Juvenile hormone pathway in honey bee larvae: A source of possible signal molecules for the reproductive behavior of Varroa destructor.

The parasitic mite Varroa destructor devastates honey bee (Apis mellifera) colonies around the world. Entering a brood cell shortly before capping, the Varroa mother feeds on the honey bee larvae. The hormones 20-hydroxyecdysone (20E) and juvenile hormone (JH), acquired from the host, have been considered to play a key role in initiating Varroa's reproductive cycle. This study focuses on differential expression of the genes involved in the biosynthesis of JH and ecdysone at six time points during the first 30 hr after cell capping in both drone and worker larvae of A. mellifera. This time frame, covering the conclusion of the honey bee brood cell invasion and the start of Varroa's ovogenesis, is critical to the successful initiation of a reproductive cycle. Our findings support a later activation of the ecdysteroid cascade in honey bee drones compared to worker larvae, which could account for the increased egg production of Varroa in A. mellifera drone cells. The JH pathway was generally downregulated confirming its activity is antagonistic to the ecdysteroid pathway during the larva development. Nevertheless, the genes involved in JH synthesis revealed an increased expression in drones. The upregulation of jhamt gene involved in methyl farnesoate (MF) synthesis came into attention since the MF is not only a precursor of JH but it is also an insect pheromone in its own right as well as JH-like hormone in Acari. This could indicate a possible kairomone effect of MF for attracting the mites into the drone brood cells, along with its potential involvement in ovogenesis after the cell capping, stimulating Varroa's initiation of egg laying.

Dissecting the genetic architecture of a stepwise infection process.

How a host fights infection depends on an ordered sequence of steps, beginning with attempts to prevent a pathogen from establishing an infection, through to steps that mitigate a pathogen's control of host resources or minimize the damage caused during infection. Yet empirically characterizing the genetic basis of these steps remains challenging. Although each step is likely to have a unique genetic and environmental signature, and may therefore respond to selection in different ways, events that occur earlier in the infection process can mask or overwhelm the contributions of subsequent steps. In this study, we dissect the genetic architecture of a stepwise infection process using a quantitative trait locus (QTL) mapping approach. We control for variation at the first line of defence against a bacterial pathogen and expose downstream genetic variability related to the host's ability to mitigate the damage pathogens cause. In our model, the water-flea Daphnia magna, we found a single major effect QTL, explaining 64% of the variance, that is linked to the host's ability to completely block pathogen entry by preventing their attachment to the host oesophagus; this is consistent with the detection of this locus in previous studies. In susceptible hosts allowing attachment, however, a further 23 QTLs, explaining between 5% and 16% of the variance, were mapped to traits related to the expression of disease. The general lack of pleiotropy and epistasis for traits related to the different stages of the infection process, together with the wide distribution of QTLs across the genome, highlights the modular nature of a host's defence portfolio, and the potential for each different step to evolve independently. We discuss how isolating the genetic basis of individual steps can help to resolve discussion over the genetic architecture of host resistance.

A gene for resistance to the Varroa mite (Acari) in honey bee (Apis mellifera) pupae.

Social insect colonies possess a range of defences which protect them against highly virulent parasites and colony collapse. The host-parasite interaction between honey bees (Apis mellifera) and the mite Varroa destructor is unusual, as honey bee colonies are relatively poorly defended against this parasite. The interaction has existed since the mid-20th Century, when Varroa switched host to parasitize A. mellifera. The combination of a virulent parasite and relatively naïve host means that, without acaricides, honey bee colonies typically die within 3 years of Varroa infestation. A consequence of acaricide use has been a reduced selective pressure for the evolution of Varroa resistance in honey bee colonies. However, in the past 20 years, several natural-selection-based breeding programmes have resulted in the evolution of Varroa-resistant populations. In these populations, the inhibition of Varroa's reproduction is a common trait. Using a high-density genome-wide association analysis in a Varroa-resistant honey bee population, we identify an ecdysone-induced gene significantly linked to resistance. Ecdysone both initiates metamorphosis in insects and reproduction in Varroa. Previously, using a less dense genetic map and a quantitative trait loci analysis, we have identified Ecdysone-related genes at resistance loci in an independently evolved resistant population. Varroa cannot biosynthesize ecdysone but can acquire it from its diet. Using qPCR, we are able to link the expression of ecdysone-linked resistance genes to Varroa's meals and reproduction. If Varroa co-opts pupal compounds to initiate and time its own reproduction, mutations in the host's ecdysone pathway may represent a key selection tool for honey bee resistance and breeding.

