Macroevolutionary persistence of heritable endosymbionts: acquisition,retention and expression of adaptive phenotypes in Spiroplasma

The phylogenetic incongruence between insects and their facultative maternally transmitted endosymbionts indicates that these infections are generally short‐lived evolutionarily. Therefore, long‐term persistence of many endosymbionts must depend on their ability to colonize and spread within new host species. At least 17 species of Drosophila are infected with endosymbiotic Spiroplasma that have various phenotypic effects. We transinfected five strains of Spiroplasma from three divergent clades into Drosophila neotestacea to test their capacity to spread in a novel host. A strain that causes male killing in Drosophila melanogaster (its native host) also does so in D. neotestacea, even though these host species diverged 40–60 mya. A strain native to D. neotestacea (designated sNeo) and the two other strains of the poulsonii clade of Spiroplasma confer resistance to wasp parasitism, suggesting that this trait may be ancestral within this clade of Spiroplasma. Conversely, no strain other than sNeo conferred resistance to the sterilizing effects of nematode parasitism, suggesting that nematode resistance is a recently derived condition. The apparent addition of nematode resistance to a Spiroplasma lineage that already confers resistance to wasp parasitism suggests endosymbionts can increase the repertoire of traits conducive to their spread. The capacity of an endosymbiont to undergo maternal transmission and express adaptive phenotypes in novel hosts, without requiring a period of host–symbiont co‐evolution, enables the spread of such symbionts immediately after the colonization of a new host. This could be critical for the macroevolutionary persistence of facultative endosymbionts whose sojourn times within individual host species are relatively brief.     

Dynamics of the continent‐wide spread of a Drosophila defensive symbiont

Facultative symbionts can represent important sources of adaptation for their insect hosts and thus have the potential for rapid spread. Drosophila neotestacea harbours a heritable symbiont, Spiroplasma, that confers protection against parasitic nematodes. We previously found a cline in Spiroplasma prevalence across central Canada, ending abruptly at the Rocky Mountains. Resampling these populations 9 years later revealed that Spiroplasma had increased substantially across the region, resembling a Fisherian wave of advance. Associations between Spiroplasma infection and host mitochondrial DNA indicate that the increase was due to local increase of Spiroplasma‐infected flies. Finally, we detected Spiroplasma west of the Rocky Mountains for the first time and showed that defence against nematodes occurs in flies with a western genetic background. Because nematode infection is common throughout D. neotestacea's range, we expect Spiroplasma to spread to the Pacific coast.     

Nonrandom associations of maternally transmitted symbionts in insects: The roles of drift versus biased cotransmission and selection

Virtually all higher organisms form holobionts with associated microbiota. To understand the biology of holobionts we need to know how species assemble and interact. Controlled experiments are suited to study interactions between particular symbionts, but they only accommodate a tiny portion of the diversity within each species. Alternatively, interactions can be inferred by testing if associations among symbionts in the field are more or less frequent than expected under random assortment. However, random assortment may not be a valid null hypothesis for maternally transmitted symbionts since drift alone can result in associations. Here, we analyse a European field survey of endosymbionts in pea aphids (Acyrthosiphon pisum), confirming that symbiont associations are pervasive. To interpret them, we develop a model simulating the effect of drift on symbiont associations. We show that drift induces apparently nonrandom assortment, even though horizontal transmissions and maternal transmission failures tend to randomise symbiont associations. We also use this model in the approximate Bayesian computation framework to revisit the association between Spiroplasma and Wolbachia in Drosophila neotestacea. New field data reported here reveal that this association has disappeared in the investigated location, yet a significant interaction between Spiroplasma and Wolbachia can still be inferred. Our study confirms that negative and positive associations are pervasive and often induced by symbiont‐symbiont interactions. Nevertheless, some associations are also likely to be driven by drift. This possibility needs to be considered when performing such analyses, and our model is helpful for this purpose.     

