Intracellular translocation of symbiotic bacteroids during late oogenesis and early embryogenesis of Bradysia tritici (syn. Sciara ocellaris) (Diptera: Sciaridae)

Abstract. The distribution of symbiotic prokaryotes (bacteroids) in ovarian follicles and young embryos of Bradysia ( Sciara ) was studied using light and electron microscopy. In mid-vitellogenic follicles (prior to oosome formation) isolated from 8-h-old midges, most symbionts were scattered in the ooplasm. Later, during oogenesis and concomitant with the formation of the oosome, symbionts aggregated at the posterior pole of the follicle. During early embryogenesis, large symbiont clusters were seen between the oolemma and oosome and lateral to the oosome. When the presumptive pole-cell nuclei moved into the oosome, the bacteroids became scattered in and around the prospective germ plasm, and many of them became incorporated into the pole cells. At the anterior pole, a nearly symbiontfree area could be recognized at first (anterior cone), but later on symbionts aggregated there (usually best seen in 1-h embryos). Thereafter, smaller symbiont clusters spread into more lateral parts of the thickened anterior cortex. Finally, the aggregates became dispersed when cleavage nuclei moved into the anterior egg cortex. The distribution of symbionts may reflect cytoplasmic streaming or other transport phenomena during development which could participate in oosome formation or in the histological differentiation of the anterior egg cortex.     

Symbiotic bacteria associated with stomach discs of human lice

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation "Candidatus Riesia pediculicola" for the louse symbionts.     

Distribution of Parthenogenesis-Inducing Symbionts in Ovaries and Eggs of Aphytis (Hymenoptera: Aphelinidae)

In parasitic Hymenoptera, severe sex ratio distortions are sometimes associated with the presence of symbiotic microorganisms. Symbiont-induced uniparental reproduction is found in several species of the genus Aphytis and is suspected in others. These intracellular bacteria (called Wolbachia) reside within the egg cytoplasm. In order to follow their vertical transmission, the distribution pattern of the symbionts in ovaries and eggs of two uniparental lines (A. lingnanensis and A. yanonensis) was studied by electron, confocal, and two-photon microscopy. The results obtained suggest that Wolbachia move from nurse cells to developing oocytes. In freshly laid eggs, the symbionts are concentrated in the posterior pole, away from the micropyle, and in later stages of embryogenesis they are found surrounding nuclei throughout the embryo. Received: 20 May 1997 / Accepted: 25 June 1997     

<Emphasis Type="Italic">Wolbachia</Emphasis> Replication and Host Cell Division in <Emphasis Type="Italic">Aedes albopictus</Emphasis>

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachias replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.     

Symbiotic Bacteria Associated with Stomach Discs of Human Lice

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation “Candidatus Riesia pediculicola” for the louse symbionts.     

Temporal changes of symbiont density and host fitness after rifampicin treatment in a whitefly of the Bemisia tabaci species complex

Microbial symbionts are essential or important partners to phloem‐feeding insects. Antibiotics have been used to selectively eliminate symbionts from their host insects and establish host lines with or without certain symbionts for investigating functions of the symbionts. In this study, using the antibiotic rifampicin we attempted to selectively eliminate certain symbionts from a population of the Middle East‐Asia Minor 1 whitefly of the Bemisia tabaci species complex, which harbors the primary symbiont “Candidatus Portiera aleyrodidarum” and two secondary symbionts “Candidatus Hamiltonella defensa” and Rickettsia. Neither the primary nor the secondary symbionts were completely depleted in the adults (F0) that fed for 48 h on a diet treated with rifampicin at concentrations of 1–100 μg/mL. However, both the primary and secondary symbionts were nearly completely depleted in the offspring (F1) of the rifampicin‐treated adults. Although the F1 adults produced some eggs (F2), most of the eggs failed to hatch and none of them reached the second instar, and consequently the rifampicin‐treated whitefly colony vanished at the F2 generation. Interestingly, quantitative polymerase chain reaction assays showed that in the rifampicin‐treated whiteflies, the density of the primary symbiont was reduced at an obviously slower pace than the secondary symbionts. Mating experiments between rifampicin‐treated and untreated adults demonstrated that the negative effects of rifampicin on host fitness were expressed when the females were treated by the antibiotic, and whether males were treated or not by the antibiotic had little contribution to the negative effects. These observations indicate that with this whitefly population it is not feasible to selectively eliminate the secondary symbionts using rifampicin without affecting the primary symbiont and establish host lines for experimental studies. However, the extinction of the whitefly colony at the second generation after rifampicin treatment indicates the potential of the antibiotic as a control agent of the whitefly pest.     

