts to infect Drosophila.1 Less studied than the other well-known endosymbiont, Wolbachia,2 the evolutionary consequences of har-boring Spiroplasma are unclear. In general, heritable endosymbi-onts can have myriad effects on their insect hosts, ranging from mutualistic to parasitic, with the potential to dramatically affect their hosts’ evolution. These bacteria are maternally transmitted, and as such their survival is intimately coupled to that of their hosts. Thus, theory predicts that they be beneficial partners; how-ever, these bacteria can also increase their own fitness at the cost of their host by manipulating the host’s reproduction to favor the production of infected females.3-6

In Drosophila, the effects of such endosymbionts are only beginning to be understood, though considerable progress has been made characterizing Wolbachia infections. According to a recent survey of GenBank, Wolbachia infects at least 36 species of Drosophila. Historically famous as a reproductive manipula-tor, Wolbachia causes such phenotypic effects as cytoplasmic incompatibility, whereby an uninfected female produces no off-spring when mated to an infected male, consequently increas-ing the infection prevalence, as infected females have no such incompatibility.2 Wolbachia can also cause male-killing, where the males die during embryogenesis, presumably to the advantage of

their infected sisters. Recently, however, several beneficial aspects of Wolbachia infection in Drosophila are under investigation: Wolbachia has been shown to confer protection against certain RNA viruses,7,8 as well as provide nutritional supplementation in some species of Drosophila.9

Similar to Wolbachia, Spiroplasma is a heritable bacterial endo-symbiont, transmitted transovarially from mother to offspring, which causes male-killing in some species of Drosophila; however, other species of Drosophila harbor Spiroplasma that do not cause male-killing, and no obvious phenotypic effects of this infec-tion have yet been found.10-12 As male-killers, Spiroplasma infec-tions in Drosophila have been known for over 50 years;12 though they were originally referred to as SROs (Sex Ratio Organisms) until technological advances allowed for their correct identifica-tion. The non-male-killing Spiroplasma, with a lack of obvious phenotype, were discovered much later,10 and only recently has the full extent of Spiroplasma infections in Drosophila started to be recognized. Extensive screening has revealed at least six-teen Drosophila species infected with genetically diverse strains of these bacteria. Within natural populations prevalence varies greatly, with an infection rate of up to 85% of non-male-killing Spiroplasma in both males and females in one Drosophila popu-lation.13 Substantial progress has been made in understanding this symbiosis by exploring the incidence and prevalence of infection, the genetic diversity of Drosophila Spiroplasma, the mechanisms of Spiroplasma infection, the modes of bacterial transmission and the fitness effects of the bacteria on their Drosophila hosts. However, further investigation of many aspects of this relation-ship is necessary to appreciate the forces driving the distribution of Spiroplasma among Drosophila as well as the evolutionary consequences of this symbiosis.

Spiroplasma are Prevalent Arthropod-Associated Bacteria

Spiroplasma are obligate host-associated bacteria and may be one of the most diverse taxa of bacteria due to their extensive host range.14 They are small, helical, motile bacteria lacking a cell wall, phylogenetically gram-positive.144 Besides Drosophila, Spiroplasma are also male-killing heritable endosymbionts in sev-eral species of ladybird beetle16-19 and a butterfly (Danaus chry-sippus).20 Spiroplasma have also been found in several species of spiders,16,21 where they may play a role in sex-ratio distortion. More commonly, Spiroplasma have been found as gut bacteria in

the Spiroplasma heritable bacterial endosymbiont of drosophila

tamara S. haselkorn

Section of Ecology; Behavior and Evolution; Division of Biological Sciences; University of California, San Diego; La Jolla, CA USA

Key words: Drosophila, Spiroplasma, Wolbachia, bacterial endosymbiont, innate immunity, bacterial infection

Correspondence to: Tamara S. Haselkorn; Email: thaselko@ucsd.eduSubmitted: 09/15/09; Revised: 11/20/09; Accepted: 12/05/09Previously published online:www.landesbioscience.com/journals/fly/article/10883

Since the discovery of the small, gram-positive bacteria, Spiro-plasma, as a sex-ratio distorting agent in drosophila over 50 years ago, substantial progress has been made in understand-ing the relationship of this bacteria with its insect host. thus far, Spiroplasma have been found as heritable endosymbionts in six-teen different species of drosophila. in some species these bac-teria cause a male-killing phenotype, where the males die during embryogenesis. in other species, however, Spiroplasma does not cause male-killing, and its fitness effects are unclear. Though recent research has identified multiple factors that affect the prevalence and transmission of Spiroplasma in drosophila populations, much work remains to fully characterize this symbiosis. Spiroplasma is the only identified heritable bacterial endosymbiont of Droso-phila, other than wolbachia, and can serve as a useful as model for elucidating the nature of insect/bacterial interactions.

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not appear to harm its insect vector; in fact, Spiroplasma can be leafhopper mutualists, conferring cold tolerance to their host.35

Incidence and Prevalence of Spiroplasma Infection in Drosophila

Sex Ratio Organisms, later identified as Spiroplasma, were first observed in four species of neotropical Drosophila in the willistoni group of the subgenus Sophophora: D. willistoni (infected with the willistoni sex ratio organism [WSRO]), D. nebulosa (NSRO), D. equinoxalis and D. paulistorum12 (Fig. 1). More recently, a male-killing Spiroplasma was found in a D. melanogaster from Brazil36 as well as a D. melanogaster from Africa.37 Prevalences of Spiroplasma infection in natural populations of D. nebulosa and D. melanogaster find a range of 1–6%, varying seasonally.12,38 Male-killing Spiroplasma infections also likely occur in the tripunctata radiation of Drosophila, in D. neocardini, D. ornatifrons and D. paraguayensis.39

