<Emphasis Type="Italic">Wolbachia</Emphasis> Infections of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed. RID= ID= <E5>Correspondence to: </E5>K. Bourtzis; <E5>email:</E5> kbourtz&commat;cc.uoi.gr Received: 9 September 2002 / Accepted: 25 September 2002     

Prevalence of <Emphasis Type="Italic">Wolbachia</Emphasis> Infection in <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Wolbachia are obligate intracellular bacteria present in reproductive tissues of many arthropod species. It has been reported that few silverleafing populations of Bemisia tabaci were positive for Wolbachia, whereas non-silverleafing populations were more likely infected with Wolbachia and all that infect B. tabaci are Wolbachia belonging to supergroup B. However, current detection methods were shown to be not sensitive enough to uncover all infections. Herein, a protocol based on polymerase chain reaction–restriction fragment length polymorphism analysis of Wolbachia 16S ribosomal DNA is presented. A systematic survey for the prevalence of Wolbachia infection in natural populations of B. tabaci using this method revealed that (1) all populations of B. tabaci tested positive for Wolbachia and the overall infection rate reached 80.5% (293 positives in 364 tests); (2) both single infection and superinfection existed within individual whiteflies tested; and (3) silverleafing populations of B. tabaci most likely harbored A Wolbachia as single infection, whereas non-silverleafing populations tend to carry B Wolbachia as superinfection. It is clear that the Wolbachia infection pattern is closely related to the genetic races of B. tabaci, and the infection frequencies are apparently much higher than those described previously. This study shows that detection methods can significantly influence estimation of Wolbachia infection. It is supposed that Wolbachia may be acting as a biotic agent promoting rapid differentiation and speciation of B. tabaci. This is the most systematic survey of Wolbachia infection within B. tabaci.     

Incidence of <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> endosymbionts in the <Emphasis Type="Italic">Osmia</Emphasis> community in Korea

Sex ratio distorting endosymbionts induce reproductive anomalies in their arthropod hosts. They have recently been paid much attention as firstly texts of evolution of host-symbiont relationships and secondly potential biological control agents to control arthropod pests. Among such organisms, Wolbachia and Cardinium bacteria are well characterized. This study aims at probing such bacteria in the Osmia community to evaluate their potential utilization to control arthropod pests. Among 17 PCR tested species, Osmia cornifrons and a parasitic fly are infected with Wolbachia and a mite species is infected with Cardinium. Phylogenetic tree analyses suggest that horizontal transfer of the bacteria occurred between phylogenetically distant hosts.     

Cytoplasmic incompatibility involving <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Wolbachia</Emphasis> in the white-backed planthopper <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae)

Cytoplasmic incompatibility (CI) is a reproductive phenotype induced by bacterial endosymbionts in arthropods. Measured as a reduction in egg hatchability resulting from the crossing of uninfected females with bacteria-infected males, CI increases the frequency of bacteria-infected hosts by restricting the fertilization opportunities of uninfected hosts in populations. Wolbachia, a type of alpha-proteobacteria, is well known as a CI inducer in a wide range of arthropod species, while Cardinium, a member of the phylum Bacteroidetes, is known to cause CI in one wasp and three spider mite species. In this study, dual infection with Cardinium and Wolbachia induced strong CI in a single host, Sogatella furcifera (Horváth), a planthopper species that is naturally infected with both bacteria. Specifically, infection with Cardinium alone was found to cause a 76 % reduction in egg development, and dual infection with Cardinium and Wolbachia a 96 % reduction, indicating that Cardinium induces CI and the dual infection raises the CI level. This study was the first to document reproductive alteration by Cardinium in a diploid host species.     

Effect of <Emphasis Type="Italic">Wolbachia</Emphasis> infection and temperature variations on the fecundity of the Uzifly <Emphasis Type="Italic">Exorista sorbillans</Emphasis> (Diptera: Tachinidae)

Wolbachia are cytoplasmically inherited endosymbionts known to cause several reproductive alterations in insects which allow their spread in host populations. In the Uzifly Exorista sorbillans, endoparasites of silkworms, the prevalence of Wolbachia is high in the field. In the present study, we investigated Wolbachia’s effects on the Uzifly fitness traits by measuring fecundity and hatching rate in crosses involving infected and cured individuals. We found evidence for positive fitness effects associated with Wolbachia infection in females which could help promote the spread of Wolbachia in E. sorbillans populations. We tested two types of treatments for removing Wolbachia, antibiotic therapy and high temperature treatment and found an influence on the reproduction: females treated by antibiotics have a lower fecundity than females cured by high temperature which could indicate a negative effect of the antibiotherapy on females’ fitness. Furthermore, the monitoring of the Uzifly populations during 2 years revealed seasonal variations of the offspring production which may be linked to temperature.     