The genetic architecture underlying diapause termination in a planktonic crustacean.

Diapause is a feature of the life cycle of many invertebrates by which unfavourable environmental conditions can be outlived. The seasonal timing of diapause allows organisms to adapt to seasonal changes in habitat suitability and thus is key to their fitness. In the planktonic crustacean Daphnia, various cues can induce the production of diapause stages that are resistant to heat, drought or freezing and contain one to two embryos in developmental arrest. Daphnia is a keystone species of many freshwater ecosystems, where it acts as the main link between phytoplankton and higher trophic levels. The correct seasonal timing of diapause termination is essential to maintain trophic interactions and is achieved via a genetically based interpretation of environmental cues like photoperiod and temperature. Field monitoring and modelling studies raised concerns on whether populations can advance their seasonal release from diapause to advances in spring phenology under global change, or if a failure to adapt will cause trophic mismatches negatively affecting ecosystem functioning. Our capacity to understand and predict the evolution of diapause timing requires information about the genetic architecture underlying this trait. In this study, we identified eight quantitative trait loci (QTLs) and four epistatic interactions that together explained 66.5% of the variation in diapause termination in Daphnia magna using QTL mapping. Our results suggest that the most significant QTL is modulating diapause termination dependent on photoperiod and is involved in three of the four detected epistatic interactions. Candidate genes at this QTL could be identified through the integration with genome data and included the presynaptic active zone protein bruchpilot. Our findings contribute to understanding the genomic control of seasonal diapause timing in an ecological relevant species.

The role of epistatic interactions underpinning resistance to parasitic Varroa mites in haploid honey bee (Apis mellifera) drones.

The Red Queen hypothesis predicts that host-parasite coevolutionary dynamics can select for host resistance through increased genetic diversity, recombination and evolutionary rates. However, in haplodiploid organisms such as the honeybee (Apis mellifera), models suggest the selective pressure is weaker than in diploids. Haplodiploid sex determination, found in A. mellifera, can allow deleterious recessive alleles to persist in the population through the diploid sex with negative effects predominantly expressed in the haploid sex. To overcome these negative effects in haploid genomes, epistatic interactions have been hypothesized to play an important role. Here, we use the interaction between A. mellifera and the parasitic mite Varroa destructor to test epistasis in the expression of resistance, through the inhibition of parasite reproduction, in haploid drones. We find novel loci on three chromosomes which explain over 45% of the resistance phenotype. Two of these loci interact only additively, suggesting their expression is independent of each other, but both loci interact epistatically with the third locus. With drone offspring inheriting only one copy of the queen's chromosomes, the drones will only possess one of two queen alleles throughout the years-long lifetime of the honeybee colony. Varroa, in comparison, completes its highly inbred reproductive cycle in a matter of weeks, allowing it to rapidly evolve resistance. Faced with the rapidly evolving Varroa, a diversity of pathways and epistatic interactions for the inhibition of Varroa reproduction could therefore provide a selective advantage to the high levels of recombination seen in A. mellifera. This allows for the remixing of phenotypes despite a fixed queen genotype.

QTL mapping of a natural genetic polymorphism for long-term parasite persistence in Daphnia populations.

Knowing the determinants of the geographic ranges of parasites is important for understanding their evolutionary ecology, epidemiology and their potential to expand their range. Here we explore the determinants of geographic range in the peculiar case of a parasite species - the microsporidian Hamiltosporidium tvaerminnensis - that has a limited geographic distribution in a wide-spread host - Daphnia magna. We conducted a quantitative trait loci (QTLs) analysis with monoclonal F2 D. magna populations originating from a cross between a susceptible northern European genotype and a resistant central European genotype. Contrary to our expectations, long-term persistence turned out to be a quantitative trait across the F2 offspring. Evidence for two QTLs, one epistatic interaction and for further minor QTL was found. This finding contrasts markedly with the previously described bimodal pattern for long-term parasite persistence in natural host genotypes across Europe and leaves open the question of how a quantitative genetic trait could determine the disjunct geographic distribution of the parasite across Europe.