SUBOPTIMAL VIRULENCE OF AN INSECT-PARASITIC NEMATODE

Recent considerations of parasite virulence have focused on the adverse effects that parasites can have on the survival of their hosts. Many parasites, however, reduce host fitness by an equally deleterious but different means, by causing partial or complete sterility of their hosts. A model of optimal parasite virulence is developed in which a quantity of host resources can be allocated to either host or parasite reproduction. Increases in parasite reproduction thus cause reductions in host fertility. The model shows that under a wide variety of ecological conditions, such parasites should completely sterilize their hosts. Only when opportunities for horizontal transmission are very limited should the parasites appropriate less than all of a host's reproductive resources. Field and laboratory evidence shows that the nematode parasite Howardula aoronymphium is relatively avirulent to one of its principal host species, Drosophila falleni, whereas it is much more virulent to D. putrida and D. neotestacea, suggesting that there may be substantial vertical transmission in D. falleni. However, epidemiological studies in the field and laboratory assays of host specificity strongly suggest that the three host species share a single parasite pool in natural populations, indicating that parasites in all three host species experience high levels of horizontal transmission. Thus, the low virulence of H. aoronymphium to D. falleni is not consistent with the model of optimal parasite virulence. It is proposed that this suboptimal virulence in D. falleni is a consequence of populations of H. aoronymphium being selected to exploit simultaneously several different host species. As a result, virulence may not be optimal in any one host. One must, therefore, consider the full range of host species in assessing a parasite's virulence.     

Association between Wolbachia and Spiroplasma within Drosophila neotestacea: an emerging symbiotic mutualism?

Interspecific mutualism can evolve when specific lineages of different species tend to be associated with each other from one generation to the next. Different maternally transmitted endosymbionts occurring within the same cytoplasmic lineage fulfil this requirement. Drosophila neotestacea is infected with maternally transmitted Wolbachia and Spiroplasma, which are cotransmitted at high frequency in natural populations. Molecular phylogenetic evidence indicates that both endosymbionts have been present in D. neotestacea for considerable evolutionary periods. Thus, conditions are suitable for the evolution of mutualism between them. In support of this possibility, there is a significant positive association between Wolbachia and Spiroplasma infection in many samples of D. neotestacea from natural populations. Theoretically, such a positive association can result from either mutualism between these endosymbionts or recent spread. Collections from present‐day populations suggest that recent spread and mutualism have both operated to generate the positive association between Wolbachia and Spiroplasma. If selection acts on the combination of these two endosymbionts, they may be in the early stages of evolution of a more complex, cooperative association.     

Interspecific transmission of endosymbiotic Spiroplasma by mites

The occurrence of closely related strains of maternally transmitted endosymbionts in distantly related insect species indicates that these infections can colonize new host species by lateral transfer, although the mechanisms by which this occurs are unknown. We investigated whether ectoparasitic mites, which feed on insect haemolymph, can serve as interspecific vectors of Spiroplasma poulsonii, a male-killing endosymbiont of Drosophila. Using Spiroplasma-specific primers for PCR, we found that mites can pick up Spiroplasma from infected Drosophila nebulosa females and subsequently transfer the infection to Drosophila willistoni. Some of the progeny of the recipient D. willistoni were infected, indicating successful maternal transmission of the Spiroplasma within the new host species. However, the transmission rate of the infection from recipient flies to their offspring was low, perhaps due to low Spiroplasma density in the recipient flies.     

Autosomal suppression and fitness costs of an old driving X chromosome in Drosophila testacea

