Multiple introgressions shape mitochondrial evolutionary history in Drosophila paulistorum and the Drosophila willistoni group.

Hybridization and the consequent introgression of genomic elements is an important source of genetic diversity for biological lineages. This is particularly evident in young clades in which hybrid incompatibilities are still incomplete and mixing between species is more likely to occur. Drosophila paulistorum, a representative of the Neotropical Drosophila willistoni subgroup, is a classic model of incipient speciation. The species is divided into six semispecies that show varying degrees of pre- and post-mating incompatibility with each other. In the present study, we investigate the mitochondrial evolutionary history of D. paulistorum and the willistoni subgroup. For that, we perform phylogenetic and comparative analyses of the complete mitochondrial genomes and draft nuclear assemblies of 25 Drosophila lines of the willistoni and saltans species groups. Our results show that the mitochondria of D. paulistorum are polyphyletic and form two non-sister clades that we name α and ß. Identification and analyses of nuclear mitochondrial insertions further reveal that the willistoni subgroup has an α-like mitochondrial ancestor and strongly suggest that both the α and ß mitochondria of D. paulistorum were acquired through introgression from unknown fly lineages of the willistoni subgroup. We also uncover multiple mitochondrial introgressions across D. paulistorum semispecies and generate novel insight into the evolution of the species.

Complex effects of environment and Wolbachia infections on the life history of Drosophila melanogaster hosts.

Wolbachia bacteria are common endosymbionts of many arthropods found in gonads and various somatic tissues. They manipulate host reproduction to enhance their transmission and confer complex effects on fitness-related traits. Some of these effects can serve to increase the survival and transmission efficiency of Wolbachia in the host population. The Wolbachia-Drosophila melanogaster system represents a powerful model to study the evolutionary dynamics of host-microbe interactions and infections. Over the past decades, there has been a replacement of the ancestral wMelCS Wolbachia variant by the more recent wMel variant in worldwide D. melanogaster populations, but the reasons remain unknown. To investigate how environmental change and genetic variation of the symbiont affect host developmental and adult life-history traits, we compared effects of both Wolbachia variants and uninfected controls in wild-caught D. melanogaster strains at three developmental temperatures. While Wolbachia did not influence any developmental life-history traits, we found that both lifespan and fecundity of host females were increased without apparent fitness trade-offs. Interestingly, wMelCS-infected flies were more fecund than uninfected and wMel-infected flies. By contrast, males infected with wMel died sooner, indicating sex-specific effects of infection that are specific to the Wolbachia variant. Our study uncovered complex temperature-specific effects of Wolbachia infections, which suggests that symbiont-host interactions in nature are strongly dependent on the genotypes of both partners and the thermal environment.

Restriction of Wolbachia Bacteria in Early Embryogenesis of Neotropical Drosophila Species via Endoplasmic Reticulum-Mediated Autophagy.

Wolbachia are maternally transmitted intracellular bacteria that are not only restricted to the reproductive organs but also found in various somatic tissues of their native hosts. The abundance of the endosymbiont in the soma, usually a dead end for vertically transmitted bacteria, causes a multitude of effects on life history traits of their hosts, which are still not well understood. Thus, deciphering the host-symbiont interactions on a cellular level throughout a host's life cycle is of great importance to understand their homeostatic nature, persistence, and spreading success. Using fluorescent and transmission electron microscopy, we conducted a comprehensive analysis of Wolbachia tropism in soma and germ line of six Drosophila species at the intracellular level during host development. Our data uncovered diagnostic patterns of infections to embryonic primordial germ cells and to particular cells of the soma in three different neotropical Drosophila species that have apparently evolved independently. We further found that restricted patterns of Wolbachia tropism are determined in early embryogenesis via selective autophagy, and their spatially restricted infection patterns are preserved in adult flies. We observed tight interactions of Wolbachia with membranes of the endoplasmic reticulum, which might play a scaffolding role for autophagosome formation and subsequent elimination of the endosymbiont. Finally, by analyzing D. simulans lines transinfected with nonnative Wolbachia, we uncovered that the host genetic background regulates tissue tropism of infection. Our data demonstrate a novel and peculiar mechanism to limit and spatially restrict bacterial infection in the soma during a very early stage of host development. IMPORTANCE All organisms are living in close and intimate interactions with microbes that cause conflicts but also cooperation between both unequal genetic partners due to their different innate interests of primarily enhancing their own fitness. However, stable symbioses often result in homeostatic interaction, named mutualism, by balancing costs and benefits, where both partners profit. Mechanisms that have evolved to balance and stably maintain homeostasis in mutualistic relationships are still quite understudied; one strategy is to "domesticate" potentially beneficial symbionts by actively controlling their replication rate below a critical and, hence, costly threshold, and/or to spatially and temporally restrict their localization in the host organism, which, in the latter case, in its most extreme form, is the formation of a specialized housing organ for the microbe (bacteriome). However, questions remain: how do these mutualistic associations become established in their first place, and what are the mechanisms for symbiont control and restriction in their early stages? Here, we have uncovered an unprecedented symbiont control mechanism in neotropical Drosophila species during early embryogenesis. The fruit fly evolved selective autophagy to restrict and control the proliferation of its intracellular endosymbiont Wolbachia in a defined subset of the stem cells as soon as the host's zygotic genome is activated.

Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies.

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host's brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

Integrative taxonomy and a new species description in the sturtevanti subgroup of the Drosophila saltans group (Diptera: Drosophilidae).

Although the biological concept of species is well established in animals, sometimes the decision about the specific status of a new species is difficult and hence requires support of an integrative analysis of several character sets. To date, the species Drosophila sturtevanti, D. magalhaesi, D. milleri and D. dacunhai, belonging to the sturtevanti subgroup of the Neotropical saltans species group, are identified mainly by the aedeagus morphology, but also present some differences in spot coloration and patterning of the female sixth tergite and in the shape and size of the spermathecae, parallel to a pattern of reproductive isolation. In the present study, we describe a novel saltans group species from French Guiana belonging to the sturtevanti subgroup. Our species designation is based on an integrative approach covering (i) aedeagi and spermathecae morphology by scanning electron microscopy, (ii) analysis of female sixth-tergite color, (iii) morphometrical analysis of aedeagi and wings, (iv) analysis of partial sequence of the COI, COII and ND4 mitochondrial genes as well as (v) intercrosses for analysis of reproductive isolation. The comparative analysis of the results on these markers with those of D. sturtevanti, D. milleri and D. dacunhai supports that this line belongs to a new species of the sturtevanti subgroup that we name Drosophila lehrmanae sp. nov. in honor of Prof. Lee Ehrman´s 85th birthday.

Age, tissue, genotype and virus infection regulate Wolbachia levels in Drosophila.

The bacterial symbiont Wolbachia can protect insects against viral pathogens, and the varying levels of antiviral protection are correlated with the endosymbiont load within the insects. To understand why Wolbachia strains differ in their antiviral effects, we investigated the factors controlling Wolbachia density in five closely related strains in their natural Drosophila hosts. We found that Wolbachia density varied greatly across different tissues and between flies of different ages, and these effects depended on the host-symbiont association. Some endosymbionts maintained largely stable densities as flies aged while others increased, and these effects in turn depended on the tissue being examined. Measuring Wolbachia rRNA levels in response to viral infection, we found that viral infection itself also altered Wolbachia levels, with Flock House virus causing substantial reductions in symbiont loads late in the infection. This effect, however, was virus-specific as Drosophila C virus had little impact on Wolbachia in all of the five host systems. Because viruses have strong tissue tropisms and antiviral protection is thought to be cell-autonomous, these effects are likely to affect the virus-blocking phenomenon. However, we were unable to find any evidence of a correlation between Wolbachia and viral titres within the same tissues. We conclude that Wolbachia levels within flies are regulated in a complex host-symbiont-virus-dependent manner and this trinity is likely to influence the antiviral effects of Wolbachia.

Parallel Sequencing of Wolbachia wCer2 from Donor and Novel Hosts Reveals Multiple Incompatibility Factors and Genome Stability after Host Transfers.