Wolbachia density and host fitness components in Muscidifurax uniraptor (Hymenoptera: pteromalidae)

Intracellular bacteria of the genus Wolbachia are found in a variety of arthropod hosts, where they cause various reproductive disorders. Attempts to study the fitness advantages and disadvantages of carrying these symbionts have yielded contradicting results. Using various doses of the antibiotic rifampicin, we were able to manipulate the density of Wolbachia in the uniparental parasitoid Muscidifurax uniraptor (Hymenoptera: Pteromalidae). The effect of different titers of the symbiont on the fecundity, reproductive rate, longevity, survival rate, and sex ratio of the host was measured. The data gathered show that following antibiotic treatments, the percentage of males rises at low doses of rifampicin and then drops again. The total sex ratio of offspring produced by treated mothers was positively correlated with the numbers of Wolbachia found in eggs laid by these females. No significant effects were detected with regard to the other studied fitness components. It is concluded that in M. uniraptor, Wolbachia are not posing any burden on the life history trait studied.     

Heritable endosymbionts of Drosophila

Although heritable microorganisms are increasingly recognized as widespread in insects, no systematic screens for such symbionts have been conducted in Drosophila species (the primary insect genetic models for studies of evolution, development, and innate immunity). Previous efforts screened relatively few Drosophila lineages, mainly for Wolbachia. We conducted an extensive survey of potentially heritable endosymbionts from any bacterial lineage via PCR screens of mature ovaries in 181 recently collected fly strains representing 35 species from 11 species groups. Due to our fly sampling methods, however, we are likely to have missed fly strains infected with sex ratio-distorting endosymbionts. Only Wolbachia and Spiroplasma, both widespread in insects, were confirmed as symbionts. These findings indicate that in contrast to some other insect groups, other heritable symbionts are uncommon in Drosophila species, possibly reflecting a robust innate immune response that eliminates many bacteria. A more extensive survey targeted these two symbiont types through diagnostic PCR in 1225 strains representing 225 species from 32 species groups. Of these, 19 species were infected by Wolbachia while only 3 species had Spiroplasma. Several new strains of Wolbachia and Spiroplasma were discovered, including ones divergent from any reported to date. The phylogenetic distribution of Wolbachia and Spiroplasma in Drosophila is discussed.     

Gut microbiota in nymph and adults of the giant mesquite bug (Thasus neocalifornicus) (Heteroptera: Coreidae) is dominated by Burkholderia acquired de novo every generation

The coreid bug Thasus neocalifornicus Brailovsky and Barrera, commonly known as the giant mesquite bug, is a ubiquitous insect of the southwestern United States. Both nymphs and adults are often found aggregated on mesquite trees (Prosopis spp.: Fabaceae) feeding on seedpods and plant sap. We characterized the indigenous bacterial populations of nymphs and adults of this species by using molecular and phylogenetic techniques and culturing methods. Results show that this insect's bacterial gut community has a limited diversity dominated by Burkholderia associates. Phylogenetic analysis by using 16s rRNA sequences suggests that these β-Proteobacteria are closely related to those symbionts obtained from other heteropteran midgut microbial communities but not to Burkholderia symbionts associated with other insect orders. These bacteria were absent from the eggs and were not found in all younger nymphs, suggesting that they are acquired after the insects have hatched. Rearing experiments of nymphs with potentially Burkholderia contaminated soil suggested that if this symbiont is not acquired, giant mesquite bugs experience higher mortality. Egg, whole-body DNA extractions of younger nymphs, and midgut DNA extractions of fifth-instar nymphs and adults also revealed the presence of α-Proteobacteria from the Wolbachia genus. However, this bacterium was also present in reproductive organs of adults, indicating that this symbiont is not specific to the gut.     

