Variations in the sensitivity of different primers for detecting Wolbachia in Anastrepha (diptera: tephritidae)

Wolbachia are endosymbiont bacteria of the family Rickettsiacea that are widespread in invertebrates and occur between 20% and 60% of Neotropical insects. These bacteria are responsible for reproductive phenomena such as cytoplasmic incompatibility, male killing, feminization and parthenogenesis. Supergroups A and B of Wolbachia are common in insects and can be identified using primers for 16S rDNA, ftsZ and wsp; these primers vary in their ability to detect Wolbachia. The ftsZ primer was the first primer used to detect Wolbachia in Anastrepha fruit flies. The primers for 16S rDNA, ftsZ and wsp and the corresponding PCR conditions have been optimized to study the distribution of Wolbachia and their effect on the biology of Anastrepha in Brazil. In this work, we examined the ability of these primers to detect Wolbachia in Anastrepha populations from three regions in the State of São Paulo, southeastern Brazil. All of the samples were positive for Wolbachia supergroup A when screened with primers for 16S A rDNA and wsp A; the wsp B primer also gave a positive result, indicating cross-reactivity. The ftsZ primer showed a poor ability to detect Wolbachia in Anastrepha and generated false negatives in 44.9% of the samples. These findings indicate that reliable PCR detection of Wolbachia requires the use of primers for 16S rDNA and wsp to avoid cross-reactions and false negatives, and that the ftsZ primer needs to be redesigned to improve its selectivity.

Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility ( het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer ( HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated ""gene dumps'' and, perhaps, simultaneously, as "" gene factories''.

FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex

P>The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.

Influence of Phase Inversion on the Formation and Stability of One-Step Multiple Emulsions

A novel method of preparation of water-in-oil-in-micelle-containing water (W/O/W(m)) Multiple emulsions using the one-step emulsification method is reported. These multiple emulsions were normal (not temporary) and stable over a 60 day test period. Previously, reported multiple emulsion by the one-step method were abnormal systems that formed at the inversion point of simple emulsion (where there is an incompatibility in the Ostwald and Bancroft theories, and typically these are O/W/O systems). Pseudoternary phase diagrams and bidimensional process-composition (phase inversion) maps were constructed to assist in process and composition optimization. The surfactants used were PEG40 hydrogenated castor oil and sorbitan oleate, and mineral and vegetables oils were investigated. Physicochemical characterization studies showed experimentally, for the First time, the significance of the ultralow surface tension point oil multiple emulsion formation by one-step via phase inversion processes. Although the significance of ultralow surface tension has been speculated previously, to the best of our knowledge, this is the first experimental confirmation. The multiple emulsion system reported here was dependent not only upon the emulsification temperature, but also upon the component ratios, therefore both the emulsion phase inversion and the phase inversion temperature were considered to fully explain their formation. Accordingly, it is hypothesized that the formation of these normal multiple emulsions is not a result of a temporary incompatibility (at the inversion point) during simple emulsion preparation, as previously reported. Rather, these normal W/O/W(m) emulsions are a result of the simultaneous occurrence of catastrophic and transitional phase inversion processes. The formation of the primary emulsions (W/O) is in accordance with the Ostwald theory and the formation of the multiple emulsions (W/O/W(m)) is in agreement with the Bancroft theory.

Polarized photoluminescence from surface-passivated lead sulfide nanocrystals

Effective surface passivation of lead sulfide (PbS) nanocrystals (NCs) in an aqueous colloidal solution has been achieved following treatment with CdS precursors. The resultant photoluminescent emission displays two distinct components, one originating from the absorption band edge and the other from above the absorption band edge. We show that both of these components are strongly polarized but display distinctly different behaviours. The polarization arising from the band edge shows little dependence on the excitation energy while the polarization of the above-band-edge component is strongly dependent on the excitation energy. In addition, time-resolved polarization spectroscopy reveals that the above-band-edge polarization is restricted to the first couple of nanoseconds, while the band edge polarization is nearly constant over hundreds of nanoseconds. We recognize an incompatibility between the two different polarization behaviours, which enables us to identify two distinct types of surface-passivated PbS NC.