The genetic basis of resistance and matching-allele interactions of a host-parasite system: The Daphnia magna-Pasteuria ramosa model.

Negative frequency-dependent selection (NFDS) is an evolutionary mechanism suggested to govern host-parasite coevolution and the maintenance of genetic diversity at host resistance loci, such as the vertebrate MHC and R-genes in plants. Matching-allele interactions of hosts and parasites that prevent the emergence of host and parasite genotypes that are universally resistant and infective are a genetic mechanism predicted to underpin NFDS. The underlying genetics of matching-allele interactions are unknown even in host-parasite systems with empirical support for coevolution by NFDS, as is the case for the planktonic crustacean Daphnia magna and the bacterial pathogen Pasteuria ramosa. We fine-map one locus associated with D. magna resistance to P. ramosa and genetically characterize two haplotypes of the Pasteuria resistance (PR-) locus using de novo genome and transcriptome sequencing. Sequence comparison of PR-locus haplotypes finds dramatic structural polymorphisms between PR-locus haplotypes including a large portion of each haplotype being composed of non-homologous sequences resulting in haplotypes differing in size by 66 kb. The high divergence of PR-locus haplotypes suggest a history of multiple, diverse and repeated instances of structural mutation events and restricted recombination. Annotation of the haplotypes reveals striking differences in gene content. In particular, a group of glycosyltransferase genes that is present in the susceptible but absent in the resistant haplotype. Moreover, in natural populations, we find that the PR-locus polymorphism is associated with variation in resistance to different P. ramosa genotypes, pointing to the PR-locus polymorphism as being responsible for the matching-allele interactions that have been previously described for this system. Our results conclusively identify a genetic basis for the matching-allele interaction observed in a coevolving host-parasite system and provide a first insight into its molecular basis.

Parasite resistance and tolerance in honeybees at the individual and social level.

Organisms living in large groups, such as social insects, are particularly vulnerable to parasite transmission. However, they have evolved diverse defence mechanisms which are not only restricted to the individual's immune response, but also include social defences. Here, we review cases of adaptations at the individual and social level in the honeybee Apis mellifera against the ectoparasitic mite Varroa destructor and the endoparasitic microsporidians Nosema ceranae and Nosema apis. They are considered important threats to honeybee health worldwide. We highlight how individual resistance may result in tolerance at the colony level and vice versa.

Host age modulates within-host parasite competition.

In many host populations, one of the most striking differences among hosts is their age. While parasite prevalence differences in relation to host age are well known, little is known on how host age impacts ecological and evolutionary dynamics of diseases. Using two clones of the water flea Daphnia magna and two clones of its bacterial parasite Pasteuria ramosa, we examined how host age at exposure influences within-host parasite competition and virulence. We found that multiply-exposed hosts were more susceptible to infection and suffered higher mortality than singly-exposed hosts. Hosts oldest at exposure were least often infected and vice versa. Furthermore, we found that in young multiply-exposed hosts competition was weak, allowing coexistence and transmission of both parasite clones, whereas in older multiply-exposed hosts competitive exclusion was observed. Thus, age-dependent parasite exposure and host demography (age structure) could together play an important role in mediating parasite evolution. At the individual level, our results demonstrate a previously unnoticed interaction of the host's immune system with host age, suggesting that the specificity of immune function changes as hosts mature. Therefore, evolutionary models of parasite virulence might benefit from incorporating age-dependent epidemiological parameters.

An SNP-based second-generation genetic map of Daphnia magna and its application to QTL analysis of phenotypic traits.

