Distribution, expression, and motif variability of ankyrin domain genes in Wolbachia pipientis

The endosymbiotic bacterium Wolbachia pipientis infects a wide range of arthropods, in which it induces a variety of reproductive phenotypes, including cytoplasmic incompatibility (CI), parthenogenesis, male killing, and reversal of genetic sex determination. The recent sequencing and annotation of the first Wolbachia genome revealed an unusually high number of genes encoding ankyrin domain (ANK) repeats. These ANK genes are likely to be important in mediating the Wolbachia-host interaction. In this work we determined the distribution and expression of the different ANK genes found in the sequenced Wolbachia wMel genome in nine Wolbachia strains that induce different phenotypic effects in their hosts. A comparison of the ANK genes of wMel and the non-CI-inducing wAu Wolbachia strain revealed significant differences between the strains. This was reflected in sequence variability in shared genes that could result in alterations in the encoded proteins, such as motif deletions, amino acid insertions, and in some cases disruptions due to insertion of transposable elements and premature stops. In addition, one wMel ANK gene, which is part of an operon, was absent in the wAu genome. These variations are likely to affect the affinity, function, and cellular location of the predicted proteins encoded by these genes.

Wolbachia density and virulence attenuation after transfer into a novel host

The factors that control replication rate of the intracellular bacterium Wolbachia pipientis in its insect hosts are unknown and difficult to explore, given the complex interaction of symbiont and host genotypes. Using a strain of Wolbachia that is known to over-replicate and shorten the lifespan of its Drosophila melanogaster host, we have tracked the evolution of replication control in both somatic and reproductive tissues in a novel host/Wolbachia association. After transinfection (the transfer of a Wolbachia strain into a different species) of the over-replicating Wolbachia popcorn strain from D. metanogaster to Drosophila simulans, we demonstrated that initial high densities in the ovaries were in excess of what was required for perfect maternal transmission, and were likely causing reductions in reproductive fitness. Both densities and fitness costs associated with ovary infection rapidly declined in the generations after transinfection. The early death effect in D. simulans attenuated only slightly and was comparable to that induced in D. metanogaster. This study reveals a strong host involvement in Wolbachia replication rates, the independence of density control responses in different tissues, and the strength of natural selection acting on reproductive fitness.

Wolbachia pipientis: bacterial density and unidirectional cytoplasmic incompatibility between infected populations of Aedes albopictus

Unidirectional cytoplasmic incompatibility is seen when certain Wolbachia-infected insect populations are crossed. Two hypotheses might explain this phenomenon: superinfections with mutually incompatible strains of Wolbachia producing incompatibility when crossed to individuals infected with only a single bacterial strain or, alternatively, a bacterial dosage model, with differences in Wolbachia densities responsible for the incompatibility. A quantitative PCR assay was set up as a general method to compare Wolbachia densities between populations. Using this assay in unidirectionally incompatible stocks of the mosquito Aedes albopictus, we have determined that densities are significantly higher in Houston than in the Mauritius and Koh Samui stocks. This is consistent with a dosage model for the observed crossing patterns, but does not rule out the possibility that superinfection is the primary cause of the incompatibility.

Bidirectional incompatibility between conspecific populations of Drosophila simulans

Cytoplasmic incompatibility (CI) describes the phenomenon whereby eggs fertilized by sperm from insects infected with a rickettsial endosymbiont fail to hatch. Unidirectional CI between conspecific populations of insects is a well documented phenomenon. Bidirectional CI has, however, only been described in mosquito populations, and recently between closely related species of parasitic wasps, where it is of interest as both an unusual form of reproductive isolation and as a potential means of insect population suppression. Here we report on the first known example of bidirectional CI between conspecific populations of Drosophila simulans. Further, we show that defects as early as the first cleavage division are associated with CI. This observation suggests that the cellular basis of CI involves disruption of processes before or during zygote formation and that CI arises from defects in the structure and/or function of the sperm during fertilization.

Morphological variation and reproductive incompatibility of three coconut-mite-associated populations of predatory mites identified as Neoseiulus paspalivorus (Acari: Phytoseiidae)

Predatory mites identified as Neoseiulus paspalivorus DeLeon (Phytoseiidae) have been considered as agents for classical biological control of the coconut mite, Aceria guerreronis Keifer (Eriophyidae), in Africa and elsewhere. Preliminary identification of geographically distinct populations as belonging to the same species (N. paspalivorus) was based on their morphological similarity. However, laboratory studies recently conducted have shown large differences in feeding behaviors and biological characteristics among individuals collected from three geographic origins: Brazil (South America), Benin and Ghana (West Africa). As morphologically similar specimens do not necessarily belong to the same species, we evaluated under laboratory conditions, reproductive compatibility between the specimens from three geographic locations to ascertain their conspecificity. Morphological measurements were also made to determine whether there is a means of discriminating between them. Inter-population crosses showed complete reproductive isolation between the three geographic populations, but interpopulation discontinuities in morphometric characters were absent. These results indicate that the tested specimens are distinct biological entities despite morphological similarity. Further molecular genetic studies are therefore proposed, including screening for endosymbionts and assessment of genetic differentiation, to determine the cause of reproductive incompatibility and to clarify the taxonomic relationship between those populations.

