Richard Stouthamer

Male-killing Wolbachia in two species of insect

The inherited bacterium Wolbachia spreads through the manipulation of host reproduction, and has been suggested to be an important factor in arthropod evolution, from host speciation to the evolution of sex–determination systems. Past work has shown that members of this group may produce cytoplasmic incompatibility, feminize genetically male hosts, and induce host parthenogenesis. Here, we report an expansion of the range of reproductive manipulations produced by members of this clade, recording Wolbachia strains that kill male hosts during embryogenesis in two host species, the ladybird Adalia bipunctata, and the butterfly Acraea encedon. Both male–killing bacteria belong to the B group of Wolbachia. However, phylogenetic analyses were unable to resolve whether the bacteria in the two species are monophyletic, or represent independent origins of male–killing among the B–group Wolbachia. We also found significant divergence within the wsp gene of Wolbachia strains found in different A. bipunctata individuals, suggesting this host species contains two Wolbachia strains, diverged in wsp sequence but monophyletic. Our observations reinforce the notion that Wolbachia may be an important agent driving arthropod evolution, and corroborates previous suggestions that male–killing behaviour is easily evolved by invertebrate symbionts.

A new Huanglongbing Species, "Candidatus Liberibacter psyllaurous," found to infect tomato and potato, is vectored by the psyllid Bactericera cockerelli (Sulc).

ABSTRACT A new huanglongbing (HLB) “Candidatus Liberibacter” species is genetically characterized, and the bacterium is designated “Candidatus Liberibacter psyllaurous.” This bacterium infects the psyllid Bactericera cockerelli and its solanaceous host plants potato and tomato, potentially resulting in “psyllid yellowing.” Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.

Cytogenetics of microbe-associated parthenogenesis and its consequences for gene flow in Trichogramma wasps

Cytogenetics and gene flow were studied in microbe-associated parthenogenetic (thelytokous) forms of three species of the genus Trichogramma (T. pretiosum, T. deion and T. nr. deion). The chromosome behaviour in newly laid eggs indicated that the mechanism allowing restoration of diploidy in unfertilized thelytokous eggs was a segregation failure of the two sets of chromosomes in the first mitotic anaphase. This results in a nucleus containing two sets of identical chromosomes. The mechanism is known as gamete duplication and results in complete homozygosity. This was confirmed by investigation of the segregation pattern of allozymes in the offspring of heterozygous thelytokous females. Contrary to the generally assumed genetic isolation of thelytokous lines, thelytokous females of these species can mate and will use the sperm to fertilize some of their eggs. These fertilized eggs give rise to females whose genome consists of one set of chromosomes from each parent. Egg fertilization and the resulting syngamy of the sperm and egg pronucleus apparently precludes the gamete duplication that would have taken place if the egg had remained unfertilized. Most field populations of Trichogramma contain both parthenogenetic (thelytokous) and sexual (arrhenotokous) forms. In the two field populations that we studied there was evidence for high levels of gene flow from the sexual (arrhenotokous) fraction to the parthenogenetic (thelytokous) fraction of the population. The implications of the cytogenetic mechanism of parthenogenesis, i.e. gamete duplication, for the mechanism of sex determination in Hymenoptera are discussed.

Incidence of a new sex–ratio–distorting endosymbiotic bacterium among arthropods

Many intracellular micro–organisms are now known to cause reproductive abnormalities and other phenomena in their hosts. The endosymbiont Wolbachia is the best known of these reproductive manipulators owing to its extremely high incidence among arthropods and the diverse host effects it has been implicated as causing. However, recent evidence suggests that another intracellular bacterium, a Cytophaga–like organism (CLO), may also induce several reproductive effects in its hosts. Here, we present the first survey of arthropod hosts for infection by the CLO. We use a sensitive hemi–nested polymerase chain reaction method to screen 223 species from 20 arthropod orders for infection by the CLO and Wolbachia. The results indicate that, although not as prevalent as Wolbachia, the CLO infects a significant number of arthropod hosts (ca. 7.2%). In addition, double infections of the CLO and Wolbachia were found in individuals of seven arthropod species. Sequencing analysis of the 16S rDNA region of the CLO indicates evidence for horizontal transmission of the CLO strains. We discuss these results with reference to future studies on host effects induced by intracellular micro–organisms.

The utility of internally transcribed spacer 2 DNA sequences of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma

The usefulness of the internally transcribed spacer 2 (ITS2) of the nuclear ribosomal gene complex is tested for providing taxonomic characters to identify Trichogramma species. The ITS2 sequences of a group of sibling species of the T. deion/T. pretiosum complexes were determined. A simple and precise identification key to the species of these assemblages was constructed using as taxonomic characters the size of the ITS2 and the difference in restriction length polymorphism of species with similarly sized ITS2. Individual wasps can be identified by amplification of their ITS2 with general primers, determining the size of the PCR product using standard agarose electrophoresis, followed in some species by a DNA-digestion with a restriction enzyme. Because this system works well for a number of closely related species we are hopeful that similar PCR-based identification can be extended to all species of the genus once their ITS2 sequences have been determined. The advantage of this identification system over the morphology-based system is that non-specialists are able to quickly and cheaply identify individual specimens. In addition, species specific primers were tested for the two most common species of these groups (i.e. T. pretiosum and T. deion). These primers can be used either as a direct identification tool or as a method to confirm the identification using the general key. The phylogeny of this group of wasps was also analyzed based on the ITS2 sequence.