Wolbachia: Invasion biology in South Pacific butterflies

Wolbachia ensdosymbionts are well known for their ability to manipulate the population biology and development of their hosts. One of the less studied outcomes of Wolbachia infection with this symbiont is the selective killing of male embryos. Recent work on butterflies living on different South Pacific islands is beginning to help us understand the complexity of the co-evolutionary interactions between these partners.

Modifying insect population age structure to control vector-borne disease

Age is a critical determinant of the ability of most arthropod vectors to transmit a range of human pathogens. This is due to the fact that most pathogens require a period of extrinsic incubation in the arthropod host before pathogen transmission can occur. This developmental period for the pathogen often comprises a significant proportion of the expected lifespan of the vector. As such, only a small proportion of the population that is oldest contributes to pathogen transmission. Given this, strategies that target vector age would be expected to obtain the most significant reductions in the capacity of a vector population to transmit disease. The recent identification of biological agents that shorten vector lifespan, such as Wolbachia, entomopathogenic fungi and densoviruses, offer new tools for the control of vector-borne diseases. Evaluation of the efficacy of these strategies under field conditions will be possible due to recent advances in insect age-grading techniques. Implementation of all of these strategies will require extensive field evaluation and consideration of the selective pressures that reductions in vector longevity may induce on both vector and pathogen.

The Genus Wolbachia

Numerous invertebrate species form long lasting symbioses with bacteria (Buchner, 1949; Buchner, 1965). One of the most common of these bacterial symbionts is Wolbachia pipientis, which has been estimated to infect anywhere from 15–75% of all insect species (Werren et al., 1995a; West et al., 1998; Jeyaprakash and Hoy, 2000; Werren and Windsor, 2000) as well as many species of arachnids, terrestrial crustaceans and filarial nematodes (O’Neill et al., 1997a; Bandi et al., 1998). In most arthropod associations, Wolbachia act as reproductive parasites manipulating the reproduction of their hosts to enhance their own vertical transmission. There appears to be little direct fitness cost to the infected host besides the costs arising from the reproductive manipulations. However instances have been reported where Wolbachia can be either deleterious (Min and Benzer, 1997; Bouchon et al., 1998) or beneficial (Girin and Boultreau, 1995; Stolk and Stouthamer, 1995; Wade and Chang, 1995; Vavre et al., 1999b; Dedeine et al., 2001) to their hosts. Wolbachia were first described as intracellular Rickettsia-like organisms (RLOs), infecting the gonad cells of the mosquito, Culex pipiens (Hertig and Wolbach, 1924), and were later named 'Wolbachia pipientis' (Hertig, 1936). It was not until the work of Yen and Barr (Yen and Barr, 1971; Yen and Barr, 1973) that Wolbachia were implicated in causing crossing incompatibilities between different mosquito populations (Laven, 1951; Ghelelovitch, 1952). When polymerase chain reaction (PCR) diagnostics for Wolbachia became available, it became clear that this agent was both extremely widespread and also responsible for a range of different reproductive phenotypes in the different hosts it infected (O’Neill et al., 1992; Rousset et al., 1992; Stouthamer et al., 1993). The most common of these are cytoplasmic incompatibility, inducing parthenogenesis, overriding host sex-determination, and male-killing (O’Neill et al., 1997a). As of the time of this writing, more than 450 different Wolbachia strains with unique gene sequences, different phenotypes, and infecting different hosts have been deposited in GenBank and the Wolbachia host database (http://www.wolbachia.sols. uq.edu.au).

