Multiple origins of obligate nematode and insect symbionts by a clade of bacteria closely related to plant pathogens.

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that Haoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont's genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations.

BACKGROUND: The drosophilid Phortica variegata is known as vector of Thelazia callipaeda, the oriental eyeworm native to Asia that has become an emergent zoonotic agent in several European regions. Unlike almost all other arthropod vectors of pathogens, only P. variegata males feed of lachrymal secretions of animals, ingesting first-stage larvae (L1) of the worm living in the orbital cavities of the host, and allowing with the same behaviour the introduction of infective L3. Despite the increased detection of T. callipaeda in many European countries, information about the length of the lachryphagous activity period of P. variegata and a deep knowledge of the environmental and climatic variables involved are still limited. METHODS: We herein present the results of a multicentre study involving five sites from four different countries (Italy, Spain, UK and USA) where canine thelaziosis is endemic and/or where it has already been ascertained the presence of P. variegata. Field data have been obtained on a fortnightly basis from mid-April to the end of November 2018 from a contemporary standardized sampling (same sampling effort and time of collection in all sites) of lachryphagous flies collected around the eyes of a human bait using an entomological net. These data have been associated to data collection of local climatic variables (day length, temperature, wind speed, barometric pressure and relative humidity). RESULTS: Overall, a total of 4862 P. variegata flies (4637 males and 224 females) were collected, with high differences in densities among the different sampling sites. Significant positive correlations were found between P. variegata male density and temperature and wind speed, while negative correlations were observed for barometric pressure and relative humidity. However, the above significant differences are confirmed in each sampling site separately only for the temperature. CONCLUSIONS: This multicentre study highlights that temperature is the major common environmental driver in describing the lachryphagous activity of P. variegata in Europe and USA and, therefore, the transmission risk of thelaziosis.

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.

Endosymbiont-based immunity in Drosophila melanogaster against parasitic nematode infection.

Associations between endosymbiotic bacteria and their hosts represent a complex ecosystem within organisms ranging from humans to protozoa. Drosophila species are known to naturally harbor Wolbachia and Spiroplasma endosymbionts, which play a protective role against certain microbial infections. Here, we investigated whether the presence or absence of endosymbionts affects the immune response of Drosophila melanogaster larvae to infection by Steinernema carpocapsae nematodes carrying or lacking their mutualistic Gram-negative bacteria Xenorhabdus nematophila (symbiotic or axenic nematodes, respectively). We find that the presence of Wolbachia alone or together with Spiroplasma promotes the survival of larvae in response to infection with S. carpocapsae symbiotic nematodes, but not against axenic nematodes. We also find that Wolbachia numbers are reduced in Spiroplasma-free larvae infected with axenic compared to symbiotic nematodes, and they are also reduced in Spiroplasma-containing compared to Spiroplasma-free larvae infected with axenic nematodes. We further show that S. carpocapsae axenic nematode infection induces the Toll pathway in the absence of Wolbachia, and that symbiotic nematode infection leads to increased phenoloxidase activity in D. melanogaster larvae devoid of endosymbionts. Finally, infection with either type of nematode alters the metabolic status and the fat body lipid droplet size in D. melanogaster larvae containing only Wolbachia or both endosymbionts. Our results suggest an interaction between Wolbachia endosymbionts with the immune response of D. melanogaster against infection with the entomopathogenic nematodes S. carpocapsae. Results from this study indicate a complex interplay between insect hosts, endosymbiotic microbes and pathogenic organisms.

Competence of Phortica variegata from the United States as an Intermediate Host of the Thelazia callipaeda Eyeworm.

Over the past 20 years, Thelazia callipaeda (the oriental eyeworm) has become endemic in Europe, infecting domestic and wild carnivores and humans. The vector of this nematode, the fruit fly Phortica variegata, has recently been discovered in the United States, and its vector competence is demonstrated for T. callipaeda in this article, therefore representing a potential new threat for infection of carnivores and humans in the United States.

Community structure of the gut microbiota in sympatric species of wild Drosophila.

Many aspects of animal ecology and physiology are influenced by the microbial communities within them. The underlying forces contributing to the assembly and diversity of gut microbiotas include chance events, host-based selection and interactions among microorganisms within these communities. We surveyed 215 wild individuals from four sympatric species of Drosophila that share a common diet of decaying mushrooms. Their microbiotas consistently contained abundant bacteria that were undetectable or at low abundance in their diet. Despite their deep phylogenetic divergence, all species had similar microbiotas, thus failing to support predictions of the phylosymbiosis hypothesis. Communities within flies were not random assemblages drawn from a common pool; instead, many bacterial operational taxonomic units (OTUs) were overrepresented or underrepresented relative to the neutral expectations, and OTUs exhibited checkerboard distributions among flies. These results suggest that selective factors play an important role in shaping the gut community structure of these flies.