Phylogeny of cytoplasmic incompatibility microorganisms in the parasitoid wasp genus Nasonia (Hymenoptera: Pteromalidae) based on 16S ribosomal DNA sequences

Cytoplasmic incompatibility results in embryo mortality in diploids, or all male offspring in haplodiploids, when individuals carrying different cytoplasmic factors are crossed. Cytoplasmic factors have been identified as intracellular micro‐organisms. Microbeinduced cytoplasmic incompatibility is found in many insect taxa and may play a role in reproductive isolation between populations. Such micro‐organisms cause bidirectional incompatibility between species of the parasitoid wasp genus Nasonia. The phylogenetic relationship of cytoplasmic incompatibility microorganisms (CIM) of different Nasonia species was analysed using their 16S ribosomal DNA (rDNA) sequence. Two 16S rDNA operons were detected in the CIM of each Nasonia species. Sequence analysis indicates that the Nasonia CIM are closely related and belong to the alpha group of the Proteobacteria.

Natural interspecific and intraspecific horizontal transfer of parthenogenesis–inducing Wolbachia in Trichogramma wasps

The intracellular bacterium Wolbachia is one of the most common symbionts in arthropods and, because of its manipulative effects on host reproduction, is assumed to be an important factor in several evolutionary processes. These bacteria are mainly vertically transmitted from mother to daughter through the egg cytoplasm, and horizontal transmission is generally assumed to be rare. Here, we show natural inter– and intraspecific horizontal transfer of parthenogenesis–inducing Wolbachia between parasitoid wasps of the genus Trichogramma. Horizontal transfer was observed when infected and uninfected larvae shared the same host egg. This is the first report, to our knowledge, on interspecific horizontal transfer of Wolbachia between closely related sympatric species. Some originally uninfected immature wasps acquired Wolbachia while inside the host egg, but not all of these newly infected females exhibited the parthenogenesis phenotype. In general, intraspecific horizontal transfer was more successful than interspecific transfer. Wolbachia underwent vertical transmission in the new species but the infection tended to be lost within several generations. Our results have important implications for understanding the evolution of Wolbachia–host associations.

Phylogeny of the arthropod endosymbiont Wolbachia based on the wsp gene

Bacteria of the genus Wolbachia (Rickettsiae) are widespread in arthropods and can induce cytoplasmic incompatibility (CI), thelytoky (T) or feminization (F) in their host. Recent research on the wsp gene of mainly CI inducing Wolbachia has shown that this gene evolves at a much faster rate than previously sequenced genes such as 16S or ftsZ. As a result this gene appears to be very useful in subdividing the Wolbachia and twelve groups have been distinguished to date. Here we extend the Wolbachia wsp data set with fifteen T‐Wolbachia, one F‐Wolbachia and three other CI‐Wolbachia strains. The results showed: (i) the addition of seven groups; (ii) no relation between host phenotype and Wolbachia phylogenetic position; and (iii) possible horizontal Wolbachia transfer between the moth Ephestia kuehniella and its parasitoid Trichogramma spp.

Horizontal transmission of parthenogenesis–inducing microbes in Trichogramma wasps

Complete parthenogenesis (thelytoky) in species of the parasitic wasp Trichogramma is usually caused by the cytoplasmically inherited bacterium Wolbachia. This symbiont induces gamete duplication, which, in these haplodiploid organisms, results in all–female broods. Antibiotic treatment ‘cures’ this condition, restoring normal sexual reproduction. Phylogenetic analysis of Wolbachia has shown that, in contrast with the strains in other host organisms (where the symbiont also induces different reproductive alterations), those in Trichogramma form a monophyletic group. This might be an indication of symbiont–host cocladogenesis. To test this, we performed comparative molecular phylogenetics on 20 parthenogenetic Trichogramma cultures and their Wolbachiae. We conclude that there is, in fact, little evidence for cocladogenesis. Instead, the phylogenetic distribution of the symbionts appears to result from occasional horizontal transmission, which probably takes place inside the hosts of Trichogramma parasitoids (usually lepidopteran eggs). This study therefore suggests that parthenogenesis is not only curable, it can sometimes be contagious also.

Influence of microbe-associated parthenogenesis on the fecundity of Trichogramma deion and T.pretiosum.

Microbe‐associated parthenogenesis (thelytoky) has been discovered in nine Trichogramma species, parasitoids of mainly lepidopteran eggs. Parthenogenetic and bisexual conspecifics co‐occur in many field populations. As an initial step to understand the dynamics of these two reproductive strategies we studied the effect of microbe‐associated parthenogenesis on fecundity. The fecundity of two parthenogenetic isofemale lines of T. pretiosum and one of T. deion was compared with bisexual lines derived from them by antibiotic treatment. In all three cases parthenogenetic females were less fecund over their lifetime than bisexual females. Also, parthenogenetic females produced fewer daughters in two cases and in one case a similar number of daughters as their respective bisexual counterparts. The lack of mating and insemination was excluded as an explanation for the reduced fecundity of parthenogenetic females, because mated and virgin parthenogenetic females produce the same number of offspring. Antibiotic treatment can also be excluded because females of field‐collected bisexual line treated with antibiotics produced the same number of offspring as untreated females. The reduced fecundity of parthenogenetic females was caused by a lower number of eggs being laid rather than by a greater developmental mortality. Parthenogenetic females produced less daughters than bisexual females when host availability was not limiting, but when host availability was severely limited, parthenogenetic females produced more daughters than the bisexual females.