Driving X chromosomes (X^Ds) bias their own transmission through males by killing Y‐bearing gametes. These chromosomes can in theory spread rapidly in populations and cause extinction, but many are found as balanced polymorphisms or as “cryptic” X^Ds shut down by drive suppressors. The relative likelihood of these outcomes and the evolutionary pathways through which they come about are not well understood. An X^D was recently discovered in the mycophagous fly, Drosophila testacea, presenting the opportunity to compare this X^D with the well‐studied X^D of its sister species, Drosophila neotestacea. Comparing features of independently evolved X^Ds in young sister species is a promising avenue towards understanding how X^Ds and their counteracting forces change over time. In contrast to the X^D of D. neotestacea, we find that the X^D of D. testacea is old, with its origin predating the radiation of three species: D. testacea, D. neotestacea and their shared sister species, Drosophila orientacea. Motivated by the suggestion that older X^Ds should be more deleterious to carriers, we assessed the effect of the X^D on both male and female fertility. Unlike what is known from D. neotestacea, we found a strong fitness cost in females homozygous for the X^D in D. testacea: a large proportion of homozygous females failed to produce offspring after being housed with males for several days. Our male fertility experiments show that although X^D male fertility is lower under sperm‐depleting conditions, X^D males have comparable fertility to males carrying a standard X chromosome under a free‐mating regime, which may better approximate conditions in wild populations of D. testacea. Lastly, we demonstrate the presence of autosomal suppression of X chromosome drive. Our results provide support for a model of X^D evolution where the dynamics of young X^Ds are governed by fitness consequences in males, whereas in older X^D systems, both suppression and fitness consequences in females likely supersede male fitness costs.     

Effect of the <Emphasis Type="Italic">Drosophila</Emphasis> endosymbiont <Emphasis Type="Italic">Spiroplasma</Emphasis> on parasitoid wasp development and on the reproductive fitness of wasp-attacked fly survivors

In a previous study, we showed that Spiroplasma, a maternally transmitted endosymbiotic bacterium of Drosophila hydei, enhances larval to adult survival of its host when exposed to oviposition attack by the parasitoid wasp Leptopilina heterotoma. The mechanism by which Spiroplasma enhances host survival has not been elucidated. To better understand this mechanism, we compared the growth of wasp larvae in Spiroplasma-infected and uninfected hosts. Our results indicate that wasp embryos in Spiroplasma-infected hosts hatch and grow normally for ~2 days, after which their growth is severely impaired, compared to wasps developing in uninfected hosts. Thus, despite their reduced ability to complete development in Spiroplasma-infected hosts, developing wasps may exert fitness costs on their hosts that are manifested after host emergence. The severity of these costs will influence the degree to which this protective mechanism contributes to the long-term persistence of Spiroplasma in D. hydei. We therefore examined survival to 10-day-old adult stage and fecundity of Spiroplasma-infected flies surviving a wasp treatment. Our results suggest detrimental effects of wasp attack on longevity of Spiroplasma-infected adult flies. However, compared to Spiroplasma-free flies exposed to wasps, Spiroplasma-infected flies exposed to wasps have ~5 times greater survival from larva to 10 day-adult. The relative fecundity of wasp-attacked Spiroplasma-infected females was ~71% that of un-attacked Spiroplasma-free females. Our combined survival and female fecundity results suggest that under high wasp parasitism, the reproductive fitness of Spiroplasma-infected flies may be ~3.5 times greater than that of uninfected females, so it is potentially relevant to the persistence of Spiroplasma in natural populations of D. hydei. Interestingly, Spiroplasma-infected males surviving a wasp attack were effectively sterile during the 3-day period examined. This observation is consistent with the expectation that, as a maternally transmitted symbiont, there is little selective pressure on Spiroplasma to enhance the reproductive fitness of its male hosts.     

Parasitism and dispersal potential of Sirex noctilio: implications for biological control

1 Sirex noctilio F. (Hymenoptera: Siricidae) is a wood‐boring wasp that attacks many pine species, including commercial trees planted throughout the world. Management of its populations is largely based on biological control using the nematode Beddingia siricidicola. Adult females are sterilized by the nematode, but are free to move and attack new trees, promoting nematode dispersal. Although generally successful, wasp management through nematode introductions has sometimes been inadequate. 2 We evaluated the effect of parasitism by B. siricidicola on flight performance of woodwasps under laboratory conditions. Using flight mills, we recorded a total of 46 flight trials over 23 h, obtained from infected and control (uninfected) females. 3 Although all wasps lost weight during flight, parasitized females were significantly smaller and suffered larger weight losses than uninfected females. In addition, total flight distance and velocity were lower in parasitized females. 4 Because nematode infection transmission relies on healthy wasps attacking trees previously visited by nematode‐bearing females, differential dispersal capacity could limit biological control success.     