Evolutionary history of <Emphasis Type="Italic">Wolbachia</Emphasis> infections in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Background  

Wolbachia are endosymbiotic bacteria that commonly infect numerous arthropods. Despite their broad taxonomic distribution, the transmission patterns of these bacteria within and among host species are not well understood. We sequenced a portion of the wsp gene from the Wolbachia genome infecting 138 individuals from eleven geographically distributed native populations of the fire ant Solenopsis invicta. We then compared these wsp sequence data to patterns of mitochondrial DNA (mtDNA) variation of both infected and uninfected host individuals to infer the transmission patterns of Wolbachia in S. invicta.     

Multiple endosymbionts in populations of the ant <Emphasis Type="Italic">Formica cinerea</Emphasis>

Background  

Many insects, including ants, are infected by maternally inherited Wolbachia endosymbiotic bacteria though other secondary endosymbionts have not been reported in ants. It has been suggested that the ability of Wolbachia to invade and remain in an ant population depends on the number of coexisting queens in a colony. We study the genetic and social structure of populations in the ant Formica cinerea which is known to have populations with either monogynous or polygynous colonies. We screen populations for several endosymbiotic bacteria to evaluate the presence of different endosymbionts, possible association between their prevalence and the social structure, and the association between endosymbiont prevalence and genetic differentiation of ant populations.     

<Emphasis Type="Italic">Wolbachia</Emphasis>-induced cytoplasmic incompatibility in Japanese populations of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

Intracellular bacteria of the genus Wolbachia (alpha Proteobacteria) induce cytoplasmic incompatibility (CI) in many arthropod species, including spider mites, but not all Wolbachia cause CI. In spider mites CI becomes apparent by a reduced egg hatchability and a lower daughter:son ratio: CI in haplodiploid organisms in general was expected to produce all-male offspring or a male-biased sex ratio without any death of eggs. In a previous study of Japanese populations of Tetranychus urticae, two out of three green-form populations tested were infected with non-CI Wolbachia strains, whereas none of six red-form populations harbored Wolbachia. As the survey of Wolbachia infection in T. urticae is still fragmentary in Japan, we checked Wolbachia infection in thirty green-form populations and 29 red-form populations collected from a wide range of Japanese islands. For Wolbachia-infected populations, we tested the effects of Wolbachia on the reproductive traits and determined the phylogenetic relationships of the different strains of Wolbachia. All but one green-form populations were infected with Wolbachia and all strains belonged to the subgroup Ori when the wsp gene was used to determine the phylogenetic relationships of different strains of Wolbachia. Six out of 29 red-form populations harbored Wolbachia and the infected strains belonged to the subgroups Ori and Bugs. Twenty-four of 29 infected green-form populations and five of six infected red-form populations induced CI among the hosts. Thus, CI-Wolbachia strains are widespread in Japan, and no geographical trend was observed in the CI-Wolbachia. Although three red-form populations harbored other intracellular bacteria Cardinium, they did not affect host reproduction.     

<Emphasis Type="Italic">Wolbachia</Emphasis> and bacteriophage WO-B density of <Emphasis Type="Italic">Wolbachia</Emphasis> a-infected <Emphasis Type="Italic">Aedes albopictus</Emphasis> mosquito

Wolbachia are maternally inherited symbiotic bacteria capable of inducing an extensive range of reproductive abnormalities in their hosts, including cytoplasmic incompatibility (CI). Its density (concentration) is likely to influence the penetrance of CI in incompatible crosses. The variations of Wolbachia density could also be linked with phage WO density. We determined the relative density (relative concentration) of prophage WO orf7 and Wolbachia (phage-to-bacteria ratio) during early developmental and adult stages of singly infected Aedes albopictus mosquito (Wolbachia A-infected) by using real-time quantitative PCR. Phage WO and Wolbachia did not develop at the same rate. Relative Wolbachia density (bacteria-to-host ratio) was high later in development (adult stages) whilst relative prophage WO density (phage-to-bacteria ratio) was low in the adult stages. Furthermore, 12-d-old adults of singly infected female mosquito had the highest Wolbachia density. In contrast, the larval stage 4 (L4) contained the highest prophage WO-B orf7 density. The association of hosts-Wolbachia-phage among diverse species is different. Thus, if phage and Wolbachia are involved in CI mechanism, the information of this association should be acquired for each specific type of organism for future use of population replacement or gene drive system.     