Infection incidence and prevalence of the non-male killing Spiroplasma in Drosophila are higher. In addition to the early dis-covery of non-male-killing Spiroplasma infecting 46% of a D. hydei population,10 recent screening of five other D. hydei populations

many species of arthropods and are assumed to be commensals, attaching to gut epithelial cells with no adverse effects.22

Several are pathogenic, however, and the transition to patho-genicity may be tied to the ability to cross the insect midgut barrier and multiply in the host hemolymph and other tissues. As pathogens, they have been well-characterized in shrimp,23 crabs24,25 and bees,26,27 causing high levels of mortality with rapid onset of s ymptoms. Routes of pathogen transmission are under current investigation, though it is speculated that the Spiroplasma are ingested,28 implicating horizontal rather than vertical transmis-sion. Bees may acquire Spiroplasma infection while foraging, as flower surfaces serve a reservoir.29

Furthermore, as agronomically important plant pathogens causing citrus stubborn disease (S. citri),30,31 and corn stunt (S. kunkelii),32,33 their small AT-rich genomes (∼1–2 Mb) have been sequenced and their mechanisms of pathogenicity are being elucidated.30,34 Plants infected with the diseases, transmitted via a leafhopper insect vector, suffer leaf yellowing, stunted growth and other deformations. Spiroplasma mutagenesis studies reveal that inactivation of the fructose utilization operon ameliorates these pathogenic effects, while inactivation of other key proteins limits the transmissibility of the disease. Spiroplasma replication does

Figure 1. drosophila species infected with Spiroplasma. Species are listed by species group, and the type of Spiroplasma infection is noted. Prevalence of infection is listed for each species where multiple individuals per population were sampled. The Drosophila phylogeny of species groups is modified from Mateos et al. 2006, originally based on Markow and o’Grady 2006.

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quinaria species group is infected13 as well as D. atripex, D. ananassae, D. simulans and D. melanogaster13,36,37,40 of the melanogaster species group in the subgenus Sophophora. Infection prevalences of the non-male-killing Spiroplasma vary greatly, with a low prevalence in D. melanogaster and D. simulans (<1%), up to a prevalence of 85% for one population of D. mojavensis.13 Sixteen species of Drosophila have been found to harbor Spiroplasma so far, and it is likely that with screening of additional Drosophila from other geographic locations more infected species will be found.

The Diversity of Spiroplasma Infecting Drosophila

Drosophila Spiroplasma were first character-ized using serological methods. Though dif-ficult to culture, the Spiroplasma infecting D. willistoni was grown in an insect cell line media and named S. poulsonii. Cultivated Spiroplasma, however, lose the ability to be vertically transmitted when reintroduced into Drosophila,41 preventing further characteriza-tion by traditional microbiological methods. Later, with the advent of molecular methods to identify bacteria using 16S ribosomal RNA gene (rRNA), S. poulsonii was found to fall in the Spiroplasma citri-poulsonii clade, closely related to Spiroplasma infecting plants. The Spiroplasma infecting two of the species of the D. willistoni group and D. melanogaster36,37,42 are very closely related. Later sequencing of the non-male-killing Spiroplasma infecting the D. hydei in Japan found that this strain was nearly identical at the 16S rRNA locus to the male-killing Spiroplasma infecting D. nebu-losa.11 Early screening hinted at a greater diver-sity of Spiroplasma infecting Drosophila,1 and a more complete sampling exposed the extent of this diversity.13,40

A multilocus phylogenetic analysis of Spiroplasma infecting 65 individuals of nine

species of Drosophila revealed that these Spiroplasma fall into four distinct clades40 (Fig. 2). The male-killing Spiroplasma of the willistoni group and the non-male-killing Spiroplasma infecting D. hydei and D. simulans fall into the poulsonii clade, with identi-cal haplotypes at the 16S rRNA locus. More rapidly evolving loci provided greater resolution, with the male-killing Spiroplasma grouping together with strong bootstrap support, separate from the non-male-killing Spiroplasma. These poulsonii Spiroplasma group most closely with the Spiroplasma found on flower surfaces (S. insolitum) and shrimp pathogens (S. penaei). The Spiroplasma infecting D. wheeleri, D. aldrichi, D. mojavensis, and four indi-viduals of D. hydei fall into the citri clade of Spiroplasma, showing

in Japan has found infection prevalences ranging from 23–66%.11 Subsequent screening of over 200 species of Drosophila in the Drosophila Species Stock Center and in more than 19 natural populations throughout southwestern North America have found seven additional species infected with Spiroplasma that do not seem to cause male-killing.1,13 This is likely an underestimate, as screens of Stock Center lines miss infections that have been lost over time due to imperfect vertical transmission, as well as any Drosophila with infected with male-killing bacteria, which could not have been established as an isofemale line. Within the repleta species group of the subgenus Drosophila there are infected D. aldrichi, D. wheeleri and D. mojavensis.1,13 The mushroom-feeding D. tenebrosa of the

Figure 2. Phylogeny of Spiroplasma infecting drosophila and other organisms. this 16s rrnA phylogeny was reconstructed using the nJ distance-based methods as described in haselkorn et al. 2009. Bootstrap support (1,000 replicates) is noted. the four major clades into which droso-phila Spiroplasma (in bold) fall are denoted with vertical bars.

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infection with such similar Spiroplasma resulted from an ancient infection followed by co-divergence.

