Daniela Schneider

Infectious speciation revisited: impact of symbiont-depletion on female fitness and mating behavior of Drosophila paulistorum.

The neotropical Drosophila paulistorum superspecies, consisting of at least six geographically overlapping but reproductively isolated semispecies, has been the object of extensive research since at least 1955, when it was initially trapped mid-evolution in flagrant statu nascendi. In this classic system females express strong premating isolation patterns against mates belonging to any other semispecies, and yet uncharacterized microbial reproductive tract symbionts were described triggering hybrid inviability and male sterility. Based on theoretical models and limited experimental data, prime candidates fostering symbiont-driven speciation in arthropods are intracellular bacteria belonging to the genus Wolbachia. They are maternally inherited symbionts of many arthropods capable of manipulating host reproductive biology for their own benefits. However, it is an ongoing debate as to whether or not reproductive symbionts are capable of driving host speciation in nature and if so, to what extent. Here we have reevaluated this classic case of infectious speciation by means of present day molecular approaches and artificial symbiont depletion experiments. We have isolated the α-proteobacteria Wolbachia as the maternally transmitted core endosymbionts of all D. paulistorum semispecies that have coevolved towards obligate mutualism with their respective native hosts. In hybrids, however, these mutualists transform into pathogens by overreplication causing embryonic inviability and male sterility. We show that experimental reduction in native Wolbachia titer causes alterations in sex ratio, fecundity, and mate discrimination. Our results indicate that formerly designated Mycoplasma-like organisms are most likely Wolbachia that have evolved by becoming essential mutualistic symbionts in their respective natural hosts; they have the potential to trigger pre- and postmating isolation. Furthermore, in light of our new findings, we revisit the concept of infectious speciation and discuss potential mechanisms that can restrict or promote symbiont-induced speciation at post- and prezygotic levels in nature and under artificial laboratory conditions.

Hidden Wolbachia diversity in field populations of the European cherry fruit fly, Rhagoletis cerasi (Diptera, Tephritidae).

The European cherry fruit fly Rhagoletis cerasi has been a field model for cytoplasmic incompatibility since the mid 1970s. Two Wolbachia strains were detected in this tephritid species and wCer2 was described as the CI inducing agent dividing European populations into two unidirectional incompatible groups, i.e. southern females produce viable offspring with northern males, whereas the reciprocal cross results in incompatibility. We detected three new Wolbachia strains by sequencing a multitude of plasmids derived from Wolbachia surface protein gene (wsp) polymerase chain reaction (PCR) products. Strain‐specific primers were developed allowing individual diagnosis without need for cloning. Hybridization of specific PCR products with a wsp oligonucleotide enhanced the detection limit significantly and revealed the presence of low‐titre infections in some strains, in different ontogenetic stages and in adults of different age. We then performed a survey of strain prevalence and infection frequency in eight European regions. wCer1 was fixed in all populations, whereas wCer2 was detected only in the South. wCer3 frequency was the lowest without a clear distribution pattern. The abundance of wCer4 was homogenous across Europe. Like wCer2, wCer5 showed significant differences in spatial distribution. Our new findings of previously undetected and recombinant Wolbachia strains in R. cerasi reveal a major caveat to the research community not to overlook hidden Wolbachia diversity in field populations. Low‐titres and geographical variability in Wolbachia diversity are expected to influence the outcome of Wolbachia population dynamics and Wolbachia‐based insect population control and may create invasion barriers for expanding and artificially introduced Wolbachia strains.

Cell invasion of Yersinia pseudotuberculosis by invasin and YadA requires protein kinase C, phospholipase C‐γ1 and Akt kinase

