Wolbachia prophage DNA adenine methyltransferase genes in different Drosophila-Wolbachia associations

Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studying of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility.     

Infection polymorphism and cytoplasmic incompatibility in Hymenoptera-Wolbachia associations

Most cases of Wolbachia infection so far documented in haplodiploid Hymenoptera are associated with parthenogenesis induction. Only three examples of Wolbachia-mediated cytoplasmic incompatibility (CI) have been reported, resulting either in haploidisation of fertilised eggs, which develop into viable males, or in their death. To better document this variability, we studied two new Wolbachia-wasp associations involving Drosophila parasitoids. In Trichopria cf. drosophilae, individuals are infected by two different Wolbachia variants, populations are nearly totally infected, and Wolbachia induces incomplete CI resulting in death of the fertilised eggs. On the other hand, Pachycrepoideus dubius harbours only one bacterial variant, populations are polymorphic for infection, and Wolbachia has no detectable effect. These two cases show that the range of variation in Wolbachia's effects in Hymenoptera is as wide as in diploids, extending from complete CI to an undetectable effect. Cases so far studied show some parallel between the strength of incompatibility, the number of Wolbachia variants infecting each wasp, and the natural infection frequency. These empirical data support theoretical models predicting evolution of CI towards lower levels, resulting in the decline and ultimate loss of infection, and place multiple infections as being an important factor in the evolution of host-Wolbachia associations.     

Cytoplasmic incompatibility and multiple symbiont infection in the ash whitefly parasitoid, Encarsia inaron

Terrestrial arthropods are commonly infected with maternally inherited symbionts that cause reproductive incompatibilities between hosts with differing infection status. Such symbionts can have major effects on the efficacy of a biological control program if releases are comprised of mixtures of differentially infected individuals. In this study, the ash whitefly parasitoid, Encarsia inaron (Hymenoptera: Aphelinidae) from Arizona was surveyed for the presence of heritable bacterial symbionts; experiments were also performed to test for two phenotypes known to be caused by Encarsia symbionts—cytoplasmic incompatibility and changes in oviposition behavior and host use. E. inaron has successfully reduced ash whitefly to non-pest status in all three locations it has been released (California, Arizona, and North Carolina) and is also notable as one of the only Encarsia species that is not autoparasitic, with both male and female wasps developing as primary parasitoids of whiteflies. We show that E. inaron is infected with both Wolbachia and Cardinium. While there was no effect of the symbionts on oviposition behavior or host use, crosses between doubly infected male wasps and uninfected females resulted in a severe reduction in the number of female offspring; male offspring production was unaffected. This study thus serves as a further warning that ascribing a phenotype to a symbiont with confidence depends on eliminating the possibility of a mixed infection, and establishes E. inaron as a useful model for dissecting Wolbachia–Cardinium interactions.     

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.     

Wolbachia infection and incompatibility dynamics in experimental selection lines

High and low levels of Wolbachia-induced cytoplasmic incompatibility (CI) were selected for in the parasitic wasp Nasonia vitripennis, in the single-infected strain Ti277. After nine generations of selection, males from lines selected for high incompatibility level (HI lines) were significantly more incompatible with uninfected females (AsymC) than the maternal strain. The reverse response, a full compatibility with AsymC, was observed in eight out of 12 lines selected for low incompatibility (LO lines), correlated with loss of Wolbachia infection. Bacterial density estimates in the eggs of some HI lines increased significantly. The procedure for line maintenance resulted in introgression of AsymC nuclear genome into the Ti277 background. Significant changes of CI level and bacterial density due to the introgression were also observed in the control lines, possibly reflecting an effect of host genotype on bacterial density and CI. After selection had been relaxed for six generations, bacterial density in the five high-infected HI lines declined back to a level comparable to the other lines. The data are consistent with the ‘bacterial dosage’ model, but with an upper threshold of bacterial infection above which there is no correlation between infection level and CI level. We further investigate the maternal transmission of bacterial density by a mother–daughter regression on bacterial density. The pattern observed is consistent with a density dependent regulation of bacterial numbers around an ‘equilibrium’ density, independent of any effects of CI. The equilibrium value is likely to be determined by both bacterial strain and host genotype.     

DDK egg-foreign sperm incompatibility in mice is not between the pronuclei

A high rate of normal postimplantation development was achieved when the pronuclei of embryos from matings of DDK females with (CBA X C57BL/6J)F1 males were transplanted into enucleated embryos of non-DDK origin. This shows that the DDK egg cytoplasm, not the maternal pronucleus, is involved in the late preimplantation-lethal incompatibility.     

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.     