The frequency of horizontal transmission has yet to be deter-mined. Possible mechanisms for intra- and inter-specific hori-zontal transmission may involve mites or ingestion. Generalist ectoparasitic mites have been shown to take up Spiroplasma from D. nebulosa and transfer it at a low frequency to D. willistoni under laboratory conditions.49 There is also one report of hori-zontal transmission of Spiroplasma by ingestion in Drosophila,50 though several attempts to repeat the experiment failed to find the same result.12,44,51 If horizontal transmission is common, then co-infections of different Spiroplasma strains in populations could lead to recombination in the Spiroplasma genomes, as is com-monly seen in Wolbachia.52 The multi-locus phylogenetic analy-sis of Drosophila Spiroplasma, however, revealed no evidence for recombination within or between loci.40 This may not indicate the absence of co-infection, since Drosophila Spiroplasma, like some other Spiroplasma, may lack the recA gene, which is nec-essary for recombination. Evidence for recombination occurring via other mechanisms, however, exists in these recA deficient Spiroplasma.53,54

While horizontal transmission is important in establishing interspecific infections, it is unclear what role, if any, horizontal transmission plays in maintaining infection within a species. It is possible that Spiroplasma may be existing as commensals, and that the infection's imperfect vertical transmission may be counterbal-anced by some horizontal transmission.55 A lack of Spiroplasma genetic variation within most Drosophila species makes it difficult to answer this question, since co-infections and/or recombina-tion would not be detected. However, within populations of D. hydei and of D. mojavensis, there is not a tight correlation between Spiroplasma infection and mitochondrial haplotype, as expected with strict vertical transmission, suggesting that horizontal trans-mission may be occurring at the population level.40

Mechanisms of Spiroplasma Infection

The maintenance and effects of male-killing Spiroplasma infec-tions have been studied extensively. In the 1960's, the Spiroplasma infecting D. nebulosa (NSRO) and D. willistoni (WSRO) were injected in the several different D. melanogaster lines to be studied in the laboratory in an attempt to understand the process of male-killing.56 While maintaining male-killing Spiroplasma infections in the lab, a non-male-killing variant arose (NSRO-A),57 which pro-vided an opportunity for comparison. Anbutsu and Fukatsu58 mea-sured the bacterial titer using quantitative PCR, of both NSRO and NSRO-A over the course of development of D. melanogaster. The titer of NSRO increased exponentially from eclosion to three-week old adults, while the titer of NSRO-A remained the same during this same time period. This significant titer difference, along with the observation that young NSRO infected female flies produce males when their Spiroplasma titers are similar to that of NSRO-A females, led them to propose the bacterial threshold density hypothesis, that a minimal bacterial titer was necessary for the occurrence of male-killing. This hypothesis is in concordance with early studies on the dynamics of Spiroplasma infections, as

approximately 3% sequence divergence at the 16S rRNA locus from the poulsonii clade, and most closely related to the plant pathogenic Spiroplasma (S. citri and S. kunkelii). The Spiroplasma infecting D. atripex and D. ananassae fall into the ixodetis clade, most closely related to S. ixodetis infecting ticks, and the male-killing Spiroplasma infecting a ladybird beetle (Anisosticta novemdecimpunctata) and butterfly (Danaus chrysippus). These ixodetis Spiroplasma show close to 10% sequence divergence at the 16S rRNA locus relative to the citri-poulsonii clade. Finally, the Spiroplasma infecting D. tenebrosa are most closely related to the ixodetis clade, but exhibit 3% sequence divergence at the 16S rRNA locus and are distinct from any other Spiroplasma sequenced thus far.

Modes and Routes of Spiroplasma Transmission

As a heritable endosymbiont, vertical transmission of Spiroplasma is a defining feature of this symbiosis. Spiroplasma are incor-porated into Drosophila oocytes early during vitellogenesis, resulting in transovarial transmission.43 For non-male-killing Spiroplasma, males as well as females are infected. The fidelity of vertical transmission of Spiroplasma can be quite high, near 100%, though it can be affected by female age, host genotype, Spiroplasma strain and temperature. When infected female D. nebulosa are mated to males of different genetic backgrounds, Spiroplasma infections were lost at different rates.12 Different lines of D. willistoni infected with dissimilar Spiroplasma strains showed an increase in infection loss with female age in one host line but not others. Rates of loss varied depending on the Spiroplasma strain, suggesting a complex interaction-phenotype between host genetic background and Spiroplasma strain.44 In several cases Spiroplasma infections in D. melanogaster, D. nebu-losa and D. hydei were lost at low temperatures (18°C),45-47 while in others loss was associated with higher temperatures.45 In the case of low temperatures, the Spiroplasma density within the fly decreases, which may be the cause of loss. At higher tempera-tures, however, density does not decrease and the reason for loss under these conditions is unclear. Interestingly, populations of D. hydei that maintain Spiroplasma at relatively high prevalences routinely experience extreme temperature fluctuations, implying the existence of a conditional fitness benefit and/or the occur-rence of re-acquisition via horizontal transmission to explain the maintenance of infection.45

Similar Spiroplasma haplotypes infect very divergent hosts, and closely related Drosophila are infected with very divergent Spiroplasma, indicating that horizontal transmission likely plays an important role in this symbiosis in establishing infections.40 The existence of Drosophila Spiroplasma falling into four distinct clades, each of which is most closely related to Spiroplasma infect-ing other organisms, suggests at least four separate introductions of Spiroplasma into Drosophila. Drosophila hydei is infected with two very different Spiroplasma, likely representing an additional introduction. Furthermore, Spiroplasma with similar or identical haplotypes infect D. melanogaster from both Brazil and Africa,37 D. simulans, D. nebulosa and D. hydei.40 As D. melanogaster and D. hydei are at least 50 million years diverged,48 it is unlikely that

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the original infection followed by replacement with a different infection.71