The application of Wolbachia in insect pest and vector control requires the establishment of genotypically stable host associations. The cytoplasmic incompatibility (CI) inducing Wolbachia strain wCer2 naturally occurs in the cherry fruit fly Rhagoletis cerasi as co-infection with other strains and was transferred to other fruit fly species by embryonic microinjections. We obtained wCer2 genome data from its native and three novel hosts, Drosophila simulans, Drosophila melanogaster, and Ceratitis capitata and assessed its genome stability, characteristics, and CI factor (cif) genes. De novo assembly was successful from Wolbachia cell-enriched singly infected D. simulans embryos, with minimal host and other bacterial genome traces. The low yield of Wolbachia sequence reads from total genomic extracts of one multiply infected R. cerasi pupa and one singly infected C. capitata adult limited de novo assemblies but was sufficient for comparative analyses. Across hosts wCer2 was stable in genome synteny and content. Polymorphic nucleotide sites were found in wCer2 of each host; however, only one nucleotide was different between R. cerasi and C. capitata, and none between replicated D. simulans lines. The wCer2 genome is highly similar to wAu (D. simulans), wMel (D. melanogaster), and wRec (Drosophila recens). In contrast to wMel and wRec (each with one cif gene pair) and wAu (without any cif genes), wCer2 has three pairs of Type I cif genes, and one Type V cifB gene without a cifA complement. This may explain previously reported CI patterns of wCer2, including incomplete rescue of its own CI modification in three novel host species.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

The effect of Wolbachia on gene expression in Drosophila paulistorum and its implications for symbiont-induced host speciation.

BACKGROUND: The Neotropical fruit fly Drosophila paulistorum (Diptera: Drosophilidae) is a species complex in statu nascendi comprising six reproductively isolated semispecies, each harboring mutualistic Wolbachia strains. Although wild type flies of each semispecies are isolated from the others by both pre- and postmating incompatibilities, mating between semispecies and successful offspring development can be achieved once flies are treated with antibiotics to reduce Wolbachia titer. Here we use RNA-seq to study the impact of Wolbachia on D. paulistorum and investigate the hypothesis that the symbiont may play a role in host speciation. For that goal, we analyze samples of heads and abdomens of both sexes of the Amazonian, Centro American and Orinocan semispecies of D. paulistorum. RESULTS: We identify between 175 and 1192 differentially expressed genes associated with a variety of biological processes that respond either globally or according to tissue, sex or condition in the three semispecies. Some of the functions associated with differentially expressed genes are known to be affected by Wolbachia in other species, such as metabolism and immunity, whereas others represent putative novel phenotypes involving muscular functions, pheromone signaling, and visual perception. CONCLUSIONS: Our results show that Wolbachia affect a large number of biological functions in D. paulistorum, particularly when present in high titer. We suggest that the significant metabolic impact of the infection on the host may cause several of the other putative and observed phenotypes. We also speculate that the observed differential expression of genes associated with chemical communication and reproduction may be associated with the emergence of pre- and postmating barriers between semispecies, which supports a role for Wolbachia in the speciation of D. paulistorum.

Symbiont-Driven Male Mating Success in the Neotropical Drosophila paulistorum Superspecies.

Microbial symbionts are ubiquitous associates of living organisms but their role in mediating reproductive isolation (RI) remains controversial. We addressed this knowledge gap by employing the Drosophila paulistorum-Wolbachia model system. Semispecies in the D. paulistorum species complex exhibit strong RI between each other and knockdown of obligate mutualistic Wolbachia bacteria in female D. paulistorum flies triggers loss of assortative mating behavior against males carrying incompatible Wolbachia strains. Here we set out to determine whether de novo RI can be introduced by Wolbachia-knockdown in D. paulistorum males. We show that Wolbachia-knockdown D. paulistorum males (i) are rejected as mates by wild type females, (ii) express altered sexual pheromone profiles, and (iii) are devoid of the endosymbiont in pheromone producing cells. Our findings suggest that changes in Wolbachia titer and tissue tropism can induce de novo premating isolation by directly or indirectly modulating sexual behavior of their native D. paulistorum hosts.

High-sensitivity detection of cryptic Wolbachia in the African tsetse fly (Glossina spp.).