Endosymbionts: factors on egg pattern formation

Elimination of the intracellular symbionts of Euscelis plebejus either by X-ray irradiation of the posterior pole of the freshly laid egg or by interruption of egg infection by application of tetracycline or lysozyme to female leafhoppers leads to the production of embryos without abdomens, ‘head-embryos’.Homogenates of symbiont-free eggs and symbiont-containing eggs in the state of invagination have a pH of 7·5±0·2 and 7·0±0·2 and an osmotic pressure (pO) of 8·3±0·2 and 7·8±0·2, respectively. The presence of symbionts leads to a decrease of both the pH and pO.These data indicate that the correct formation of the posterior gradient, necessary for normal abdomen development, is dependent on the presence of endosymbionts at the posterior pole. It is possible that the symbionts change the pH and pO of the posterior gradient. These results are consistent with a hypothetical model of early differentiation of the Euscelis egg.     

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.     

Virulence, multiple infections and regulation of symbiotic population in the Wolbachia-Asobara tabida symbiosis

The density and regulation of microbial populations are important factors in the success of symbiotic associations. High bacterial density may improve transmission to the next generation, but excessive replication could turn out to be costly to the host and result in higher virulence. Moreover, differences in virulence may also depend on the diversity of symbionts. Using the maternally transmitted symbiont Wolbachia, we investigated how bacterial density and diversity are regulated and influence virulence in host insects subject to multiple infection. The model we used was the wasp Asobara tabida that naturally harbors three different Wolbachia strains, of which two are facultative and induce cytoplasmic incompatibility, whereas the third is necessary for the host to achieve oogenesis. Using insect lines infected with different subsets of Wolbachia strains, we show that: (i) some traits of A. tabida are negatively affected by Wolbachia; (ii) the physiological cost increases with the number of co-infecting strains, which also corresponds to an increase in the total bacterial density; and (iii) the densities of the two facultative Wolbachia strains are independent of one another, whereas the obligatory strain is less abundant when it is alone, suggesting that there is some positive interaction with the other strains.     

Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species.     

A bacterial symbiont in the Bacteroidetes induces cytoplasmic incompatibility in the parasitoid wasp Encarsia pergandiella

Vertically transmitted symbionts of arthropods have been implicated in several reproductive manipulations of their hosts. These include cytoplasmic incompatibility (CI), parthenogenesis induction in haplodiploid species (PI), feminization and male killing. One symbiont lineage in the alpha-Proteobacteria, Wolbachia, is the only bacterium known to cause all of these effects, and has been thought to be unique in causing CI, in which the fecundity of uninfected females is reduced after mating with infected males. Here, we provide evidence that an undescribed symbiont in the Bacteroidetes group causes CI in a sexual population of the parasitic wasp Encarsia pergandiella. Wasps were crossed in all four possible combinations of infected and uninfected individuals. In the cross predicted to be incompatible, infected (I) males x uninfected (U) females, progeny production was severely reduced, with these females producing only 12.6% of the number of progeny in other crosses. The incompatibility observed in this haplodiploid species was the female mortality type; dissections showed that most progeny from the incompatible cross died as eggs. The 16S rDNA sequence of this symbiont is 99% identical to a parthenogenesis-inducing symbiont in other Encarsia, and 96% identical to a feminizing symbiont in haplodiploid Brevipalpus mites. Thus, this recently discovered symbiont lineage is capable of inducing three of the four principal manipulations of host reproduction known to be caused by Wolbachia.     