The factors underlying the host specificity of Spiroplasma are not well understood. Many male-killing Spiroplasma infections were transferred to new Drosophila hosts, including D. melano-gaster and D. pseudoobscura among others, and the male-killing phenotype was often still expressed.12 In some instances, however, the Spiroplasma infection reduced the viability, fecundity, and fertility of their artificial D. melanogaster host.60 In many cases, such as for the D. melanogaster NSRO, these newly established infections could be maintained easily in the lab. Other times such transfections were not so easily established, as when the non-male killing Spiroplasma of D. hydei was transferred to D. melanogaster. No male-killing was observed, but the flies suffered early mortal-ity.11 Bacterial strain also plays a role, as the WSRO Spiroplasma transfections into different species appear much less stable than NSRO transfections.12

It is unclear what role the Drosophila immune system plays in maintenance of this symbiosis. Transfer of several different types of non-Drosophila Spiroplasma into D. pseudoobscura led to vari-ous results, including clearance of Spiroplasma infection, replica-tion within host with no adverse effects, and replication within host and pathogenic effects, depending on the Spiroplasma type.51 Only one study has examined immune response to Spiroplasma infection, and while Spiroplasma infection did not elicit an immune response, bacterial titer was suppressed by ectopic activa-tion of antimicrobial genes.74 It is unknown if Spiroplasma merely escapes detection because it lacks typical immune elicitors such as a bacterial cell wall, or if it interacts in any way with the host immune system.

Spiroplasma appear to be most prevalent in the Drosophila hemolymph. Early studies determined the presence of infection by observing the Spiroplasma in the hemolymph under dark field microscopy.12 When the Spiroplasma titers of different Drosophila host tissues, including the hemolymph, fat body, intestine, and ovary, were measured using quantitative PCR, Spiroplasma pro-liferation in the extracellular environment of the hemolymph most closely matched that of the whole body Spiroplasma dynamics. The bacteria were present, though, at lower levels in the other host tissues.75 These infection dynamics are in contrast to those of Wolbachia infection, which is primarily intracellular and not found in the insect hemolymph. When Wolbachia and Spiroplasma coinfect flies, the density of Spiroplasma is unaffected by Wolbachia, whereas the density of Wolbachia is lowered by the presence of Spiroplasma.76 Thus, the extent to which Wolbachia and Spiroplasma coinfections occur in natural populations may be low. Montenegro et al.36 found four individuals of D. mela-nogaster from Brazil that were co-infected; however, coinfection of the non-male-killing Spiroplasma and Wolbachia has not been observed in natural populations screened to date.

Other Fitness Effects and Evolutionary Implications

Male-killing infections in insects have a range of effects and evo-lutionary implications. Infections at high prevalences causing population-level sex ratio distortions can lead to changes in host

observed increases in Spiroplasma presence in the hemolymph correlated with decreases in the production of male offspring.59 Spiroplasma titers in the naturally occurring non-male killing D. hydei were also shown to be lower than the male-killing infections in adults one week after eclosion.11

Though the precise male-killing mechanism is unknown, many techniques have been employed to gain a better understand-ing. Early studies using electron microscopy established that male death occurred during early embryogenesis before gastrulation, noting irregular movement of chromosomes during meiosis in infected embryos,60 with a lack of cell differentiation in the pri-mordial nervous system tissues.61,62 Recent studies have identified the initial target as the sex-determining pathway, since when any of the five interacting genes of the dosage compensation complex are inactivated, male-killing does not occur.63 In naturally-infected D. nebulosa, male-killing occurs during a narrow developmental time period following the formation of the dosage compensa-tion complex, with widespread apoptosis and arrested develop-ment 10–12 hours into embryonic development.64 The NSRO Spiroplasma strain in D. melanogaster, however, caused both the early male-killing, as described above, as well as late male-killing (death during the late larval stages).65 The occurrence of early ver-sus late male-killing depended on maternal host age, and may be related to the density of Spiroplasma as well as whether or not the Spiroplasma strain is studied in its natural host.

Host genetic background also affects the Spiroplasma male-killing phenotype. In many instances, Drosophila harboring male-killing infections have been maintained in the laboratory using males from an uninfected line, and the efficiency of male-killing was maintained near 100% for many generations. However, this efficiency of male-killing varied, depending on the male strain used.12 To further explore this effect, Kageyama et al.66 crossed Spiroplasma-infected D. melanogaster females to males of ten dif-ferent genetic backgrounds, and found male-killing suppressed in two lines. Spiroplasma infection, though, was maintained at high bacterial densities, suggesting the existence of suppressors acting on the male-killing effectors directly.

Phage, or other bacterial extrachromosomal elements, may play an important role in this symbiosis.41,67,68 Some evidence sug-gests they may be involved in the male-killing mechanism itself. In one artificial experimenal transfer of WSRO to D. robusta, lev-els of Spiroplasma in the hemolymph decreased to non-detectable levels, while male-killing still occurred, implying that the male-lethal agent may be separate from the bacteria.68,69 The differ-ent Spiroplasma strains, both male-killing and non-male killing, harbor different combinations of two to four different phage. There are, however, no obvious correlations between presence of certain phage and the male-killing phenotype.10,70-72 Phage may also affect the distribution of Spiroplasma among Drosophila, as these viruses lyse Spiroplasma from different Drosophila species or geographically distinct Spiroplasma haplotypes of the same spe-cies.12,70 For different combinations of male-killing (S. poulsonii) haplotypes, artificial super-infection resulted in various amounts of bacterial lysing, observable using a dark field microscope and mixing hemolymph samples on a slide.12,73 Co-infection of two different Spiroplasma in vivo sometimes resulted in clearance of

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other constraints. Additionally, there may be costs to harboring Spiroplasma that are not yet apparent, as has been demonstrated for other facultative endosymbionts.83

Alternatively, many of the above studies imply the existence of one or several conditional fitness benefits that are not obvi-ous under standard laboratory conditions. Examples of condi-tional fitness benefits observed in other bacterial endosymbionts include nutritional supplementation,84 resistance to parasitoid wasps85 or predators,86 resistance to RNA viruses,7 and thermal tolerance.84 Such fitness benefits could have large ecological and evolutionary consequences, allowing Drosophila to successfully explore new habitats and extend their geographic range. Fitness effects due to interactions with other bacteria, such as Wolbachia, with known fitness effects, may also be biologically significant. For Spiroplasma, their effects on their hosts need to be explored under such stresses and conditions to determine if any such fitness benefits exist.

