A butterfly egg-killing hypersensitive response in Brassica nigra is controlled by a single locus, PEK, containing a cluster of TIR-NBS-LRR receptor genes.

Knowledge of plant recognition of insects is largely limited to a few resistance (R) genes against sap-sucking insects. Hypersensitive response (HR) characterizes monogenic plant traits relying on R genes in several pathosystems. HR-like cell death can be triggered by eggs of cabbage white butterflies (Pieris spp.), pests of cabbage crops (Brassica spp.), reducing egg survival and representing an effective plant resistance trait before feeding damage occurs. Here, we performed genetic mapping of HR-like cell death induced by Pieris brassicae eggs in the black mustard Brassica nigra (B. nigra). We show that HR-like cell death segregates as a Mendelian trait and identified a single dominant locus on chromosome B3, named PEK (Pieris  egg- killing). Eleven genes are located in an approximately 50 kb region, including a cluster of genes encoding intracellular TIR-NBS-LRR (TNL) receptor proteins. The PEK locus is highly polymorphic between the parental accessions of our mapping populations and among B. nigra reference genomes. Our study is the first one to identify a single locus potentially involved in HR-like cell death induced by insect eggs in B. nigra. Further fine-mapping, comparative genomics and validation of the PEK locus will shed light on the role of these TNL receptors in egg-killing HR.

Insect egg-killing: a new front on the evolutionary arms-race between brassicaceous plants and pierid butterflies.

Evolutionary arms-races between plants and insect herbivores have long been proposed to generate key innovations such as plant toxins and detoxification mechanisms that can drive diversification of the interacting species. A novel front-line of plant defence is the killing of herbivorous insect eggs. We test whether an egg-killing plant trait has an evolutionary basis in such a plant-insect arms-race. Within the crucifer family (Brassicaceae), some species express a hypersensitive response (HR)-like necrosis underneath butterfly eggs (Pieridae) that leads to eggs desiccating or falling off the plant. We studied the phylogenetic distribution of this trait, its egg-killing effect on and elicitation by butterflies, by screening 31 Brassicales species, and nine Pieridae species. We show a clade-specific induction of strong, egg-killing HR-like necrosis mainly in species of the Brassiceae tribe including Brassica crops and close relatives. The necrosis is strongly elicited by pierid butterflies that are specialists of crucifers. Furthermore, HR-like necrosis is linked to PR1 defence gene expression, accumulation of reactive oxygen species and cell death, eventually leading to egg-killing. Our findings suggest that the plants' egg-killing trait is a new front on the evolutionary arms-race between Brassicaceae and pierid butterflies beyond the well-studied plant toxins that have evolved against their caterpillars.

Parasitic wasp-associated symbiont affects plant-mediated species interactions between herbivores.

Microbial mutualistic symbiosis is increasingly recognised as a hidden driving force in the ecology of plant-insect interactions. Although plant-associated and herbivore-associated symbionts clearly affect interactions between plants and herbivores, the effects of symbionts associated with higher trophic levels has been largely overlooked. At the third-trophic level, parasitic wasps are a common group of insects that can inject symbiotic viruses (polydnaviruses) and venom into their herbivorous hosts to support parasitoid offspring development. Here, we show that such third-trophic level symbionts act in combination with venom to affect plant-mediated interactions by reducing colonisation of subsequent herbivore species. This ecological effect correlated with changes induced by polydnaviruses and venom in caterpillar salivary glands and in plant defence responses to herbivory. Because thousands of parasitoid species are associated with mutualistic symbiotic viruses in an intimate, specific relationship, our findings may represent a novel and widespread ecological phenomenon in plant-insect interactions.

Synergistic effects of direct and indirect defences on herbivore egg survival in a wild crucifer.

Evolutionary theory of plant defences against herbivores predicts a trade-off between direct (anti-herbivore traits) and indirect defences (attraction of carnivores) when carnivore fitness is reduced. Such a trade-off is expected in plant species that kill herbivore eggs by exhibiting a hypersensitive response (HR)-like necrosis, which should then negatively affect carnivores. We used the black mustard (Brassica nigra) to investigate how this potentially lethal direct trait affects preferences and/or performances of specialist cabbage white butterflies (Pieris spp.), and their natural enemies, tiny egg parasitoid wasps (Trichogramma spp.). Both within and between black mustard populations, we observed variation in the expression of Pieris egg-induced HR. Butterfly eggs on plants with HR-like necrosis suffered lower hatching rates and higher parasitism than eggs that did not induce the trait. In addition, Trichogramma wasps were attracted to volatiles of egg-induced plants that also expressed HR, and this attraction depended on the Trichogramma strain used. Consequently, HR did not have a negative effect on egg parasitoid survival. We conclude that even within a system where plants deploy lethal direct defences, such defences may still act with indirect defences in a synergistic manner to reduce herbivore pressure.

Geographic and temporal variations in population dynamics of Ixodes ricinus and associated Borrelia infections in The Netherlands.