Symbiont‐conferred reproduction and fitness benefits can favour their host occurrence

Double infections of Wolbachia and Spiroplasma are frequent in natural populations of Tetranychus truncatus, a polyphagous mite species that has been a dominant species in China since 2009. However, little is known about the causes and ecological importance of such coexistences. In this study, we established T. truncatus strains with different infection types and then inferred the impact of the two endosymbionts on host reproduction and fitness. Double infection induced cytoplasmic incompatibility, which was demonstrated by reduction in egg hatchability of incompatible crosses. However, doubly infected females produced more eggs relative to other strains. Wolbachia and Spiroplasma did not affect host survival, whereas doubly infected females and males developed faster than other strains. Such reproduction and fitness benefits provided by double infections may be associated with the lower densities of each symbiont, and the quantitative results also confirmed competition between Wolbachia and Spiroplasma in doubly infected females. These symbiont‐conferred beneficial effects maintain stable prevalence of the symbionts and also help drive T. truncatus outbreaks in combination with other environmental factors.     

Rapid evolution of host specificity in a parasitic nematode

Summary In eastern North America, the nematodeHowardula aoronymphium parasitizes four species of mushroom-breedingDrosophila:D. falleni andD. recens of the quinaria species group, andD. putrida andD. testacea of the testacea group. One strain ofH. aoronymphium, designated Mendon-87, was initially capable of infecting all four of these host species. After less than 3 years in laboratory culture usingD. falleni as the sole host, this strain had completely lost the ability to infectD. putrida. Two other nematode strains parasitizedD. falleni andD. putrida at equal rates. These results demonstrate the existence of genetic variation for host specificity within this nematode species. More importantly, they show that host specificity can evolve rapidly when only one host is available for parasitization. Ecological conditions are such that natural populations ofH. aoronymphium may comprise numerous host races, lineages incapable of parasitizing the full range of host species. However, I argue that such host races are probably ephemeral and thus unlikely to persist long enough to undergo speciation.     

Nematode parasitism in a northern European drosophilid community

Many factors may influence the structure of invertebrate communities. Among these is the presence of parasites which attack some or all members of a guild and potentially promote coexistence of competitor species. We assessed the prevalence of nematode (Allantonematidae) parasitism in Dutch woodland drosophilids (Diptera). Nematodes were found in 6 of the 18 drosophilid species sampled (percentage parasitism in parentheses): Drosophila phalerata (16%), D. kuntzei (5.1%), D. immigrans (0.5%), D. testacea (1.2%) and D. transversa (2.8%) were all parasitized by Howardula aoronymphium and D. subobscura (3%) was parasitized by Parazitylenchus diplogenus. This is the first report of nematode parasitism of D. immigrans and D. transversa. There were no important seasonal trends in percentage parasitism. We explored the consequences of nematode parasitism for individual drosophilids. Nematodes did not exert an important influence on the wing length (adult body size) of the drosophilids we sampled, but egg loads (fecundities) of female D. phalerata, D. subobscura and D. kuntzei were reduced by nematode parasitism. Parasitism rates were positively correlated with relative host abundance, in Dutch and other communities, suggesting that species diversity is promoted by a disproportionately high parasitism of more common host species.     