Male rescue maintains low frequency parthenogenesis-inducing <Emphasis Type="Italic">Wolbachia</Emphasis> infection in <Emphasis Type="Italic">Trichogramma</Emphasis> populations

Parthenogenesis-inducing (PI) Wolbachia bacteria are reproductive parasites that cause infected (W ⁺) female haplodiploid parasitoids to produce daughters without fertilization by males. Theoretically, PI Wolbachia infection should spread to fixation within Trichogramma populations as males are no longer required to produce female offspring. Infections in some naturally occurring Trichogramma populations are, however, maintained at frequencies ranging from 4 to 26%. Here we describe discrete equation models to examine if the PI Wolbachia infection in Trichogramma populations can be maintained at relatively low frequencies by mating regularity. Model outcomes suggest the probability of W ⁺ females mating could stabilize Wolbachia infection frequency at low levels in Trichogramma populations. The primary mechanism maintaining low-level PI Wolbachia infection in Trichogramma populations is reducing the survivorship from egg to adult in infected relative to uninfected females. The model successfully demonstrates that the relatively low PI Wolbachia infection frequency in host populations can be maintained by fertilization, or male rescue, of infected eggs, which avoids potentially hazardous gamete duplication that occurs during Wolbachia-induced parthenogenesis.     

Iranian <Emphasis Type="Italic">Trichogramma</Emphasis>: ITS2 DNA characterization and natural <Emphasis Type="Italic">Wolbachia</Emphasis> infection

Egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae) are important natural enemies of many lepidopterous pests in agricultural and forest areas. Because the use of indigenous Trichogramma populations/species can significantly increase the biological control efficiency, the characterization of endemic populations and precise species identification is important. In this study, Trichogramma wasps were collected from parasitized eggs of several lepidopteran pests in Northern and Central Iran. Seven Trichogramma species were identified based on the morphology and the nucleotide sequence, size and restriction profile (PCR-RFLP) of the internal transcribed spacer 2 (ITS2) region of the rDNA of T. brassicae Bezdenko, T. cacoeciae Marchal, T. embryophagum Hartig, T. evanescens Westwood, T. euproctidis Girault, T. pintoi Voegelé and T. tshumakovae Sorokina. This is the first report of T. euproctidis in Iran. Additionally, natural populations were surveyed for the prevalence of Wolbachia. Two populations of T. brassicae were found to be infected by a single Wolbachia strain which was identified by using a multi-locus sequencing approach.     

The endosymbiont <Emphasis Type="Italic">Wolbachia</Emphasis> in Eurasian populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The endosymbiotic α-proteobacteria Wolbachia is widely spread among arthropods and Filariidae nematodes. This bacterium is transmitted vertically via a transovarian route. Wolbachia is a cause of several reproductive abnormalities in the host species. We analyzed the isofemale lines created using flies collected from Drosophila melanogaster natural populations for infection with the endosymbiont Wolbachia. Wolbachia were genotyped according to five variable markers: the presence of insertion sequence IS5 in two loci, the copy number of two minisatellite repeats, and an inversion. Overall, 665 isofemale lines isolated from the populations of D. melanogaster from Ukraine, Belarus, Moldova, Caucasus, Central Asia, Ural, Udmurtia, Altai, West and East Siberia, and Far East in 1974 through 2005 were used in the work. The samples from Ukrainian, Altaian, and Middle Asian populations were largest. The infection rate of D. melanogaster populations from Middle Asia, Altaian, and Eastern Europe (Ukraine, Moldavia, and Belarus) with Wolbachia amounted to 64, 56, and 39%, respectively. The D. melanogaster population from the Caucasus displayed heterogeneity in the genotypes of this cytoplasmic infection. The Wolbachia genotype wMel, detected in all the populations studied, was the most abundant. The genotype wMelCS2 was always present in the populations from Middle Asia and Altai and was among the rare variants in the D. melanogaster populations from the Eastern Europe. Single instances of the Wolbachia genotype wMelCS occurred in a few flies from the Central Asian and Altai populations, but was not found this genotype in the other regions.     