Incompatibility of Macroscopic Local Realism with Quantum Mechanics in Measurements with Macroscopic Uncertainties

We show that quantum mechanics predicts a contradiction with local hidden variable theories for photon number measurements which have limited resolving power, to the point of imposing an uncertainty in the photon number result which is macroscopic in absolute terms. We show how this can be interpreted as a failure of a new premise, macroscopic local realism.

Host age effect and expression of cytoplasmic incompatibility in field populations of Wolbachia-superinfected Aedes albopictus

The Asian tiger mosquito, Aedes albopictus (Skuse), is a known vector of dengue in South America and Southeast Asia. It is naturally superinfected with two strains of Wolbachia endosymbiont that are able to induce cytoplasmic incompatibility (CI). In this paper, we report the strength of CI expression in crosses involving field-caught males. CI expression was found to be very strong in all crosses between field males and laboratory-reared uninfected or wAlbA infected young females. In addition, crossing experiments with laboratory colonies showed that aged super- infected males could express strong CI when mated with young uninfected or wAlbA infected females. These results provide additional evidence that the CI properties of Wolbachia infecting Aedes albopictus are well suited for applied strategies that seek to utilise Wolbachia for host population modification.

Wolbachia infection and expression of cytoplasmic incompatibility in Armigeres subalbatus (Diptera: Culicidae)

Polymerase chain reaction screening revealed that Armigeres subalbatus (Coquillett), a vector of filariasis, was infected with the intracellular bacteria Wolbachia. Laboratory crosses between infected males and uninfected females resulted in less than half the number of offspring than control crosses between uninfected individuals when young (2- to 3-d-old) males were used in the cross. However, incompatibility was lost when old (14- to 17-d-old) males were used. Field-collected females did not show detectable cytoplasmic incompatibility, and this may be because of the age at which males mate in the field. We used head pigment fluorescence levels to age field males collected from mating swarms, and found that 25-63% of swarming males were older than 13 d. Male age may be one factor influencing the observed low levels of cytoplasmic incompatibility detected in the field.

A stable triple Wolbachia infection in Drosophila with nearly additive incompatibility effects

Drosophila simulans strains infected with three different Wolbachia strains were generated by experimental injection of a third symbiont into a naturally double-infected strain. This transfer led to a substantial increase in total Wolbachia density in the host strain. Each of the three symbionts was stably transmitted in the presence of the other two. Triple-infected males were incompatible with double-infected females. No evidence was obtained for interference between modification effects of the different Wolbachia strains in males. Some incompatibility was observed between triple-infected males and females. However, this incompatibility reaction is not a specific property of triple-infected flies, because it was also observed in double-infected strains.

Wolbachia infections and the expression of cytoplasmic incompatibility in Drosophila sechellia and D. mauritiana

Various stocks of Drosophila mauritiana and D. sechellia were found to be infected with Wolbachia, a Rickettsia-like bacterium that is known to cause cytoplasmic incompatibility and other reproductive abnormalities in arthropods. Testing for the expression of cytoplasmic incompatibility in these two species showed partial incompatibility in D. sechellia but no expression of incompatibility in D. mauritiana. To determine whether absence of cytoplasmic incompatibility in D. mauritiana was due to either the bacterial or host genome, we transferred bacteria from D. mauritiana into an uninfected strain of D. simulans, a host species known to express high levels of incompatibility with endogenous Wolbachia. We also performed the reciprocal transfer of the natural D. simulans Riverside infection into a tetracycline-treated stock of D. mauritiana. In each case, the ability to express incompatibility was unaltered by the different host genetic background. These experiments indicate that in D. simulans and D. mauritiana expression of the cytoplasmic incompatibility phenotype is determined by the bacterial strain and that D. mauritiana harbors a neutral strain of Wolbachia.

16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects

Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the α-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggests that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized.

Cytoplasmic incompatibility in Drosophila populations: influence of assortative mating on symbiont distribution

Cytoplasmic incompatibility is known to occur between strains of both Drosophila simulans and D. melanogaster. Incompatibility is associated with the infection of Drosophila with microorganismal endosymbionts. This paper reports survey work conducted on strains of D. simulans and D. melanogaster from diverse geographical locations finding that infected populations are relatively rare and scattered in their distribution. The distribution of infected populations of D. simulans appears to be at odds with deterministic models predicting the rapid spread of the infection through uninfected populations. Examination of isofemale lines from four localities in California where populations appear to be polymorphic for the infection failed to find evidence for consistent assortative mating preferences between infected and uninfected populations that may explain the basis for the observed polymorphism.

Structural analysis and molecular model of a self-incompatibility RNase from wild tomato

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S-3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S-3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S-3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S-3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.