Effects of co-occurring Wolbachia and Spiroplasma endosymbionts on the Drosophila immune response against insect pathogenic and non-pathogenic bacteria.

BACKGROUND: Symbiotic interactions between microbes and animals are common in nature. Symbiotic organisms are particularly common in insects and, in some cases, they may protect their hosts from pathogenic infections. Wolbachia and Spiroplasma endosymbionts naturally inhabit various insects including Drosophila melanogaster fruit flies. Therefore, this symbiotic association is considered an excellent model to investigate whether endosymbiotic bacteria participate in host immune processes against certain pathogens. Here we have investigated whether the presence of Wolbachia alone or together with Spiroplasma endosymbionts in D. melanogaster adult flies affects the immune response against the virulent insect pathogen Photorhabdus luminescens and against non-pathogenic Escherichia coli bacteria. RESULTS: We found that D. melanogaster flies carrying no endosymbionts, those carrying both Wolbachia and Spiroplasma, and those containing Wolbachia only had similar survival rates after infection with P. luminescens or Escherichia coli bacteria. However, flies carrying both endosymbionts or Wolbachia only contained higher numbers of E. coli cells at early time-points post infection than flies without endosymbiotic bacteria. Interestingly, flies containing Wolbachia only had lower titers of this endosymbiont upon infection with the pathogen P. luminescens than uninfected flies of the same strain. We further found that the presence of Wolbachia and Spiroplasma in D. melanogaster up-regulated certain immune-related genes upon infection with P. luminescens or E. coli bacteria, but it failed to alter the phagocytic ability of the flies toward E. coli inactive bioparticles. CONCLUSION: Our results suggest that the presence of Wolbachia and Spiroplasma in D. melanogaster can modulate immune signaling against infection by certain insect pathogenic and non-pathogenic bacteria. Results from such studies are important for understanding the molecular basis of the interactions between endosymbiotic bacteria of insects and exogenous microbes.

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.

Recent genome reduction of Wolbachia in Drosophila recens targets phage WO and narrows candidates for reproductive parasitism.

Wolbachia are maternally transmitted endosymbionts that often alter their arthropod hosts' biology to favor the success of infected females, and they may also serve as a speciation microbe driving reproductive isolation. Two of these host manipulations include killing males outright and reducing offspring survival when infected males mate with uninfected females, a phenomenon known as cytoplasmic incompatibility. Little is known about the mechanisms behind these phenotypes, but interestingly either effect can be caused by the same Wolbachia strain when infecting different hosts. For instance, wRec causes cytoplasmic incompatibility in its native host Drosophila recens and male killing in D. subquinaria. The discovery of prophage WO elements in most arthropod Wolbachia has generated the hypothesis that WO may encode genes involved in these reproductive manipulations. However, PCR screens for the WO minor capsid gene indicated that wRec lacks phage WO. Thus, wRec seemed to provide an example where phage WO is not needed for Wolbachia-induced reproductive manipulation. To enable investigation of the mechanism of phenotype switching in different host backgrounds, and to examine the unexpected absence of phage WO, we sequenced the genome of wRec. Analyses reveal that wRec diverged from wMel approximately 350,000 years ago, mainly by genome reduction in the phage regions. While it lost the minor capsid gene used in standard PCR screens for phage WO, it retained two regions encompassing 33 genes, several of which have previously been associated with reproductive parasitism. Thus, WO gene involvement in reproductive manipulation cannot be excluded and reliance on single gene PCR should not be used to rule out the presence of phage WO in Wolbachia. Additionally, the genome sequence for wRec will enable transcriptomic and proteomic studies that may help elucidate the Wolbachia mechanisms of altered reproductive manipulations associated with host switching, perhaps among the 33 remaining phage genes.

Wolbachia do not live by reproductive manipulation alone: infection polymorphism in Drosophila suzukii and D. subpulchrella.

Drosophila suzukii recently invaded North America and Europe. Populations in Hawaii, California, New York and Nova Scotia are polymorphic for Wolbachia, typically with <20% infection frequency. The Wolbachia in D. suzukii, denoted wSuz, is closely related to wRi, the variant prevalent in continental populations of D. simulans. wSuz is also nearly identical to Wolbachia found in D. subpulchrella, plausibly D. suzukii's sister species. This suggests vertical Wolbachia transmission through cladogenesis ('cladogenic transmission'). The widespread occurrence of 7-20% infection frequencies indicates a stable polymorphism. wSuz is imperfectly maternally transmitted, with wild infected females producing on average 5-10% uninfected progeny. As expected from its low frequency, wSuz produces no cytoplasmic incompatibility (CI), that is, no increased embryo mortality when infected males mate with uninfected females, and no appreciable sex-ratio distortion. The persistence of wSuz despite imperfect maternal transmission suggests positive fitness effects. Assuming a balance between selection and imperfect transmission, we expect a fitness advantage on the order of 20%. Unexpectedly, Wolbachia-infected females produce fewer progeny than do uninfected females. We do not yet understand the maintenance of wSuz in D. suzukii. The absence of detectable CI in D. suzukii and D. subpulchrella makes it unlikely that CI-based mechanisms could be used to control this species without transinfection using novel Wolbachia. Contrary to their reputation as horizontally transmitted reproductive parasites, many Wolbachia infections are acquired through introgression or cladogenesis and many cause no appreciable reproductive manipulation. Such infections, likely to be mutualistic, may be central to understanding the pervasiveness of Wolbachia among arthropods.