To understand the effects of Spiroplasma on Drosophila, it is apparent that this symbiosis must be studied in the context of the natural ecology of both organisms. The ecology of many of the infected Drosophila species is well known (reviewed in ref. 87), and the dynamics of Spiroplasma infection may become obvious when studied in their natural habitat. Moreover, the interactions may vary depending on Spiroplasma strain and Drosophila host, given the diversity of Drosophila species infected and the variety of Spiroplasma strains. Furthermore, the genetic and genomic tools of both Drosophila and Spiroplasma provide a great opportunity to make major advances in the understanding of the interactions of an insect host and its heritable endosymbiont; to further understand how these bacteria can evade the host immune system and survive in the hemolymph, as well as explore how such bacteria control their replication and enter different host tissues. With the advent of new sequencing technologies, comparative genomic analysis of Spiroplasma infecting Drosophila, as well as Spiroplasma of other hosts, will become more feasible. Genomic analysis can elucidate the factors affecting the evolution and adaptation of these bacteria to their diverse hosts, including the identification of genes that may be involved in their mutualistic or pathogenic lifestyles as well as those involved in their various modes of transmission. Such advances, in combination with previous research, will help resolve the mysteries surrounding Spiroplasma infection and further elu-cidate its evolutionary importance in Drosophila.

Note added in proof

A recent study by Jaenike et al.88 has found an additional spe-cies of Drosophila infected with non-male-killing Spiroplasma, D. neotestacea. In natural populations of this species co-infections of Spiroplasma and Wolbachia are common and may reflect a mutualistic interaction between the two bacteria

Acknowledgements

I would like to thank Dr. Therese Markow for guidance during the preparation of this manuscript, Dr. Luciano Matzkin and Sarah Johnson for useful discussion, as well as Dr. John Jaenike and an anonymous reviewer for very helpful comments.

sexual selection and mating behavior, as well as select for male-killing resistance genes. They can decrease population density, lowering the effective population size and reducing genetic varia-tion by cutting offspring production of infected females in half. At the extreme, these infections could cause population extinc-tion.77 Yet this is a phenotype caused by multiple bacterial taxa,78 and such male-killing infections will spread if they increase fit-ness of infected females. This occurs if daughters benefit from the death of their brothers, either by gaining more access to limited resources (reduced larval competition), or through the preven-tion of inbreeding.78,79 Given that current sampling indicates that male-killing Spiroplasma prevalence in Drosophila is low, the evo-lutionary implications are difficult to discern. The low prevalence may reflect the ecology and life history of the infected Drosophila species, in which infected females may not receive fitness benefits from male-killing and suggests that male-killing may not be the driving force maintaining this symbiosis.

Attempts to measure the fitness effects of the male-killing Spiroplasma in Drosophila in laboratory settings have produced ambiguous results. D. nebulosa females infected with their natu-ral male-killing Spiroplasma strains mate two days sooner than uninfected females.80 Studies by Ebbert44 showed a similar shift in the timing of reproduction, the effect of which varied with Drosophila line and Spiroplasma strain, but no overall increase in the production of offspring. In fact, there was an overall decrease in fertility and lifespan in infected females. The possible benefit of early reproduction may occur under crowded conditions, though laboratory experiments exploring this situation found infected flies to be at a disadvantage.81 Early reproduction, however, may be advantageous during periods of population expansion. In D. melanogaster there was no measurable fitness effect of Spiroplasma infection on larval competitiveness and adult fecundity, at both low and high larval density conditions.82 No early reproduction in these Spiroplasma-infected lines was observed. Similarly, no apparent differences in survival or reproduction were detected in D. hydei infected with non-male-killing Spiroplasma, and curing D. hydei of Spiroplasma infection resulted in no fitness decrease.11

Given that the fidelity of vertical transmission is imperfect, and that evident fitness effects on the host under laboratory conditions are limited or negative, the persistence of Spiroplasma infections in natural populations remains a mystery. It is possible, as men-tioned previously, that Spiroplasma may be existing as commen-sals, and that the infections imperfect vertical transmission may be counterbalanced by some horizontal transmission.55 Further exploration of the role and mechanisms of horizontal transmis-sion in natural populations could elucidate this possibility. For example, if acquisition by ingestion were possible, then rotting fruits or cacti, which serve as both feeding and larval development sites for many arthropods, could serve as an infection reservoir for Drosophila. Incidence and prevalence in some Drosophila species and not others may reflect either chance acquisition or differences in species immune systems, such that some species are resistant to infection. Species-specific ecology may play a role, possibly preventing Spiroplasma replication due to habitat temperature or

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41. Williamson DL, Sakaguchi B, Hackett KJ, Whitcomb RF, Tully JG, Carle P, et al. Spiroplasma poulsonii sp. nov., a new species associated with male-lethality in Drosophila willistoni, a neotropical species of fruit fly. Int J Syst Bacteriol 1999; 49:611-8.

42. Bentley JK, Hinds G, Hurst GD. The male-killing Spiroplasma of Drosophila nebulosa and Drosophila willistoni have identical ITS sequences. Drosophila Information Service 2002; 85:63-5.

