Ken Kraaijeveld

Wolbachia is a maternally inherited bacterium that manipulates the reproduction of its host. Recent studies have shown that male-killing strains can induce cytoplasmic incompatibility (CI) when introgressed into a resistant host. Phylogenetic studies suggest that transitions between CI and other Wolbachia phenotypes have also occurred frequently, raising the possibility that latent CI may be widespread among Wolbachia. Here, we investigate whether a parthenogenesis-inducing Wolbachia strain can also induce CI. Parthenogenetic females of the parasitoid wasp Asobara japonica regularly produce a small number of males that may be either infected or not. Uninfected males were further obtained through removal of the Wolbachia using antibiotics and from a naturally uninfected strain. Uninfected females that had mated with infected males produced a slightly, but significantly more male-biased sex ratio than uninfected females that had mated with uninfected males. This effect was strongest in females that mated with males that had a relatively high Wolbachia titer. Quantitative PCR indicated that infected males did not show higher ratios of nuclear versus mitochondrial DNA content. Wolbachia therefore does not cause diploidization of cells in infected males. While these results are consistent with CI, other alternatives such as production of abnormal sperm by infected males cannot be completely ruled out. Overall, the effect was very small (9%), suggesting that if CI is involved it may have degenerated through the accumulation of mutations.

ABSTRACT Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts by, for example, inducing parthenogenesis. In most cases of Wolbachia-induced parthenogenesis, the infection is fixed and the entire host population consists of females. In the absence of males and sexual reproduction, genes involved in sexual reproduction are not actively maintained by selection. Accumulation of neutral mutations or selection against maintenance of sexual traits may lead to their loss or deterioration. In addition, females may lose the ability to reproduce sexually due to ‘functional virginity mutations’ that may spread concomitantly with the Wolbachia infection through a population. The parasitoid wasp Tetrastichus coeruleus (Nees) (Hymenoptera: Eulophidae) forms an ideal model to study the decay of sexual functionality, because it has both Wolbachia-infected, parthenogenetic populations and uninfected, sexual populations. We compared several components of sexual functionality of arrhenotokous (sexual) and thelytokous (parthenogenetic) T. coeruleus females. First, we tested whether arrhenotokous and thelytokous females were equally attractive and receptive to males. Second, we examined whether mating is costly to females by measuring the life span of mated and virgin females. Last, we studied the morphology of the spermathecae of arrhenotokous and thelytokous females. Mated females had shorter life spans than virgin females, showing that mating carried a fitness cost. Two sexual traits of thelytokous females have degraded compared to arrhenotokous females. Arrhenotokous and thelytokous females were equally attractive to males, but thelytokous females were unreceptive to males. Furthermore, there was a clear difference in spermathecal morphology between arrhenotokous and thelytokous females. Our data do not allow distinction between the various potential causes of such degradation. Although the longevity cost of mating may indicate selection against the maintenance of costly sexual traits, accumulation of neutral mutations, functional virginity mutations, manipulation by Wolbachia, and/or the genetic distance between the two populations may all have contributed to the decay of sexual traits in thelytokous females.

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen-allergic patients. Current pollen monitoring methods are microscope-based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost-effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next-generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.

Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts. These manipulations are expected to have consequences on the population genetics of the host, such as heterozygosity levels, genetic diversity and gene flow. The parasitoid wasp Tetrastichus coeruleus has populations that are infected with parthenogenesis-inducing Wolbachia and populations that are not infected. We studied the population genetics of T. coeruleus between and within Wolbachia-infected and uninfected populations, using nuclear microsatellites and mitochondrial DNA. We expected reduced genetic diversity in both DNA types in infected populations. However, migration and gene flow could introduce new DNA variants into populations. We therefore paid special attention to individuals with unexpected (genetic) characteristics. Based on nuclear and mitochondrial DNA, two genetic clusters were evident: a thelytokous cluster containing all Wolbachia-infected, parthenogenetic populations and an arrhenotokous cluster containing all uninfected, sexual populations. Nuclear and mitochondrial DNA did not exhibit concordant patterns of variation, although there was reduced genetic diversity in infected populations for both DNA types. Within the thelytokous cluster, there was nuclear DNA variation, but no mitochondrial DNA variation. This nuclear DNA variation may be explained by occasional sex between infected females and males, by horizontal transmission of Wolbachia, and/or by novel mutations. Several females from thelytokous populations were uninfected and/or heterozygous for microsatellite loci. These unexpected characteristics may be explained by migration, by inefficient transmission of Wolbachia, by horizontal transmission of Wolbachia, and/or by novel mutations. However, migration has not prevented the build-up of considerable genetic differentiation between thelytokous and arrhenotokous populations.