Dynamics of multiple symbiont density regulation during host development: tsetse fly and its microbial flora

Symbiotic associations often enhance hosts' physiological capabilities, allowing them to expand into restricted terrains, thus leading to biological diversification. Stable maintenance of partners is essential for the overall biological system to succeed. The viviparous tsetse fly (Diptera: Glossinidae) offers an exceptional system to examine factors that influence the maintenance of multiple symbiotic organisms within a single eukaryotic host. This insect harbours three different symbionts representing diverse associations, coevolutionary histories and transmission modes. The enterics, obligate mutualist Wigglesworthia and beneficial Sodalis, are maternally transmitted to the intrauterine larvae, while parasitic Wolbachia infects the developing oocyte. In this study, the population dynamics of these three symbionts were examined through host development and during potentially disruptive events, including host immune challenge, the presence of third parties (such as African trypanosomes) and environmental perturbations (such as fluctuating humidity levels). While mutualistic partners exhibited well-regulated density profiles over different host developmental stages, parasitic Wolbachia infections varied in individual hosts. Host immune status and the presence of trypanosome infections did not impact the steady-state density levels observed for mutualistic microbes in either sex, while these factors resulted in an increase in Wolbachia density in males. Interestingly, perturbation of the maternal environment resulted in the deposition of progeny harbouring greater overall symbiont loads. The regulation of symbiont density, arising from coadaptive processes, may be an important mechanism driving inter-specific relations to ensure their competitive survival and to promote specialization of beneficial associations.     

Wolbachia distribution and cytoplasmic incompatibility during sperm development: the cyst as the basic cellular unit of CI expression

The growth and distribution of the intracellular microbe Wolbachia pipientis during spermatogenesis in several different host/symbiont genetic combinations in Drosophila melanogaster and Drosophila simulans is described. Considerable intra- and inter-strain variation in Wolbachia density and tissue distribution was observed. Wolbachia were found inside spermatocytes and spermatids or within the somatic cyst cells surrounding the germ cells. Some strains displayed both tissue distributions. High rates of cytoplasmic incompatibility (CI) are correlated with high levels of Wolbachia only when spermatocytes and/or spermatids harbor the microbe. Wolbachia infection of somatic cyst cells, although sometimes present at high levels, did not result in significant CI expression. CI-inducing Wolbachia strains within D. simulans showed no distinguishable differences in distribution or density within infected spermatids. To dissect the relative contribution of host and symbiont to the expression of CI, Wolbachia from various host strains known to exhibit varying levels of CI were introgressed into new uninfected host genetic backgrounds. These introgression experiments confirm that the mod(+)/mod(-) phenotype is an intrinsic Wolbachia trait and is not determined by host factors. The level of sperm modification in those lines harboring Wolbachia capable of modifying sperm, however, is influenced by host genetic background. These results form the basis of the Wolbachia Infected Spermatocyte/Spermatid Hypothesis (WISSH). According to WISSH, Wolbachia infection in spermatocytes and then spermatids during sperm development is required for CI expression.     

Maternally inherited male-killing microorganisms may confound interpretation of mitochondrial DNA variability

Variation in mitochondrial DNA is often used to trace the evolutionary history of populations and species. We here discuss the effect of infection with cytoplasmically inherited male-lethal symbionts on mitochondrial genome evolution. Male-lethal symbionts spread when killing male hosts increases the lifetime reproductive success of sibling female hosts. This increase in the survivorship of daughters from individuals bearing a male-killer will produce a concomitant increase in the frequency of mitotypes associated with the male-killer. If horizontal transmission of the microoorganism is rare and population sizes not very small, then linkage disequilibrium between microorganism and particular mitotypes will result in a reproduction of within-population mitochondrial variability both because of a selective sweep during the spread of such a micro-organism, and also at equilibrium. Male-killing symbionts may thus confound the use of mtDNA variability in estimation of population parameters. We discuss the differences between the effects of male-killers and the cytoplasmic incompatibility-inducing symbiont Wolbachia , and the possibility that estimation of gene flow between populations may also be confounded by symbiont presence.     