43. Niki Y. Ultrastructural study of the Sex Ratio Organism (SRO) transmission into oocytes during oogenesis in Drosophila melanogaster. Jpn J Genet 1988; 63:11-21.

44. Ebbert MA. The interaction phenotype in the Drosophila willistoni-Spiroplasma Symbiosis. Evolution 1991; 45:971-88.

45. Anbutsu H, Goto S, Fukatsu T. High and low tem-peratures differently affect infection density and vertical transmission of male-killing Spiroplasma symbionts in Drosophila hosts. Appl Environ Microbiol 2008; 74:6053-9.

46. Montenegro H, Klaczko LB. Low temperature cure of a male killing agent in Drosophila melanogaster. J Invertebr Pathol 2003; 86:50-1.

47. Osaka R, Nomura M, Watada M, Kageyama D. Negative effects of low temperatures on the vertical transmission and infection density of a Spiroplasma endosymbiont in Drosophila hydei. Curr Microbiol 2008; 57:335-9.

48. Tamura K, Subramanian S, Kumar S. Temporal pat-terns of fruit fly (Drosophila) evolution revealed by mutation clocks. Mol Biol Evol 2004; 21:36-44.

49. Jaenike J, Polak M, Fiskin A, Helou M, Minhas M. Interspecific transmission of endosymbiotic Spiroplasma by mites. Biol Lett 2007; 3:23-5.

50. Carvalho GG, da Cruz M. Transfer of the “sexratio” factor in Drosophila willistoni by ingestion. Science 1962; 138:51.

51. Williamson DL. Studies on the pathogenicity of Spiroplasma for Drosophila pseudoobscura. Annales de microbiologie 1984; 135:157-62.

52. Baldo L, Bordenstein S, Wernegreen JJ, Werren JH. Widespread recombination throughout Wolbachia genomes. Mol Biol Evol 2006; 23:437-49.

53. Marais A, Bove JM, Renaudin J. Characterization of the recA gene regions of Spiroplasma citri and Spiroplasma melliferum. J Bacteriol 1996; 178:7003-9.

54. Marais A, Bove JM, Renaudin J. Spiroplasma citri virus SpV1-derived cloning vector: Deletion forma-tion by illegitimate and homologous recombination in a Spiroplasmal host strain which probably lacks a functional recA gene. J Bacteriol 1996; 178:862-70.

55. Anderson RM, May RM. The population dynamics of microparasites and their invertebrate hosts. Phil Trans R Soc Lond B 1981; 291:451-524.

56. Sakaguchi B, Poulson DF. Interspecific transfer of the “sex-ratio” condition from Drosophila willistoni to D. melanogaster. Genetics 1963; 48:841-61.

57. Yamada M, Nawa S, Watanabe T. A mutant of SR organism (SRO) in Drosophila that does not kill the nost males. Jpn J Genet 1982; 57.

58. Anbutsu H, Fukatsu T. Population dynamics of male-killing and non-male-killing Spiroplasma in Drosophila melanogaster. Appl Environ Microbiol 2003; 69:1428-34.

59. Williamson DL. Kinetic studies of “sex-ratio” spiro-chetes in Drosophila melanogaster Meigen females. J Invertebr Pathol 1965; 7:493-501.

60. Counce SJ, Poulson DF. Developmental effects of the sex ratio agent in embryos of Drosophila willistoni. J Exp Zool 1962; 151:17-31.

61. Koana T, Miyake T. Effects of the sex ratio organism on in vitro differentiation of Drosophila embryonic cells. Genetics 1983; 104:113-22.

62. Tsuchiyama S, Sakaguchi B, Oishi K. Analysis of gynandromorph survivals in Drosophila melanogaster infected with the male-killing SR organisms. Genetics 1978; 89:711-21.

21. Goodacre SL, Martin OY, Thomas CFG, Hewitt GM. Wolbachia and other endosymbiont infections in spi-ders. Mol Ecol 2006; 15:517-27.

22. Bove JM. Spiroplasma: Infections agents of plants, arthropods and vertebrates. Wiener Klinische Wochenschrift 1997; 109:604-12.

23. Nunan LM, Lightner DV, Oduori MA, Gasparich GE. Spiroplasma penaei sp nov., associated with mortalities in Penaeus vannamei, Pacific white shrimp. Int J Syst Evol Microbiol 2005; 55:2317-22.

24. Wang W, Gu W, Ding Z, Ren Y, Chen J, Hou Y. A novel Spiroplasma pathogen causing systemic infec-tion in the crayfish Procambarus clarkii (Crustacea: Decapod), in China. FEMS Microbiol Lett 2005; 249:131-7.

25. Wang W, Rong L, Gu W, Du K, Chen J. Study on experimental infections of Spiroplasma from the Chinese mitten crab in crayfish, mice and embryonated chickens. Res Microbiol 2003; 154:677-80.

26. Clark TB. Spiroplasma sp. a new pathogen in honey bees. J Invertebr Pathol 1977; 29:112-3.

27. Mouches C, Menara A, Tully JG, Rose DL, McCoy RE, Clarle-Junca P, et al. Spiroplasma apis, a new species from the honeybee (Apis mellifera). Ann Inst Microbiol (Paris) 1983; 134:383-97.

28. Heres A, Lightner DV. Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on mul-tilocus sequence analysis. J Invertebr Pathol 2010; 103:30-5.

29. Clark TB. Spiroplasma: Diversity of arthropod res-ervoirs and host-parasite relationships. Science 1982; 217:57-9.

30. Bove JM, Renaudin J, Saillard C, Foissac X, Garnier M. Spiroplasma citri, a plant pathogenic molligute: rela-tionships with its two hosts, the plant and the leafhop-per vector. Annu Rev Phytopathol 2003; 41:483-500.