In a countrywide investigation of the ecological factors that contribute to Lyme borreliosis risk, a longitudinal study on population dynamics of the sheep tick Ixodes ricinus and their infections with Borrelia burgdorferi sensu lato (s.l.) was undertaken at 24 sites in The Netherlands from July 2006 to December 2007. Study sites were mature forests, dune vegetations, or new forests on land reclaimed from the sea. Ticks were sampled monthly and nymphal ticks were investigated for the presence of Borrelia spp. I. ricinus was the only tick species found. Ticks were found in all sites, but with significant spatial and temporal variations in density between sites. Peak densities were found in July and August, with lowest tick numbers collected in December and January. In some sites, questing activities of I. ricinus nymphs and adults were observed in the winter months. Mean monthly Borrelia infections in nymphs varied from 0% to 29.0% (range: 0%-60%), and several sites had significantly higher mean nymphal Borrelia infections than others. Four genospecies of Borrelia burgdorferi s.l. were found, with B. afzelii being dominant at most sites. Borrelia infection rates in nymphal ticks collected in July, September, and November 2006 were significantly higher (23.7%, p<0.01) than those in the corresponding months of 2007 (9.9%). The diversity in Borrelia genospecies between sites was significantly different (p<0.001). Habitat structure (tree cover) was an effective discriminant parameter in the determination of Borrelia infection risk, as measured by the proportion of nymphal ticks infected with B. burgdorferi s.l. Thickness of the litter layer and moss cover were positively related to nymphal and adult tick densities. The study shows that Borrelia-infected ticks are present in many forest and dune areas in The Netherlands and suggests that in such biotopes, which are used for a wide variety of recreational activities, the infection risk is high.

Comparative AFLP reveals paternal sex ratio chromosome specific DNA sequences in the parasitoid wasp Trichogramma kaykai.

The parasitoid wasp Trichogramma kaykai with a haplo-diploid sex determination has a B chromosome called the paternal sex ratio (PSR) chromosome that confers paternal genome loss during early embryogenesis, resulting in male offspring. So far, it is not well known whether the PSR chromosome has unique DNA sequence characteristics. By comparative AFLP fingerprinting of genomic DNA from wasps with and without the PSR chromosome, we isolated DNA from PSR-specific bands. Fourteen of such DNA fragments were analysed to confirm their PSR specificity. Seven were sequenced and two (PT-AFLP 1 and PT-AFLP1 3) were identified as parts of retrotransposon genes based on BLAST searches. Internal primers designed from a third AFLP fragment allowed PCR amplification of a PSR chromosome specific marker, which can be used to screen for the PSR trait in male wasps. Southern analysis revealed a dispersed repetitive nature of this third sequence in the T. kaykai genome, suggesting that it is part of a transposon. A fourth AFLP fragment (PT-AFLP 5) appears to be a large repetitive sequence on the PSR chromosome. This sequence is also found in the genome of both T. kaykai and the closely related species Trichogramma deion, but its distribution on the PSR chromosome strongly resembles that of T. deion rather than that of T. kaykai. Our results provide further insight into the repetitive nature of sequences comprising B chromosomes and their similarities with their host and closely related species.

Genetic differentiation between resistance phenotypes in the phytophagous flea beetle, Phyllotreta nemorum.

The flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae) is genetically polymorphic for resistance against the defences of one of its host plants, Barbarea vulgaris R.Br. (Brassicales: Brassicaceae). Whereas resistant flea beetles are able to use B. vulgaris as well as other cruciferous plants as food, non-resistant beetles cannot survive on B. vulgaris. This limitation to host plant use of non-resistant beetles could potentially lead to asymmetric gene flow and some degree of genetic isolation between the different resistance-genotypes. Therefore, we studied the extent of genetic differentiation at neutral allozyme loci between samples of flea beetles that were collected at different locations and first tested for resistance phenotype. Since earlier work has shown a weak, but significant, effect of geographical distance between the samples on their genetic differentiation, in the present study variation at the neutral allozyme loci in P. nemorum was partitioned between geographical distance and resistance-phenotype. Both sources independently contributed statistically significantly to population differentiation. Thus, there appears to be a limitation to genetic exchange between the resistant and non-resistant flea beetles when corrections are made for their geographic differentiation. This is consistent with the presence of some degree of host race formation in this flea beetle.

Variations in Ixodes ricinus density and Borrelia infections associated with cattle introduced into a woodland in The Netherlands.

The effect of introduced large herbivores on the abundance of Ixodes ricinus ticks and their Borrelia infections was studied in a natural woodland in The Netherlands. Oak and pine plots, either ungrazed or grazed by cattle, were selected. Ticks were collected weekly by blanket dragging. Borrelia infections were determined by PCR and restriction fragment length polymorphism. Rodent densities were estimated using mark-release-recapture methods. On occasion, the cattle were inspected for tick infestations. Meteorological data were recorded for each habitat. Significantly more ticks were collected in the ungrazed woodland than in the grazed woodland. The ungrazed oak habitat had higher tick densities than the pine habitat, while in the grazed habitats, tick densities were similar. Borrelia infection rates ranged from zero in larvae to 26% in nymphs to 33% in adult ticks, and B. afzelii, B. burgdorferi sensu stricto, B. garinii, and B. valaisiana were the species involved. Coinfections were found in five ticks. There was no effect of the presence of cattle on Borrelia infections in the ticks. In the ungrazed area, Borrelia infections in nymphs were significantly higher in the oak habitat than in the pine habitat. More mice were captured in the ungrazed area, and these had a significantly higher tick burden than mice from the grazed area. Tick burden on cattle was low. The results suggest that grazing has a negative effect on small rodents as well as on ticks but not on Borrelia infections. Implications of these results for management of woodland reserves and risk of Lyme disease are discussed.

Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress.

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.