Cytoplasmic incompatibility following somatic cell fusion inGriffithsia pacifica Kylin,a red alga

Summary Somatic cell fusion between two isolates ofG. pacifica is followed by a cytoplasmic incompatibility reaction (CIR) in the cytoplasm donated by only one of the isolates. This CIR is characterized by the aggregation, fusion and lysis of chloroplasts of the sensitive strain; the chloroplasts of the other strain are unaffected. In addition, the nuclei of both strains retain a normal distribution during the fusion and lysis events. Cell elongation and nuclear division stop in CIR-affected cells. The CIR begins in the hybrid cell and then appears sequentially in adjacent cells of the sensitive strain; this transfer occurs only between living cells which share a crosswall. There is a lag between hybrid cell formation and the initiation of the CIR. This lag is more than 3 times as long at 17 C than at 24 C; over this range, the rate of movement of the CIR along a filament is temperature-insensitive. Thus it appears that a temperature dependent process, perhaps the synthesis of CIR-inducing agents, is required for the initiation of the CIR; subsequent movement of such agents appears to occur by diffusion.Abbreviations CIR Cytoplasmic incompatibility reaction - HC hybrid cell - 1st SC first shoot cell - 2nd SC second shoot cell - 3rd SC third shoot cell - Pac-PP Puerto Penasco isolate - Pac-BC British Columbia isolate This research was supported in part by National Science Foundation Grant PCM 7823240 to SDW; NICHD Developmental Biology Training Grant ST 32 HDO 71835, Traineeship to DJK and a State of Washington Graduate Opportunities for Women and Minorities to DJK.     

A recessive soybean cyst nematode allele for incompatibility with soybean PI 88287

The objective of this research was to characterise the degree of dominance of a soybean cyst nematode (Heterodera glycines) allele for incompatibility which interacts with a recessive soybean (Glycine max) allele for incompatibility to prevent the formation of cysts. Crosses of inbred nematode populations were made and the F, and F, populations evaluated for the numbers of cysts they could produce on several soybean lines. The nematode gene for avirulence interacts with the one recessive gene for resistance in soybean line PI 88287 and also appears to be recessive. This is the first example of a recessive-recessive gene-for-gene interaction; genes for avirulence and resistance are usually dominant. The difficulties of doing definitive genetic studies with cyst nematodes are discussed.     

Cytoplasmic incompatibility in allopatric field populations of the small brown planthopper, Laodelphax striatellus, in Japan

The small brown planthopper, Laodelphax striatellus (Homoptera, Delphacidae), shows reproductive cytoplasmic incompatibility between laboratory-cultured strains. Wild planthoppers, were crossed for the purpose of examining the incompatibility principle in allopatric field populations. On the basis of the incompatibility recorded in field populations, Japanese planthopper populations were divided into two groups: one from the north-eastern part and one from the western part of Japan. Crosses between the females of the former group and males of the latter gave infertile eggs, showing unidirectional incompatibility. A small number of eggs from incompatible pairs, however, developed normally.

---

Résumé Laodelphax striatellus présente une incompatibilité cytoplasmique reproductive entre souches de laboratoire. Des individus de la nature ont été croisés pour examiner l'incompatibilité entre populations allopatriques récoltées dans les champs. Sur la base de l'incompatibilité observée avec les populations naturelles, les populations japonaises ont été divisées en deux groupes: un pour la partie nordest du Japon, l'autre pour la partie occidentale. Les croisements entre femelles du premier groupe et mâles du second ont donné des oeufs stériles, montrant une incompatibilité unilatérale. Un petit nombre d'oeufs de couples incompatibles se sont cependant développés normalement.

     

Influence of aging on cytoplasmic incompatibility, sperm modification and Wolbachia density in Culex pipiens mosquitoes

Wolbachia are maternally inherited endocellular bacteria, widespread in invertebrates and capable of altering several aspects of host reproduction. Cytoplasmic incompatibility (CI) is commonly found in arthropods and induces hatching failure of eggs from crosses between Wolbachia-infected males and uninfected females (or females infected by incompatible strains). Several factors such as bacterial and host genotypes or bacterial density contribute to CI strength and it has been proposed, mostly from Drosophila data, that older males have a lower Wolbachia load in testes which, thus, induces a lighter CI. Here, we challenge this hypothesis using different incompatible Culex pipiens mosquito strains and show that CI persists at the same intensity throughout the mosquito life span. Embryos from incompatible crosses showed even distributions of abortive phenotypes over time, suggesting that host ageing does not reduce the sperm-modification induced by Wolbachia. CI remained constant when sperm was placed in the spermathecae of incompatible females, indicating that sperm modification is also stable over time. The capacity of infected females to rescue CI was independent of age. Last, the density of Wolbachia in whole testes was highly strain-dependent and increased dramatically with age. Taken together, these data stress the peculiarity of the C.pipiens/Wolbachia interaction and suggest that the bacterial dosage model should be rejected in the case of this association.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Curing of four different plasmids in Yersinia pestis using plasmid incompatibility

Aims: Plasmids are critical for the pathogenicity of Yersinia pestis. In order to carry out a systematic investigation of their role in pathogenesis, we cured plasmids from Y. pestis. Methods and Results: Each plasmid’s replicon of Y. pestis was cloned into plasmid pEX18Gm containing a counter‐selectable sacB gene, and was then introduced into Y. pestis strain 201 by electroporation. Strains containing recombinant plasmids were cultivated under antibiotic selection. The resultant plasmid‐curing colonies, identified by specific polymerase chain reactions, were then cured off pEX18Gm under sucrose pressure. This method was used to successfully cure all four plasmids of Y. pestis, singly or in different combinations. Conclusions: Naturally evolving plasmids in Y. pestis are difficult to remove by conventional curing methods. We employed a method based on plasmid incompatibility to cure the plasmids from Y. pestis, which confirmed the efficacy of this method for curing plasmids with different types of replicons from one bacterium. Significance and Impact of the Study: There have been no reports on the curing of multiple plasmids by using replication mechanisms from one bacterium with this technique. In the present study, we were able to successfully apply this methodology to cure four plasmids from Y. pestis, confirming its feasibility.     