31. Markham PG, Townsend R, Bar-Joseph M, Daniels MJ, Plaskitt A, Meddins BM. Spiroplasma are the causal agents of citrus little-leaf disease. Ann Appl Biol 1974; 78:49-57.

32. Davis RE, Worley JF, Whitcomb RF, Ishijima T, Steere RL. Helical flilaments produced by a mycoplasma-like organism associated with corn stunt disease. Science 1972; 176:521-3.

33. Hackett KJ, Clark TB. Ecology of Spiroplasma. In: Whitcomb RF, Tully JG, eds. The Mycoplasmas, Vol V: Spiroplasma, Acholeplasmas and Mycoplasmas of Plant and Arthropods. San Diego, CA: Academic Press 1989; 113-200.

34. Calavan EC, Bove JM. Ecology of Spiroplasma citri. In: Whitcomb RF, Tully JG, eds. The Mycoplasmas, Vol V: Spiroplasma, Acholeplasmas and Mycoplasmas of Plant and Arthropods. San Diego, CA: Academic Press 1989; 425-87.

35. Ebbert MA, Nault LR. Survival in Dalbulus leafhop-per vectors improves after exposure to maize stunting pathogens. Entomologia Experimentalis Et Applicata 2001; 100:311-24.

36. Montenegro H, Solferini VN, Klaczko LB, Hurst GDD. Male-killing Spiroplasma naturally infect-ing Drosophila melanogaster. Insect Mol Biol 2005; 14:281-7.

37. Pool JE, Wong A, Aquadro CF. Finding of male-killing Spiroplasma infecting Drosophila melanogaster in Africa implies transatlantic migration of this endosymbiont. Heredity 2006; 97:27-32.

38. Marques EJ, de Magalhaes LE. Drosophila Information Service 1973; 50:87.

39. Montenegro H, Hatadani LM, Medeiros HF, Klaczko LB. Male killing in three species of the tripunctata radiation of Drosophila (Diptera: Drosophilidae). J Zool Sys Evol Res 2006; 44:130-5.

40. Haselkorn TS, Markow TA, Moran NA. Multiple introductions of the Spiroplasma bacterial endosymbi-ont into Drosophila. Mol Ecol 2009; 18:1294-305.

References1. Mateos M, Castrezana SJ, Nankivell BJ, Estes AM,

Markow TA, Moran NA. Heritable endosymbionts of Drosophila. Genetics 2006; 174:363-76.

2. Werren JH, Baldo L, Clark ME. Wolbachia: mas-ter manipulators of invertebrate biology. Nature Rev Microbiol 2008; 6:741-51.

3. Anderson RM, May RM. Coevolution of Hosts and Parasites. Parasitology 1982; 85:411-26.

4. Bull JJ, Molineux IJ, Rice WR. Selection of Benevolence in a Host-Parasite System. Evolution 1991; 45:875-82.

5. Ewald PW. Host-Parasite Relations, Vectors and the Evolution of Disease Severity. Ann Rev Ecol Sys 1983; 14:465-85.

6. O’Neill S, Hoffmann A, Werren JH. Influential Passengers: Inherited Microorganisms and Arthropod Reproduction. Oxford University Press: New York 1997.

7. Hedges LM, Brownlie JC, O’Neill SL, Johnson KN. Wolbachia and virus protection in insects. Science 2008; 322:702.

8. Teixeira L, Ferreira A, Ashburner M. The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. PLoS Biol 2008; 6:2.

9. Brownlie JC, Cass BN, Riegler M, Witsenburg JJ, Iturbe-Ormaetxe I, McGraw EA, et al. Evidence for metabolic provisioning by a common invertebrate endosymbiont, Wolbachia pipientis, during periods of nutritional stress. PLoS Path 2009; 5:1000368.

10. Ota T, Kawabe M, Oishi K, Poulson DF. Non-male-killing Spiroplasma in Drosophila hydei. J Hered 1979; 70:211-3.

11. Kageyama D, Anbutsu H, Watada M, Hosokawa T, Shimada M, Fukatsu T. Prevalence of a non-male-kill-ing Spiroplasma in natural populations of Drosophila hydei. Appl Environ Microbiol 2006; 72:6667-73.

12. Williamson DL, Poulson DF. Sex Ratio Organisms (Spiroplasma) of Drosophila in The Mycoplasmas III: Plant and Insect Mycoplasmas, Academic Press, New York 1979; 3:176-206.

13. Watts T, Haselkorn TS, Moran NA, Markow TA. Variable incidence of Spiroplasma infections in natural populations of Drosophila species. PloS One 2009; 4:5703.

14. Gasparich GE, Whitcomb RF, Dodge D, French FE, Glass J, Williamson DL. The genus Spiroplasma and its non-helical descendants: phylogenetic clas-sification, correlation with phenotype and roots of the Mycoplasma mycoides clade. Int J Sys Evol Microbiol 2004; 54:893-918.

15. Woese CR, Maniloff J, Zablen LB. Phylogenetic analy-sis of the mycoplasmas. Proc Natl Acad Sci USA 1980; 77:494-8.

16. Duron O, Bouchon D, Boutin S, Bellamy L, Zhou L, Engelstadter J, et al. The diversity of reproductive parasites among arthropods: Wolbachia do not walk alone. BMC Biol 2008; 6:27.

17. Hurst GDD, von der Schulenburg JHG, Majerus TMO, Bertrand D, Zakharov IA, Baungaard J, et al. Invasion of one insect species, Adalia bipunctata, by two different male-killing bacteria. Insect Mol Biol 1999; 8:133-9.

18. Majerus TMO, von der Schulenburg JHG, Majerus MEN, Hurst GDD. Molecular identification of a male-killing agent in the ladybird Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Insect Mol Biol 1999; 8:551-5.