The outer membrane proteins YadA and invasin of Yersinia pseudotuberculosis promote invasion into mammalian cells through β1‐integrins and trigger the production of interleukin (IL)‐8. FAK, c‐Src and the PI3 kinase were previously found to be important for both YadA‐ and invasin‐promoted uptake. Here, we demonstrate that two different downstream effectors of PI3 kinase, Akt and phospholipase Cγ1 are required for efficient cell invasion. Inhibition of Akt or phospholipase C‐γ (PLC‐γ)1 by pharmaceutical agents as well as reduced expression of the isoforms Akt1 and Akt2, and of PLC‐γ1 by RNA interference decreased entry of YadA‐ and Inv‐expressing bacteria significantly. In addition, we report that the conventional protein kinases C (PKC)α and ‐β, positioned downstream of PLC‐γ1, are activated upon Inv‐ or YadA‐promoted cell entry. They colocalize with intracellular bacteria and their depletion by siRNA treatment also resulted in a strong reduction of cell entry. In contrast, neither Akt nor PLC‐γ1, and the PKCs are essential for YadA‐ and Inv‐mediated IL‐8 synthesis and release. We conclude that YadA and invasin of Y. pseudotuberculosis both trigger similar signal transduction pathways during integrin‐mediated phagocytosis into epithelial cells, which lead to the activation of Akt, PLC‐γ1, PKCα and ‐β downstream of PI3 kinase, separate from the MAPK‐dependent pathway that triggers IL‐8 production.

Global Wolbachia prevalence, titer fluctuations and their potential of causing cytoplasmic incompatibilities in tsetse flies and hybrids of Glossina morsitans subgroup species.

Graphical abstract Highlights ► VNTRs are highly diagnostic tools for fingerprinting Wolbachia in tsetse flies. ► Multiple infections, free and nuclear insertions into host chromosomes, do exist. ► Some infections can escape detection via hiding as low-titer infections. ► In hybrids Wolbachia can transform into pathogens by loss of replication control.

Transgenerational Transmission of the Glossina pallidipes Hytrosavirus Depends on the Presence of a Functional Symbiome

The vertically transmitted endosymbionts (Sodalis glossinidius and Wigglesworthia glossinidia) of the tsetse fly (Diptera: Glossinidae) are known to supplement dietary deficiencies and modulate the reproductive fitness and the defense system of the fly. Some tsetse fly species are also infected with the bacterium, Wolbachia and with the Glossina hytrosavirus (GpSGHV). Laboratory-bred G. pallidipes exhibit chronic asymptomatic and acute symptomatic GpSGHV infection, with the former being the most common in these colonies. However, under as yet undefined conditions, the asymptomatic state can convert to the symptomatic state, leading to detectable salivary gland hypertrophy (SGH+) syndrome. In this study, we investigated the interplay between the bacterial symbiome and GpSGHV during development of G. pallidipes by knocking down the symbionts with antibiotic. Intrahaemocoelic injection of GpSGHV led to high virus titre (109 virus copies), but was not accompanied by either the onset of detectable SGH+, or release of detectable virus particles into the blood meals during feeding events. When the F1 generations of GpSGHV-challenged mothers were dissected within 24 h post-eclosion, SGH+ was observed to increase from 4.5% in the first larviposition cycle to >95% in the fourth cycle. Despite being sterile, these F1 SGH+ progeny mated readily. Removal of the tsetse symbiome, however, suppressed transgenerational transfer of the virus via milk secretions and blocked the ability of GpSGHV to infect salivary glands of the F1 progeny. Whereas GpSGHV infects and replicates in salivary glands of developing pupa, the virus is unable to induce SGH+ within fully differentiated adult salivary glands. The F1 SGH+ adults are responsible for the GpSGHV-induced colony collapse in tsetse factories. Our data suggest that GpSGHV has co-evolved with the tsetse symbiome and that the symbionts play key roles in the virus transmission from mother to progeny.

Allele Intersection Analysis: A Novel Tool for Multi Locus Sequence Assignment in Multiply Infected Hosts

Wolbachia are wide-spread, endogenous α-Proteobacteria of arthropods and filarial nematodes. 15–75% of all insect species are infected with these endosymbionts that alter their hosts reproduction to facilitate their spread. In recent years, many insect species infected with multiple Wolbachia strains have been identified. As the endosymbionts are not cultivable outside living cells, strain typing relies on molecular methods. A Multi Locus Sequence Typing (MLST) system was established for standardizing Wolbachia strain identification. However, MLST requires hosts to harbour individual and not multiple strains of supergroups without recombination. This study revisits the applicability of the current MLST protocols and introduces Allele Intersection Analysis (AIA) as a novel approach. AIA utilizes natural variations in infection patterns and allows correct strain assignment of MLST alleles in multiply infected host species without the need of artificial strain segregation. AIA identifies pairs of multiply infected individuals that share Wolbachia and differ in only one strain. In such pairs, the shared MLST sequences can be used to assign alleles to distinct strains. Furthermore, AIA is a powerful tool to detect recombination events. The underlying principle of AIA may easily be adopted for MLST approaches in other uncultivable bacterial genera that occur as multiple strain infections and the concept may find application in metagenomic high-throughput parallel sequencing projects.