Incidence of the endosymbionts <Emphasis Type="Italic">Wolbachia</Emphasis>, <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Spiroplasma</Emphasis> in phytoseiid mites and associated prey

Endosymbiotic bacteria that potentially influence reproduction and other fitness-related traits of their hosts are widespread in insects and mites and their appeal to researchers’ interest is still increasing. We screened 20 strains of 12 agriculturally relevant herbivorous and predatory mite species for infection with Wolbachia, Cardinium and Spiroplasma by the use of PCR. The majority of specimens originated from Austria and were field collected or mass-reared. Eight out of 20 strains (40%) tested, representing seven of 12 mite species (58%), carried at least one of the three bacteria. We found Wolbachia in the herbivorous spider mites Tetranychus urticae and Bryobia rubrioculus, with the former also carrying Spiroplasma and the latter also carrying Cardinium. Cardinium was furthermore found in two populations of the predatory mite Euseius finlandicus and the spider mite Eotetranychus uncatus. Spiroplasma was detected in the predatory mite Neoseiulus californicus. All bacteria positive PCR products were sequenced, submitted to GenBank and analyzed in BLAST queries. We found high similarities to complete identity with bacteria found in the same and different mite species but also with bacteria found in insect species like ladybirds, butterflies and minute pirate bugs, Orius. We discuss the significance of potential (multiple) infections with the investigated bacteria for biological control.     

Post-pollination hybridization barriers in <Emphasis Type="Italic">Nicotiana</Emphasis> section <Emphasis Type="Italic">Alatae</Emphasis>

Nicotiana section Alatae contains eight species with variable flower sizes and morphologies. Section members readily hybridize in the glasshouse, but no hybrids have been observed in natural sympatric and parapatric populations. To investigate interspecific crossing relationships with respect to mechanisms preventing hybridization, all members of section Alatae were intercrossed in a complete diallel. We found positive correlation between the pistil length of the pollen donor and interspecific seed set relative to the conspecific cross. Pollen tube growth rate and pollen donor pistil length were positively correlated as well. Furthermore, pollen from short-pistil members of section Alatae could only grow a maximum distance proportional to, but greater than, their own pistil lengths. Our results show that pollen tube growth capacity (i.e., rate and distance), provides a hybridization barrier in long-pistil species × short-pistil species crosses. We also found another hybridization barrier not specifically related to pollen tube growth capacity in short-pistil species × long-pistil species. Taken together, these barriers can generally be described by a ‘pistil-length mismatch’ rule; in section Alatae, pollen has the most success fertilizing ovules from species with pistil lengths similar to their own. This rule could contribute to hybridization barriers in Section Alatae because the species display dramatically different pistil lengths. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Strains Typing in Different Geographic Population Spider, <Emphasis Type="Italic">Hylyphantes Graminicola</Emphasis> (Linyphiidae)

The Wolbachia endosymbiont of spiders has not been extensively examined. In order to investigate the distribution, evolutionary history, and reproductive phenotype of Wolbachia in spiders in China, we tested 11 geographic populations of Hylyphantes graminicola. Wolbachia infection has been detected in each population. 10 Wolbachia strains have been characterized by multilocus sequence typing (MLST). Phylogenetic analyses indicated that eight Wolbachia strains in H. graminicola belonged to supergroup B, and two belonged to supergroup A. No correlation existed between Wolbachia diversity and host’s geographic distance. The significant correlation was observed between pairwise distance of H. graminicola COI and genetic divergence of associated Wolbachia strains. We also found that Wolbachia infection frequencies in hosts varied over geographic space.     

Survey of <Emphasis Type="Italic">Wolbachia</Emphasis> and Its Phage WO in the Uzifly <Emphasis Type="Italic">Exorista sorbillans</Emphasis> (Diptera: Tachinidae)

Wolbachia are cytoplasmically inherited alpha-proteobacteria well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts they infect. Despite their obligate intracellular lifestyle which usually protects bacteria from phage infection, Wolbachia harbor a widespread temperate phage called WO. Evidences of horizontal phage transfers indicate that this phage could promote genetic exchanges between strains leading to evolutionary changes in the genomes of Wolbachia, and could be involved in the phenotypes these bacteria induced. In this study, we report the survey of Wolbachia and WO phage infections in 20 populations of the Uzifly Exorista sorbillans, a tachinid endoparasite of silkworm Bombyx mori, collected from different geographic regions of India. Previous studies demonstrated that Wolbachia is associated with positive reproductive fitness effects in this species. Polymerase chain reaction using the ftsZ gene encoding for a Wolbachia cell division protein and the orf7 capsid protein gene of the phage showed that all flies checked were infected by Wolbachia and its phage WO. Phylogenetic analyses based on the Wolbachia surface protein gene revealed 100% of double infections by the arthropod supergroups A and B. These results can serve as a valuable basis for understanding the evolution of Wolbachia bacteria and may provide information about the dynamics of Wolbachia–host associations. This knowledge could be exploited for the use of Wolbachia for effective control strategies of the Uzifly, a serious menace of the silkworm B. mori.     