Evidence for a global Wolbachia replacement in Drosophila melanogaster

Wolbachia are maternally inherited intracellular α-Proteobacteria found in numerous arthropod and filarial nematode species [1, 2 and 3]. They influence the biology of their hosts in many ways. In some cases, they act as obligate mutualists and are required for the normal development and reproduction of the host [4 and 5]. They are best known, however, for the various reproductive parasitism traits that they can generate in infected hosts. These include cytoplasmic incompatibility (CI) between individuals of different infection status, the parthenogenetic production of females, the selective killing of male embryos, and the feminization of genetic males [1 and 2]. Wolbachia infections of Drosophila melanogaster are extremely common in both wild populations and long-term laboratory stocks [6, 7 and 8]. Utilizing the newly completed genome sequence of Wolbachia pipientis wMel [9], we have identified a number of polymorphic markers that can be used to discriminate among five different Wolbachia variants within what was previously thought to be the single clonal infection of D. melanogaster. Analysis of long-term lab stocks together with wild-caught flies indicates that one of these variants has replaced the others globally within the last century. This is the first report of a global replacement of a Wolbachia strain in an insect host species. The sweep is at odds with current theory that cannot explain how Wolbachia can invade this host species given the observed cytoplasmic incompatibility characteristics of Wolbachia infections in D. melanogaster in the field [6].

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 replication and host cell division in Aedes albopictus

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, Wolbachiarsquos 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.

Wolbachia pipientis: intracellular infection and pathogenesis in Drosophila

Wolbachia pipientis is a vertically transmitted, obligate intracellular symbiont of arthropods. The bacterium is best known for its ability to manipulate host reproductive biology where it can induce cytoplasmic incompatibility, parthenogenesis, feminization and male-killing. In addition to the various reproductive phenotypes it generates through interaction with host reproductive tissue it is also known to infect somatic tissues. However, relatively little is known about the consequences of infection of these tissues with the exception that in some hosts Wolbachia acts as a classical mutualist and in others a pathogen, dramatically shortening adult insect lifespan. Manipulation experiments have demonstrated that the severity of Wolbachia-induced effects on the host is determined by a combination of host genotype, Wolbachia strain, host tissue localization, and interaction with the environment. The recent completion of the whole genome sequence of Wolbachia pipientis wMel strain indicates that it is likely to use a type IV secretion system to establish and maintain infection in its host. Moreover, an unusual abundance of genes encoding proteins with eukaryotic-like ankyrin repeat domains suggest a function in the various described phenotypic effects in hosts.

The potential of virulent Wolbachia to modulate disease transmission by insects

A virulent strain of Wolbachia has recently been identified in Drosophila that drastically reduces adult lifespan. It has been proposed that this phenotype might be introduced into insect disease vector populations to reduce pathogen transmission. Here we model the requirements for spread of such an agent and the associated reduction in disease transmission. First, a simulation of mosquito population age structure was used to describe the age distribution of mosquitoes transmitting dengue virus. Second, given varying levels of cytoplasmic incompatibility and fecundity effect, the maximum possible longevity reduction that would allow Wolbachia to invade was obtained. Finally, the two models were combined to estimate the reduction in disease transmission according to different introduction frequencies. With strong CI and limited effect of fecundity, an introduction of Wolbachia with an initial frequency of 0.4 could result in a 60–80% reduction of transmitting mosquitoes. Greater reductions are possible at higher initial release rates.

Molecular phylogeny of Wolbachia endosymbionts in southeast asian mosquitoes (Diptera: Culicidae) based on wsp gene sequences

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts. Insect-associated Wolbachia form a monophyletic clade in the α-Proteobacteria and recently have been separated into two supergroups (A and B) and 19 groups. Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia. Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences. Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup. Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups.

Wolbachia infections of tephritid fruit flies: molecular evidence for five distinct strains in a single host species

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and can induce cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing or feminization in their hosts. Here, we report phylogenetic relationships of Wolbachia in tephritid fruit flies based on wsp gene sequences. We also report, for the first time, five distinct strains of Wolbachia in Bactrocera ascita sp. B. Four of the five Wolbachia strains found in this species were in the same groups as those found in other tephritid fruit flies, suggesting possible horizontal transmission of Wolbachia from other fruit flies into B. ascita sp. B. The unreliability of wsp-specific group primers demonstrated in this study suggests that these primers might be useful only for preliminary identification of Wolbachia. Final determination of group affiliation needs to be verified with wsp sequence data.