Infectious adaptation: potential host range of a defensive endosymbiont in Drosophila.

Maternally transmitted symbionts persist over macroevolutionary timescales by undergoing occasional lateral transfer to new host species. To invade a new species, a symbiont must survive and reproduce in the new host, undergo maternal transmission, and confer a selective benefit sufficient to overcome losses due to imperfect maternal transmission. Drosophila neotestacea is naturally infected with a strain of Spiroplasma that restores fertility to nematode-parasitized females, which are otherwise sterilized by parasitism. We experimentally transferred Spiroplasma from D. neotestacea to four other species of mycophagous Drosophila that vary in their ability to resist and/or tolerate nematode parasitism. In all four species, Spiroplasma achieved within-host densities and experienced rates of maternal transmission similar to that in D. neotestacea. Spiroplasma restored fertility to nematode-parasitized females in one of these novel host species. Based on estimates of maternal transmission fidelity and the expected benefit of Spiroplasma infection in the wild, we conclude that Spiroplasma has the potential to spread and become abundant within Drosophila putrida, which is broadly sympatric with D. neotestacea and in which females are rendered completely sterile by nematode parasitism. Thus, a major adaptation within D. putrida could arise via lateral transmission of a heritable symbiont from D. neotestacea.

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.

Cryptic Onchocerca species infecting North American cervids, with implications for the evolutionary history of host associations in Onchocerca.

Parasites in the genus Onchocerca infect humans, ruminants, camels, horses, suids, and canids, with effects ranging from relatively benign to debilitating. In North America, Onchocerca cervipedis is the sole species known to infect cervids, while at least 5 Onchocerca species infect Eurasian cervids. In this study, we report the discovery of a cervid-parasitizing Onchocerca only distantly related to O. cervipedis. To reconstruct the phylogenetic history of the genus Onchocerca, we used newly acquired DNA sequence from O. cervipedis (from moose in Northwest Territories, Canada) and from the newly discovered species (from white-tailed deer in upstate New York), as well as previously published sequences. Ancestral host reconstructions suggest that host switches have been common throughout the evolutionary history of Onchocerca, and that bovid- and cervid-parasitizing species have been particularly important sources of descendant species. North America cervids might therefore serve as a source for Onchocerca invasions into new hosts. Given the high density of deer populations, the potential for zoonotic infections may also exist. Our discovery of a new Onchocerca species with relatively limited sampling suggests that the diversity of Onchocerca associated with cervids in North America may be greater than previously thought, and surveys utilizing molecules and morphology are necessary.

Maintenance of a male-killing Wolbachia in Drosophila innubila by male-killing dependent and male-killing independent mechanisms.

Many maternally inherited endosymbionts manipulate their host's reproduction in various ways to enhance their own fitness. One such mechanism is male killing (MK), in which sons of infected mothers are killed by the endosymbiont during development. Several hypotheses have been proposed to explain the advantages of MK, including resource reallocation from sons to daughters of infected females, avoidance of inbreeding by infected females, and, if transmission is not purely maternal, the facilitation of horizontal transmission to uninfected females. We tested these hypotheses in Drosophila innubila, a mycophagous species infected with MK Wolbachia. There was no evidence of horizontal transmission in the wild and no evidence Wolbachia reduced levels of inbreeding. Resource reallocation does appear to be operative, as Wolbachia-infected females are slightly larger, on average, than uninfected females, although the selective advantage of larger size is insufficient to account for the frequency of infection in natural populations. Wolbachia-infected females from the wild-although not those from the laboratory-were more fecund than uninfected females. Experimental studies revealed that Wolbachia can boost the fecundity of nutrient-deprived flies and reduce the adverse effect of RNA virus infection. Thus, this MK endosymbiont can provide direct, MK-independent fitness benefits to infected female hosts in addition to possible benefits mediated via MK.

Population genetics of beneficial heritable symbionts.

Most species of insects are infected with heritable, facultative symbionts. Such symbionts first appear within a host lineage as a result of lateral transfer from other host species. Although some facultative symbionts are reproductive parasites and thus adversely affect the transmission of host nuclear genes, there is growing evidence that many are beneficial to their hosts by, for example, conferring protection from natural enemies. The origin, spread, and maintenance of such symbionts bears many similarities to, as well as important differences from, the process of adaptive evolution by beneficial nuclear mutations. The time is ripe for the development of a coherent theory of the 'population genetics' of beneficial heritable symbionts.