BACKGROUND: In African tsetse flies Glossina, spp. detection of bacterial symbionts such as Wolbachia is challenging since their prevalence and distribution are patchy, and natural symbiont titers can range at levels far below detection limit of standard molecular techniques. Reliable estimation of symbiont infection frequency, especially with regard to interrelations between symbionts and their potential impact on host biology, is of pivotal interest in the context of future applications for the control and eradication of Glossina-vectored African trypanosomosis. The presence or absence of symbionts is routinely screened with endpoint polymerase chain reaction (PCR), which has numerous advantages, but reaches its limits, when detecting infections at natural low titer. To not only determine presence of native tsetse symbionts but also to localize them to specific host tissues, fluorescence in situ hybridization (FISH) can be applied. However, classic FISH assays may not detect low-titer infections due to limitations in sensitivity. RESULTS: We have compared classic endpoint PCR with high-sensitivity blot-PCR. We demonstrate that the latter technique allows for clear detection of low-titer Wolbachia in the morsitans and palpalis groups while classic endpoint PCR does not. In order to localize Wolbachia in situ in high and low-titer Glossina species, we applied high-end Stellaris® rRNA-FISH. We show that with this high sensitivity method, even low amounts of Wolbachia can be traced in specific tissues. Furthermore, we highlight that more tissues and organs than previously recorded are infested with Wolbachia in subspecies of the morsitans and palpalis groups. CONCLUSIONS: Our results demonstrate that overall symbiont infection frequencies as well as the presence in specific host tissues may be underestimated when using low-sensitivity methods. To better understand the complex interrelation of tsetse flies and their native symbionts plus the pathogenic trypanosomes, it is important to consider application of a broader range of high-sensitivity detection tools.

Effect of antibiotic treatment and gamma-irradiation on cuticular hydrocarbon profiles and mate choice in tsetse flies (Glossina m. morsitans).

BACKGROUND: Symbiotic microbes represent a driving force of evolutionary innovation by conferring novel ecological traits to their hosts. Many insects are associated with microbial symbionts that contribute to their host's nutrition, digestion, detoxification, reproduction, immune homeostasis, and defense. In addition, recent studies suggest a microbial involvement in chemical communication and mating behavior, which can ultimately impact reproductive isolation and, hence, speciation. Here we investigated whether a disruption of the microbiota through antibiotic treatment or irradiation affects cuticular hydrocarbon profiles, and possibly mate choice behavior in the tsetse fly, Glossina morsitans morsitans. Four independent experiments that differentially knock down the multiple bacterial symbionts of tsetse flies were conducted by subjecting tsetse flies to ampicillin, tetracycline, or gamma-irradiation and analyzing their cuticular hydrocarbon profiles in comparison to untreated controls by gas chromatography - mass spectrometry. In two of the antibiotic experiments, flies were mass-reared, while individual rearing was done for the third experiment to avoid possible chemical cross-contamination between individual flies. RESULTS: All three antibiotic experiments yielded significant effects of antibiotic treatment (particularly tetracycline) on cuticular hydrocarbon profiles in both female and male G. m. morsitans, while irradiation itself had no effect on the CHC profiles. Importantly, tetracycline treatment reduced relative amounts of 15,19,23-trimethyl-heptatriacontane, a known compound of the female contact sex pheromone, in two of the three experiments, suggesting a possible implication of microbiota disturbance on mate choice decisions. Concordantly, both female and male flies preferred non-treated over tetracycline-treated flies in direct choice assays. CONCLUSIONS: While we cannot exclude the possibility that antibiotic treatment had a directly detrimental effect on fly vigor as we are unable to recolonize antibiotic treated flies with individual symbiont taxa, our results are consistent with an effect of the microbiota, particularly the obligate nutritional endosymbiont Wigglesworthia, on CHC profiles and mate choice behavior. These findings highlight the importance of considering host-microbiota interactions when studying chemical communication and mate choice in insects.

Wolbachia modifies thermal preference in Drosophila melanogaster.

Environmental variation can have profound and direct effects on fitness, fecundity, and host-symbiont interactions. Replication rates of microbes within arthropod hosts, for example, are correlated with incubation temperature but less is known about the influence of host-symbiont dynamics on environmental preference. Hence, we conducted thermal preference (Tp ) assays and tested if infection status and genetic variation in endosymbiont bacterium Wolbachia affected temperature choice of Drosophila melanogaster. We demonstrate that isogenic flies infected with Wolbachia preferred lower temperatures compared with uninfected Drosophila. Moreover, Tp varied with respect to three investigated Wolbachia variants (wMel, wMelCS, and wMelPop). While uninfected individuals preferred 24.4°C, we found significant shifts of -1.2°C in wMel- and -4°C in flies infected either with wMelCS or wMelPop. We, therefore, postulate that Wolbachia-associated Tp variation within a host species might represent a behavioural accommodation to host-symbiont interactions and trigger behavioural self-medication and bacterial titre regulation by the host.