Protection against a fungal pathogen conferred by the aphid facultative endosymbionts Rickettsia and Spiroplasma is expressed in multiple host genotypes and species and is not influenced by co‐infection with another symbiont

Many insects harbour facultative endosymbiotic bacteria, often more than one type at a time. These symbionts can have major effects on their hosts' biology, which may be modulated by the presence of other symbiont species and by the host's genetic background. We investigated these effects by transferring two sets of facultative endosymbionts (one Hamiltonella and Rickettsia, the other Hamiltonella and Spiroplasma) from naturally double‐infected pea aphid hosts into five novel host genotypes of two aphid species. The symbionts were transferred either together or separately. We then measured aphid fecundity and susceptibility to an entomopathogenic fungus. The pathogen‐protective phenotype conferred by the symbionts Rickettsia and Spiroplasma varied among host genotypes, but was not influenced by co‐infection with Hamiltonella. Fecundity varied across single and double infections and between symbiont types, aphid genotypes and species. Some host genotypes benefit from harbouring more than one symbiont type.     

Variable Incidence of Spiroplasma Infections in Natural Populations of Drosophila Species

Spiroplasma is widespread as a heritable bacterial symbiont in insects and some other invertebrates, in which it sometimes acts as a male-killer and causes female-biased sex ratios in hosts. Besides Wolbachia, it is the only heritable bacterium known from Drosophila, having been found in 16 of over 200 Drosophila species screened, based on samples of one or few individuals per species. To assess the extent to which Spiroplasma infection varies within and among species of Drosophila, intensive sampling consisting of 50–281 individuals per species was conducted for natural populations of 19 Drosophila species. Infection rates varied among species and among populations of the same species, and 12 of 19 species tested negative for all individuals. Spiroplasma infection never was fixed, and the highest infection rates were 60% in certain populations of D. hydei and 85% in certain populations of D. mojavensis. In infected species, infection rates were similar for males and females, indicating that these Spiroplasma infections do not confer a strong male-killing effect. These findings suggest that Spiroplasma has other effects on hosts that allow it to persist, and that environmental or host variation affects transmission or persistence leading to differences among populations in infection frequencies.     

Can maternally inherited endosymbionts adapt to a novel host? Direct costs of Spiroplasma infection,but not vertical transmission efficiency,evolve rapidly after horizontal transfer into D. melanogaster

Maternally inherited symbionts are common in arthropods and many have important roles in host adaptation. The observation that specific symbiont lineages infect distantly related host species implies new interactions are commonly established by lateral transfer events. However, studies have shown that symbionts often perform poorly in novel hosts. We hypothesized selection on the symbiont may be sufficiently rapid that poor performance in a novel host environment is rapidly ameliorated, permitting symbiont maintenance. Here, we test this prediction for a Spiroplasma strain transinfected into the novel host Drosophila melanogaster. In the generations immediately following transinfection, the symbiont had low transmission efficiency to offspring and imposed severe fitness costs on its host. We observed that effects on host fitness evolved rapidly, being undetectable after 17 generations in the novel host, whereas vertical transmission efficiency was poorly responsive over this period. Our results suggest that long-term symbiosis may more readily be established in cases where symbionts perform poorly in just one aspect of symbiosis.     

Does host outcrossing disrupt compatibility with heritable symbionts?

Vertically transmitted microbes are common in macro‐organisms and can enhance host defense against environmental stress. Because vertical transmission couples host and symbiont lineages, symbionts may become specialized to host species or genotypes. Specialization and contrasting reproductive modes of symbiotic partners could create incompatibilities between inherited symbionts and novel host genotypes when hosts outcross or hybridize. Such incompatibilities could manifest as failed colonization or poor symbiont growth in host offspring that are genetically dissimilar from their maternal host. Moreover, outcrossing between host species could influence both host and symbiont reproductive performance. We tested these hypotheses by manipulating outcrossing between populations and species of two grasses, Elymus virginicus and E. canadensis, that host vertically transmitted fungal endophytes (genus Epichloё). In both greenhouse and field settings, we found that host–symbiont compatibility was robust to variation in host genetic background, spanning within‐population, between‐population and between‐species crosses. Symbiont transmission into the F₁ generation was generally high and weakly affected by host outcrossing. Furthermore, endophytes grew equally well in planta regardless of host genetic background and transmitted at high frequencies into the F₂ generation. However, outcrossing, especially inter‐specific hybridization, reduced reproductive fitness of the host, and thereby the symbiont. Our results challenge the hypothesis that host genetic recombination, which typically exceeds that of symbionts, is a disruptive force in heritable symbioses. Instead, symbionts may be sufficiently generalized to tolerate ecologically realistic variation in host outcrossing.     