Wolbachia-mediated persistence of mtDNA from a potentially extinct species.

Drosophila quinaria is polymorphic for infection with Wolbachia, a maternally transmitted endosymbiont. Wolbachia-infected individuals carry mtDNA that is only distantly related to the mtDNA of uninfected individuals, and the clade encompassing all mtDNA haplotypes within D. quinaria also includes the mtDNA of several other species of Drosophila. Nuclear gene variation reveals no difference between the Wolbachia-infected and uninfected individuals of D. quinaria, indicating that they all belong to the same interbreeding biological species. We suggest that the Wolbachia and the mtDNA with which it is associated were derived via interspecific hybridization and introgression. The sequences in the Wolbachia and the associated mtDNA are ≥6% divergent from those of any known Drosophila species. Thus, in spite of nearly complete species sampling, the sequences from which these mitochondria were derived remain unknown, raising the possibility that the donor species is extinct. The association between Wolbachia infection and mtDNA type within D. quinaria suggests that Wolbachia may be required for the continued persistence of the mtDNA from an otherwise extinct Drosophila species. We hypothesize that pathogen-protective effects conferred by Wolbachia operate in a negative frequency-dependent manner, thus bringing about a stable polymorphism for Wolbachia infection.

Defensive endosymbionts: a cryptic trophic level in community ecology.

Maternally transmitted endosymbionts are widespread among insects, but how they are maintained within host populations is largely unknown. Recent discoveries show that some endosymbionts protect their hosts from pathogens or parasites. Spiroplasma, an endosymbiont of Drosophila neotestacea, protects female hosts from the sterilizing effects of parasitism by the nematode Howardula aoronymphium. Here, we show that Spiroplasma spreads rapidly within experimental populations of D. neotestacea subject to Howardula parasitism, but is neither strongly favored nor selected against in the absence of Howardula. In a reciprocal experiment, Howardula declined steadily to extinction in populations of Spiroplasma-infected flies, whereas in populations of uninfected flies, the prevalence of Howardula parasitism increased to c. 100%. Thus, Spiroplasma and Howardula exhibit effectively consumer-resource trophic dynamics. The recent spread of Spiroplasma in natural populations of D. neotestacea coincides with a decline in the prevalence of Howardula parasitism in the wild.

Adaptation via symbiosis: recent spread of a Drosophila defensive symbiont.

Recent studies have shown that some plants and animals harbor microbial symbionts that protect them against natural enemies. Here we demonstrate that a maternally transmitted bacterium, Spiroplasma, protects Drosophila neotestacea against the sterilizing effects of a parasitic nematode, both in the laboratory and the field. This nematode parasitizes D. neotestacea at high frequencies in natural populations, and, until recently, almost all infections resulted in complete sterility. Several lines of evidence suggest that Spiroplasma is spreading in North American populations of D. neotestacea and that a major adaptive change to a symbiont-based mode of defense is under way. These findings demonstrate the profound and potentially rapid effects of defensive symbionts, which are increasingly recognized as major players in the ecology of species interactions.

The mushroom habitat as an ecological arena for global exchange of Wolbachia.

Wolbachia infect a variety of arthropod and nematode hosts, but in arthropods, host phylogenetic relationships are usually poor predictors of strain similarity. This suggests that new infections are often established by horizontal transmission. To gain insight into the factors affecting the probability of horizontal transmission among host species, we ask how host phylogeny, geographical distribution and ecology affect patterns of Wolbachia strain similarity. We used multilocus sequence typing (MLST) to characterize Wolbachia strain similarity among dipteran hosts associated with fleshy mushrooms. Wolbachia Supergroup A was more common than Supergroup B in Diptera, and also more common in mycophagous than non-mycophagous Diptera. Within Supergroup A, host family within Diptera had no effect on strain similarity, and there was no tendency for Wolbachia strains from sympatric host species to be more similar to one another than to strains from hosts in different biogeographical realms. Supergroup A strains differed between mycophagous and non-mycophagous Diptera more than expected by chance, suggesting that ecological associations can facilitate horizontal transmission of Wolbachia within mycophagous fly communities. For Supergroup B, there were no significant associations between strain similarity and host phylogeny, biogeography, or ecology. We identified only two cases in which closely related hosts carried closely related Wolbachia strains, evidence that Wolbachia-host co-speciation or early introgression can occur but may not be a major contributor to overall strain diversity. Our results suggest that horizontal transmission of Wolbachia can be influenced by host ecology, thus leading to partial restriction of Wolbachia strains or strain groups to particular guilds of insects.

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.