19. Tinsley MC, Majerus ME. A new male-killing parasitism: Spiroplasma bacteria infect the ladybird beetle Anisosticta novemdecimpunctata (Coleoptera: Coccinellidae). Parasitology 2006; 132:757-65.

20. Jiggins FM, Hurst GDD, Jiggins CD, Von der Schulenburg JHG, Majerus MEN. The butterfly Danaus chrysippus is infected by a male-killing Spiroplasma bac-terium. Parasitology 2000; 120:439-46.

www.landesbioscience.com Fly 87

76. Goto S, Anbutsu H, Fukatsu T. Asymmetrical interac-tions between Wolbachia and Spiroplasma endosymbi-onts coexisting in the same insect host. Appl Environ Microbiol 2006; 72:4805-10.

77. Hatcher MJ, Taneyhill DE, Dunn AM, Tofts C. Population dynamics under parasitic sex ratio distor-tion. Theor Popul Biol 1999; 56:11-28.

78. Hurst GDD, Jiggins FM. Male-killing bacteria in insects: Mechanisms, incidence and implications. Emerg Infect Dis 2000; 6:329-36.

79. Engelstadter J, Hurst GD. The impact of male-killing bacteria on host evolutionary processes. Genetics 2007; 175:245-54.

80. Malogolowkin-Cohen C, Rodrigues-Pereira MA. Sexual drive of normal and SR flies of Drosophila nebu-losa. Evolution 1975; 29:579-80.

81. Ebbert MA. Variable effects of crowding on Drosophila hosts of male-lethal and non-male-lethal Spiroplasma in laboratory populations. Heredity 1995; 74:227-40.

82. Montenegro H, Petherwick AS, Hurst GDD, Klaczko LB. Fitness effects of Wolbachia and Spiroplasma in Drosophila melanogaster. Genetica 2006; 127:207-15.

83. Oliver K, Campos J, Moran N, Hunter M. Population dynamics of defensive symbionts in aphids. Proc Biol Sci 2008; 275:293-9.

84. Chen DQ, Montllor CB, Purcell AH. Fitness effects of two facultative endosymbiotic bacteria on the pea aphid, Acyrthosiphon pisum, and the blue alfalfa aphid, A. kondoi. Entomologia Experimentalis Et Applicata 2000; 95:315-23.

85. Oliver KM, Russell JA, Moran NA, Hunter MS. Facultative bacterial symbionts in aphids confer resis-tance to parasitic wasps. Proc Natl Acad Sci USA 2003; 100:1803-7.

86. Piel J, Hofer I, Hui DQ. Evidence for a symbiosis island involved in horizontal acquisition of pederin biosynthetic capabilities by the bacterial symbiont of Paederus fuscipes beetles. J Bacteriol 2004; 186:1280-6.

87. Markow TA, O’Grady PM. Drosophila biology in the genomic age. Genetics 2007; 177:1269-76.

88. Jaenike J, Stahlhut JK, Boelio LM, Unckless RL. Association between Wolbachia and Spiroplasma within Drosophila neotestacea: an emerging symbiotic mutualism? Mol Ecol 2010; 19:414-25.

63. Veneti Z, Bentley JK, Koana T, Braig HR, Hurst GDD. A functional dosage compensation complex required for male killing in Drosophila. Science 2005; 307:1461-3.

64. Bentley JK, Veneti Z, Heraty J, Hurst GD. The pathology of embryo death caused by the male-killing Spiroplasma bacterium in Drosophila nebulosa. BMC Biol 2007; 5:9.

65. Kageyama D, Anbutsu H, Shimada M, Fukatsu T. Spiroplasma infection causes either early or late male killing in Drosophila, depending on maternal host age. Die Naturwissenschaften 2007; 94:333-7.

66. Kageyama D, Anbutsu H, Shimada M, Fukatsu T. Effects of host genotype against the expression of Spiroplasma-induced male killing in Drosophila mela-nogaster. Heredity 2009; 102:475-82.

67. Oishi K. Spirochaete-mediated abnormal sex-ratio (SR) condition in Drosophila: a second virus associated with spirochaetes and its use in the study of the SR condi-tion. Genet Res 1971; 18:45-56.

68. Williamson DL. The sex-ratio spirochete in Drosophila robusta. Jpn J Genet 1969; 44:36-41.

69. Williamson DL. Atypical transovarial transmission of sex ratio spirochetes by Drosophila robusta Sturtevant. J Exp Zool 1966; 161:425-9.

70. Cohen AJ, Williamson DL, Oishi K. Spv3 Viruses of Drosophila Spiroplasma. Israel J Med Sci 1987; 23:429-33.

71. Oishi K, Poulson DF. Virus associated with Sr-Spirochetes of Drosophila nebulosa. Proc Natl Acad Sci USA 1970; 67:1565-72.

72. Oishi K, Poulson DF, Williamson DL. Virus-mediated change in clumping properties of Drosophila SR Spiroplasma. Curr Microbiol 1984; 10:153-8.

73. Sakaguchi B, Oishi K, Kobayash S. Interference between sex-ratio agents of Drosophila willistoni and Drosophila nebulosa. Science 1965; 147:160-2.

74. Hurst GDD, Anbutsu H, Kutsukake M, Fukatsu T. Hidden from the host: Spiroplasma bacteria infecting Drosophila do not cause an immune response, but are suppressed by ectopic immune activation. Insect Mol Biol 2003; 12:93-7.

75. Anbutsu H, Fukatsu T. Tissue-specific infection dynam-ics of male-killing and nonmale-killing Spiroplasma in Drosophila melanogaster. FEMS Microbiol Ecol 2006; 57:40-6.