Strain-specific regulation of intracellular Wolbachia density in multiply infected insects

Vertically transmitted symbionts suffer a severe reduction in numbers when they pass through host generations, resulting in genetic homogeneity or even clonality of their populations. Wolbachia endosymbionts that induce cytoplasmic incompatibility in their hosts depart from this rule, because cytoplasmic incompatibility actively maintains multiple infection within hosts. Hosts and symbionts are thus probably under peculiar selective pressures that must shape the way intracellular bacterial populations are regulated. We studied the density and location of Wolbachia within adult Leptopilina heterotoma, a haplodiploid wasp that is parasitic on Drosophila and that is naturally infected with three Wolbachia strains, but for which we also obtained one simply infected and two doubly infected lines. Comparison of these four lines by quantitative polymerase chain reaction using a real-time detection system showed that total Wolbachia density varies according to the infection status of individuals, while the specific density of each Wolbachia strain remains constant regardless of the presence of other strains. This suggests that Wolbachia strains do not compete with one another within the same host individual, and that a strain-specific regulatory mechanism is operating. We discuss the regulatory mechanisms that are involved, and how this process might have evolved as a response to selective pressures acting on both partners.     

Parasitoid bacterial symbionts as markers of within‐host competitive outcomes: superparasitoid advantage and sex ratio bias

1. Bacterial symbionts have the potential to alter insect fitness, which could influence insect competitive ability. To investigate this possibility, within‐host competitions were staged between individuals of the parasitoid, Encarsia inaron Walker, that were differentially infected with the bacterial symbionts Cardinium and Wolbachia. 2. When parasitoids of different infection status parasitised the same whitefly host, there was no evidence that symbiont infection influenced the outcome of competition. 3. Using symbionts as markers, a significant advantage for the second (superparasitising) wasp was detected when eggs were deposited within 4 h of one another, probably as a result of ovicidal behaviour by the superparasitoid, but not when eggs were deposited 1 day apart. Additionally, the emerged sex ratio of superparasitoid offspring was male biased when eggs were deposited 4 h after the initial eggs, but had an even sex ratio when deposited 1 day later. 4. The present study demonstrates the potential utility of symbiont infection as a marking system for investigating within‐host competition among parasitoids.     

Distribution of the bacterial symbiont Cardinium in arthropods

Abstract 'Candidatus Cardinium', a recently described bacterium from the Bacteroidetes group, is involved in diverse reproduction alterations of its arthropod hosts, including cytoplasmic incompatibility, parthenogenesis and feminization. To estimate the incidence rate of Cardinium and explore the limits of its host range, 99 insect and mite species were screened, using primers designed to amplify a portion of Cardinium 16S ribosomal DNA (rDNA). These arthropods were also screened for the presence of the better-known reproductive manipulator, Wolbachia. Six per cent of the species screened tested positive for Cardinium, compared with 24% positive for Wolbachia. Of the 85 insects screened, Cardinium was found in four parasitic wasp species and one armoured scale insect. Of the 14 mite species examined, one predatory mite was found to carry the symbiont. A phylogenetic analysis of all known Cardinium 16S rDNA sequences shows that distantly related arthropods can harbour closely related symbionts, a pattern typical of horizontal transmission. However, closely related Cardinium were found to cluster among closely related hosts, suggesting host specialization and horizontal transmission among closely related hosts. Finally, the primers used revealed the presence of a second lineage of Bacteroidetes symbionts, not related to Cardinium, in two insect species. This second symbiont lineage is closely allied with other arthropod symbionts, such as Blattabacterium, the primary symbionts of cockroaches, and male-killing symbionts of ladybird beetles. The combined data suggest the presence of a diverse assemblage of arthropod-associated Bacteroidetes bacteria that are likely to strongly influence their hosts' biology.