Two different XY-quadrivalent associations and impairment of fertility in men

One sterile and one subfertile man with balanced reciprocal autosomal translocations, a t(9;15), and a t(14;21), were analyzed using whole mount pachytene spreads, histology, and semen analyses. In both probands with different types of quadrivalent complexes lack of pairing near the translocation breakpoints and significant associations with XY bivalents were found. Variability in the frequency of XY-quadrivalent contacts and an increase in late pachytene to 52% in t(9;15) and 90% in t(14;21) could be observed. The lower rate was associated with reduced postmeiotic spermatogenesis and the higher rate with complete spermatogenic arrest. In both translocation carriers the XY-quadrivalent association is considered to be the main cause for testicular malfunction rather than nonpaired segments in the multivalent complexes.     

Adaptive evolution of sperm bindin tracks egg incompatibility in neotropical sea urchins of the genus Echinometra

Bindin is a gamete recognition protein known to control species-specificsperm-egg adhesion and membrane fusion in sea urchins. Previousanalyses have shown that diversifying selection on bindin aminoacid sequence is found when gametically incompatible speciesare compared, but not when species are compatible. The presentstudy analyzes bindin polymorphism and divergence in the threeclosely related species of Echinometra in Central America: E.lucunter and E. viridis from the Caribbean, and E. vanbruntifrom the eastern Pacific. The eggs of E. lucunter have evolveda strong block to fertilization by sperm of its neotropicalcongeners, whereas those of the other two species have not.As in the Indo-West Pacific (IWP) Echinometra, the neotropicalspecies show high intraspecific bindin polymorphism in the samegene regions as in the IWP species. Maximum likelihood analysisshows that many of the polymorphic codon sites are under mildpositive selection. Of the fixed amino acid replacements, mosthave accumulated along the bindin lineage of E. lucunter. Weanalyzed the data with maximum likelihood models of variationin positive selection across lineages and codon sites, and withmodels that consider sites and lineages simultaneously. Ourresults show that positive selection is concentrated along theE. lucunter bindin lineage, and that codon sites with aminoacid replacements fixed in this species show by far the highestsignal of positive selection. Lineage-specific positive selectionparalleling egg incompatibility provides support that adaptiveevolution of sperm proteins acts to maintain recognition ofbindin by changing egg receptors. Because both egg incompatibilityand bindin divergence are greater between allopatric speciesthan between sympatric species, the hypothesis of selectionagainst hybridization (reinforcement) cannot explain why adaptiveevolution has been confined to a single lineage in the AmericanEchinometra. Instead, processes acting to varying degrees withinspecies (e.g., sperm competition, sexual selection, and sexualconflict) are more promising explanations for lineage-specificpositive selection on bindin.     

Wolbachia-induced unidirectional cytoplasmic incompatibility and the stability of infection polymorphism in parapatric host populations

Wolbachia are intracellular, maternally inherited bacteria that are widespread among arthropods and commonly induce a reproductive incompatibility between infected male and uninfected female hosts known as unidirectional cytoplasmic incompatibility (CI). If infected and uninfected populations occur parapatrically, CI acts as a post-zygotic isolation barrier. We investigate the stability of such infection polymorphisms in a mathematical model with two populations linked by migration. We determine critical migration rates below which infected and uninfected populations can coexist. Analytical solutions of the critical migration rate are presented for mainland-island models. These serve as lower estimations for a more general model with two-way migration. The critical migration rate is positive if either Wolbachia causes a fecundity reduction in infected female hosts or its transmission is incomplete, and is highest for intermediate levels of CI. We discuss our results with respect to local adaptations of the Wolbachia host, speciation, and pest control.     

Cytoplasmic/nuclear localization of tuberin in different cell lines

Summary. Tuberous sclerosis (TSC) is an autosomal dominantly inherited disease affecting 1 in 6000 individuals. The TSC gene products, hamartin and tuberin, form a complex, of which tuberin is assumed to be the functional component being involved in a wide variety of different cellular processes. Tuberin has been demonstrated to be localized to both, the cytoplasm and the nucleus. The cytoplasmic/nuclear localization of tuberin is known to be regulated by the serine/threonine protein kinase Akt. Akt also regulates the cytoplasmic/nuclear localization of the cyclin-dependent kinase inhibitor p27. In this study the localization of these two Akt-regulated proteins was analysed in different cell lines.