Genetic characterization of <Emphasis Type="Italic">Wolbachia</Emphasis> from Great Salt Lake brine flies

Wolbachia are intracellular prokaryotic endosymbionts associated with a wide distribution of arthropod and nematode hosts. Their association ranges from parasitism to mutualism, and there is growing evidence that Wolbachia can have dramatic effects on host reproduction, physiology, and immunity. Although all Wolbachia are currently considered as single species, W. pipientis, phylogenetic studies reveal about a dozen monophyletic groups, each designated as a supergroup. This study uses 16S rRNA gene sequences to examine the genetic diversity of Wolbachia present in three species of Great Salt Lake brine flies, Cirrula hians, Ephydra gracilis, and Mosillus bidentatus. The brine fly Wolbachia sequences are highly similar, with an average nucleotide sequence divergence among the three species of 0.00174. The brine fly Wolbachia form a monophyletic group that is affiliated with a subset of supergroup B, indicating that this supergroup may be more diverse than previously thought. These findings expand the phylogenetic diversity of Wolbachia and extend their host range to taxa adapted to a hypersaline environment.     

Molecular Evidence of <Emphasis Type="Italic">Wolbachia</Emphasis> Infection in Natural Populations of Tropical Odonates

Wolbachia are endosymbiotic bacteria that cause reproductive alterations in numerous arthropod species. Using a PCR-based method, we found that, out of 33 odonate species, four species were infected with Wolbachia. This finding represents the first record of Wolbachia infection in tropical odonates. Identical wsp gene sequences were found in the Wolbachia-infected common odonate species, Agriocnemis f. femina, collected from different locations in Thailand. The infection frequencies in several natural populations suggest that replacement of uninfected populations by Wolbachia-infected ones has recently occurred in this damselfly species.Received: 12 November 2002 / Accepted: 13 December 2002     

Identification and Characterization of <Emphasis Type="Italic">Wolbachia</Emphasis> in <Emphasis Type="Italic">Solenopsis saevissima</Emphasis> Fire Ants (Hymenoptera: Formicidae) in Southeastern Brazil

The genus Solenopsis appears to have evolved and radiated very rapidly in South America and then spread throughout the rest of the continent. As part of the expansion process, distribution patterns and different degrees of geographic isolation among populations of S. saevissima can be observed. We have investigated the presence of Wolbachia in 52 colonies and 1623 individuals in southeastern Brazil. Detection of Wolbachia infection was based on amplification of the 16S rRNA and wsp genes by polymerase chain reactions. Wolbachia was found in only one of the four locations investigated and it was observed that the populations were polymorphic for infection. The infection level observed increased during the period of screening. In particular, double infection (16SWA and B) increased from 44% in 2005 to 90% in 2006. The A-group of Wolbachia from the wsp sequences was determined by sequencing. However, two variant wsp sequences were detected in Wolbachia present in these populations. The alignment of our sequences with those deposited in GenBank indicated significant differences in relation to homologous sequences. Phylogenetic relationships were inferred using parsimony, and confidence intervals were estimated by bootstrapping. Then the divergence of the Wolbachia of S. saevissima in the populations studied with other variants allowed us to verify that wSS1 and dwSS2 formed a distinct clade within the A-group (>75%). These results can be useful in studies on the dynamics of ant populations.     

Negative Evidence of <Emphasis Type="Italic">Wolbachia</Emphasis> in the Predaceous Mite <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis>

The cytoplasmically inherited bacterium Wolbachia is widespread in arthropod species and has been repeatedly detected in the predaceous mite Phytoseiulus persimilis. Our original goal was to assess the prevalence of Wolbachia infection in P. persimilis and the potential fitness consequences for this host. To accomplish that goal, seven P. persimilis strains were obtained from Europe, Africa and the USA and reared on the phytophagous mite Tetranychus urticae as prey. After preliminary results showed that the T. urticae used was infected with Wolbachia, the minimum starvation time of the predators to prevent false positive results from undigested prey was determined. We tested DNA samples by PCR (polymerase chain reaction) after starving the predators or feeding them Wolbachia-free T. urticae for various periods. Those experiments showed that Wolbachia could not be detected after 16 h at 25 °C and 48 h at 20 °C. To verify the results of the PCR analyses, we furthermore conducted crossing experiments with antibiotic-treated and untreated individuals. No indications of Wolbachia effects were recorded. Additionally, we screened live eggs of four of the seven strains reared in our laboratory and alcohol samples of 10 other P. persimilis strains for the occurrence of Wolbachia by PCR, none of which tested positive. Synthesis of our study and previous reports suggests that infection of P. persimilis with Wolbachia is extremely rare and of minor importance. We discuss the significance of our findings for future studies on the presence of Wolbachia in predaceous arthropods.