Insertion sequence polymorphism and genomic rearrangements uncover hidden Wolbachia diversity in Drosophila suzukii and D. subpulchrella.

Ability to distinguish between closely related Wolbachia strains is crucial for understanding the evolution of Wolbachia-host interactions and the diversity of Wolbachia-induced phenotypes. A useful model to tackle these issues is the Drosophila suzukii - Wolbachia association. D. suzukii, a destructive insect pest, harbor a non-CI inducing Wolbachia 'wSuz' closely related to the strong CI-inducing wRi strain. Multi locus sequence typing (MLST) suggests presence of genetic homogeneity across wSuz strains infecting European and American D. suzukii populations, although different Wolbachia infection frequencies and host fecundity levels have been observed in both populations. Currently, it is not clear if these differences are due to cryptic wSuz polymorphism, host background, geographical factors or a combination of all of them. Here, we have identified geographical diversity in wSuz in D. suzukii populations from different continents using a highly diagnostic set of markers based on insertion sequence (IS) site polymorphism and genomic rearrangements (GR). We further identified inter-strain diversity between Wolbachia infecting D. suzukii and its sister species D. subpulchrella (wSpc). Based on our results, we speculate that discernible wSuz variants may associate with different observed host phenotypes, a hypothesis that demands future investigation. More generally, our results demonstrate the utility of IS and GRs in discriminating closely related Wolbachia strains.

Short and long-term evolutionary dynamics of subtelomeric piRNA clusters in Drosophila.

Two Telomeric Associated Sequences, TAS-R and TAS-L, form the principal subtelomeric repeat families identified in Drosophila melanogaster. They are PIWI-interacting RNA (piRNA) clusters involved in repression of Transposable Elements. In this study, we revisited TAS structural and functional dynamics in D. melanogaster and in related species. In silico analysis revealed that TAS-R family members are composed of previously uncharacterized domains. This analysis also showed that TAS-L repeats are composed of arrays of a region we have named "TAS-L like" (TLL) identified specifically in one TAS-R family member, X-TAS. TLL were also present in other species of the melanogaster subgroup. Therefore, it is possible that TLL represents an ancestral subtelomeric piRNA core-cluster. Furthermore, all D. melanogaster genomes tested possessed at least one TAS-R locus, whereas TAS-L can be absent. A screen of 110 D. melanogaster lines showed that X-TAS is always present in flies living in the wild, but often absent in long-term laboratory stocks and that natural populations frequently lost their X-TAS within 2 years upon lab conditioning. Therefore, the unexpected structural and temporal dynamics of subtelomeric piRNA clusters demonstrated here suggests that genome organization is subjected to distinct selective pressures in the wild and upon domestication in the laboratory.

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.

Symbionts commonly provide broad spectrum resistance to viruses in insects: a comparative analysis of Wolbachia strains.

In the last decade, bacterial symbionts have been shown to play an important role in protecting hosts against pathogens. Wolbachia, a widespread symbiont in arthropods, can protect Drosophila and mosquito species against viral infections. We have investigated antiviral protection in 19 Wolbachia strains originating from 16 Drosophila species after transfer into the same genotype of Drosophila simulans. We found that approximately half of the strains protected against two RNA viruses. Given that 40% of terrestrial arthropod species are estimated to harbour Wolbachia, as many as a fifth of all arthropods species may benefit from Wolbachia-mediated protection. The level of protection against two distantly related RNA viruses--DCV and FHV--was strongly genetically correlated, which suggests that there is a single mechanism of protection with broad specificity. Furthermore, Wolbachia is making flies resistant to viruses, as increases in survival can be largely explained by reductions in viral titer. Variation in the level of antiviral protection provided by different Wolbachia strains is strongly genetically correlated to the density of the bacteria strains in host tissues. We found no support for two previously proposed mechanisms of Wolbachia-mediated protection--activation of the immune system and upregulation of the methyltransferase Dnmt2. The large variation in Wolbachia's antiviral properties highlights the need to carefully select Wolbachia strains introduced into mosquito populations to prevent the transmission of arboviruses.

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

BACKGROUND: Detecting 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. RESULTS: We 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 repeat motif) discovered in the genome of wMel (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. CONCLUSIONS: Simple 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).

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.

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 (10(9) 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.