Uncovering Wolbachia Diversity upon Artificial Host Transfer

The common endosymbiotic Wolbachia bacteria influence arthropod hosts in multiple ways. They are mostly recognized for their manipulations of host reproduction, yet, more recent studies demonstrate that Wolbachia also impact host behavior, metabolic pathways and immunity. Besides their biological and evolutionary roles, Wolbachia are new potential biological control agents for pest and vector management. Importantly, Wolbachia-based control strategies require controlled symbiont transfer between host species and predictable outcomes of novel Wolbachia-host associations. Theoretically, this artificial horizontal transfer could inflict genetic changes within transferred Wolbachia populations. This could be facilitated through de novo mutations in the novel recipient host or changes of haplotype frequencies of polymorphic Wolbachia populations when transferred from donor to recipient hosts. Here we show that Wolbachia resident in the European cherry fruit fly, Rhagoletis cerasi, exhibit ancestral and cryptic sequence polymorphism in three symbiont genes, which are exposed upon microinjection into the new hosts Drosophila simulans and Ceratitis capitata. Our analyses of Wolbachia in microinjected D. simulans over 150 generations after microinjection uncovered infections with multiple Wolbachia strains in trans-infected lines that had previously been typed as single infections. This confirms the persistence of low-titer Wolbachia strains in microinjection experiments that had previously escaped standard detection techniques. Our study demonstrates that infections by multiple Wolbachia strains can shift in prevalence after artificial host transfer driven by either stochastic or selective processes. Trans-infection of Wolbachia can claim fitness costs in new hosts and we speculate that these costs may have driven the shifts of Wolbachia strains that we saw in our model system.

More than fishing in the dark: PCR of a dispersed sequence produces simple but ultrasensitive Wolbachia detection

BackgroundDetecting intracellular bacterial symbionts can be challenging when they persist at very low densities. Wolbachia, a widespread bacterial endosymbiont of invertebrates, is particularly challenging. Although it persists at high titers in many species, in others its densities are far below the detection limit of classic end-point Polymerase Chain Reaction (PCR). These low-titer infections can be reliably detected by combining PCR with DNA hybridization, but less elaborate strategies based on end-point PCR alone have proven less sensitive or less general.ResultsWe introduce a multicopy PCR target that allows fast and reliable detection of A-supergroup Wolbachia - even at low infection titers - with standard end-point PCR. The target is a multicopy motif (designated ARM: A-supergroup r epeat m otif) discovered in the genome of w Mel (the Wolbachia in Drosophila melanogaster). ARM is found in at least seven other Wolbachia A-supergroup strains infecting various Drosophila, the wasp Muscidifurax and the tsetse fly Glossina. We demonstrate that end-point PCR targeting ARM can reliably detect both high- and low-titer Wolbachia infections in Drosophila, Glossina and interspecific hybrids.ConclusionsSimple end-point PCR of ARM facilitates detection of low-titer Wolbachia A-supergroup infections. Detecting these infections previously required more elaborate procedures. Our ARM target seems to be a general feature of Wolbachia A-supergroup genomes, unlike other multicopy markers such as insertion sequences (IS).

Restricted Distribution and Lateralization of Mutualistic Wolbachia in the Drosophila Brain

Microbial symbionts are universal entities of all living organisms that can significantly affect host fitness traits in manifold ways but, even more fascinating, also their behaviour. Although better known from parasitic symbionts, we currently lack any cases where ‘neurotrophic’ symbionts have co‐evolved mutualistic behavioural interactions from which both partners profit. By theory, most mutualistic associations have originated from ancestral parasitic ones during their long‐term co‐evolution towards a cost–benefit equilibrium. To manipulate host behaviour in a way where both partners benefit in a reciprocal manner, the symbiont has to target and remain restricted to defined host brain regions to minimize unnecessary fitness costs. By using the classic Drosophila paulistorum model system we demonstrate that (i) mutualistic Wolbachia are restricted to various Drosophila brain areas, (ii) form bacteriocyte‐like structures within the brain, (iii) exhibit strictly lateral tropism, and (iv) finally propose that their selective neuronal infection affects host sexual behaviour adaptively.