Digest: Fitness costs of Spiroplasma infection in pea aphids*

Endosymbionts sometimes help their hosts resist parasites, but does infection of pea aphids (Acyrthosiphon pisum) with different strains of the endosymbiont Spiroplasma confer fitness benefits that offset the costs? Mathé‐Hubert et al. found that across four life‐history traits, Spiroplasma infection induced negative effects on host fitness when compared to controls. Only two of 12 strains of Spiroplasma showed a marginal protective effect against host parasitism by Aphidius ervi, implying Spiroplasma infection is almost entirely detrimental to pea aphid host fitness.     

Molecular tracking of individual host use in the Shiny Cowbird – a generalist brood parasite

Generalist parasites exploit multiple host species at the population level, but the individual parasite's strategy may be either itself a generalist or a specialist pattern of host species use. Here, we studied the relationship between host availability and host use in the individual parasitism patterns of the Shiny Cowbird Molothrus bonariensis, a generalist avian obligate brood parasite that parasitizes an extreme range of hosts. Using five microsatellite markers and an 1120‐bp fragment of the mtDNA control region, we reconstructed full‐sibling groups from 359 cowbird eggs and chicks found in nests of the two most frequent hosts in our study area, the Chalk‐browed Mockingbird Mimus saturninus and the House Wren Troglodytes aedon. We were able to infer the laying behavior of 17 different females a posteriori and found that they were mostly faithful to a particular laying area and host species along the entire reproductive season and did not avoid using previously parasitized nests (multiple parasitism) even when other nests were available for parasitism. Moreover, we found females using the same host nest more than once (repeated parasitism), which had not been previously reported for this species. We also found few females parasitizing more than one host species. The use of an alternative host was not related to the main hosts' nest availability. Overall, female shiny cowbirds use a spatially structured and host species specific approach for parasitism, but they do so nonexclusively, resulting in both detectable levels of multiple parasitism and generalism at the level of individual parasites.     

Male killing Spiroplasma protects Drosophila melanogaster against two parasitoid wasps

Maternally transmitted associations between endosymbiotic bacteria and insects are diverse and widespread in nature. Owing to imperfect vertical transmission, many heritable microbes have evolved compensational mechanisms to enhance their persistence in host lineages, such as manipulating host reproduction and conferring fitness benefits to host. Symbiont-mediated defense against natural enemies of hosts is increasingly recognized as an important mechanism by which endosymbionts enhance host fitness. Members of the genus Spiroplasma associated with distantly related Drosophila hosts are known to engage in either reproductive parasitism (i.e., male killing) or defense against natural enemies (the parasitic wasp Leptopilina heterotoma and a nematode). A male-killing strain of Spiroplasma (strain Melanogaster Sex Ratio Organism (MSRO)) co-occurs with Wolbachia (strain wMel) in certain wild populations of the model organism Drosophila melanogaster. We examined the effects of Spiroplasma MSRO and Wolbachia wMel on Drosophila survival against parasitism by two common wasps, Leptopilina heterotoma and Leptopilina boulardi, that differ in their host ranges and host evasion strategies. The results indicate that Spiroplasma MSRO prevents successful development of both wasps, and confers a small, albeit significant, increase in larva-to-adult survival of flies subjected to wasp attacks. We modeled the conditions under which defense can contribute to Spiroplasma persistence. Wolbachia also confers a weak, but significant, survival advantage to flies attacked by L. heterotoma. The host protective effects exhibited by Spiroplasma and Wolbachia are additive and may provide the conditions for such cotransmitted symbionts to become mutualists. Occurrence of Spiroplasma-mediated protection against distinct parasitoids in divergent Drosophila hosts suggests a general protection mechanism.