BACKGROUND: Although Daphnia is increasingly recognized as a model for ecological genomics and biomedical research, there is, as of yet, no high-resolution genetic map for the genus. Such a map would provide an important tool for mapping phenotypes and assembling the genome. Here we estimate the genome size of Daphnia magna and describe the construction of an SNP array based linkage map. We then test the suitability of the map for life history and behavioural trait mapping. The two parent genotypes used to produce the map derived from D. magna populations with and without fish predation, respectively and are therefore expected to show divergent behaviour and life-histories. RESULTS: Using flow cytometry we estimated the genome size of D. magna to be about 238 mb. We developed an SNP array tailored to type SNPs in a D. magna F2 panel and used it to construct a D. magna linkage map, which included 1,324 informative markers. The map produced ten linkage groups ranging from 108.9 to 203.6 cM, with an average distance between markers of 1.13 cM and a total map length of 1,483.6 cM (Kosambi corrected). The physical length per cM is estimated to be 160 kb. Mapping infertility genes, life history traits and behavioural traits on this map revealed several significant QTL peaks and showed a complex pattern of underlying genetics, with different traits showing strongly different genetic architectures. CONCLUSIONS: The new linkage map of D. magna constructed here allowed us to characterize genetic differences among parent genotypes from populations with ecological differences. The QTL effect plots are partially consistent with our expectation of local adaptation under contrasting predation regimes. Furthermore, the new genetic map will be an important tool for the Daphnia research community and will contribute to the physical map of the D. magna genome project and the further mapping of phenotypic traits. The clones used to produce the linkage map are maintained in a stock collection and can be used for mapping QTLs of traits that show variance among the F2 clones.

Local adaptation of sex induction in a facultative sexual crustacean: insights from QTL mapping and natural populations of Daphnia magna.

Dormancy is a common adaptation in invertebrates to survive harsh conditions. Triggered by environmental cues, populations produce resting eggs that allow them to survive temporally unsuitable conditions. Daphnia magna is a crustacean that reproduces by cyclical parthenogenesis, alternating between the production of asexual offspring and the sexual reproduction of diapausing eggs (ephippia). Prior to ephippia production, males (necessary to ensure ephippia fertilization) are produced parthenogenetically. Both the production of ephippia and the parthenogenetic production of males are induced by environmental factors. Here, we test the hypothesis that the induction of D. magna resting egg production shows a signature of local adaptation. We postulated that Daphnia from permanent ponds would produce fewer ephippia and males than Daphnia from intermittent ponds and that the frequency and season of habitat deterioration would correlate with the timing and amount of male and ephippia production. To test this, we quantified the production of males and ephippia in clonal D. magna populations in several different controlled environments. We found that the production of both ephippia and males varies strongly among populations in a way that suggests local adaptation. By performing quantitative trait locus mapping with parent clones from contrasting pond environments, we identified nonoverlapping genomic regions associated with male and ephippia production. As the traits are influenced by two different genomic regions, and both are necessary for successful resting egg production, we suggest that the genes for their induction co-evolve.

The expression and evolution of virulence in multiple infections: the role of specificity, relative virulence and relative dose.

BACKGROUND: Multiple infections of the same host by different strains of the same microparasite species are believed to play a crucial role during the evolution of parasite virulence. We investigated the role of specificity, relative virulence and relative dose in determining the competitive outcome of multiple infections in the Daphnia magna-Pasteuria ramosa host-parasite system. RESULTS: We found that infections by P. ramosa clones (single genotype) were less virulent and produced more spores than infections by P. ramosa isolates (possibly containing multiple genotypes). We also found that two similarly virulent isolates of P. ramosa differed considerably in their within-host competitiveness and their effects on host offspring production when faced with coinfecting P. ramosa isolates and clones. Although the relative virulence of a P. ramosa isolate/clone appears to be a good indicator of its competitiveness during multiple infections, the relative dose may alter the competitive outcome. Moreover, spore counts on day 20 post-infection indicate that the competitive outcome is largely decided early in the parasite's growth phase, possibly mediated by direct interference or apparent competition. CONCLUSIONS: Our results emphasize the importance of epidemiology as well as of various parasite traits in determining the outcome of within-host competition. Incorporating realistic epidemiological and ecological conditions when testing theoretical models of multiple infections, as well as using a wider range of host and parasite genotypes, will enable us to better understand the course of virulence evolution.

The first-generation Daphnia magna linkage map.

BACKGROUND: Daphnia magna is a well-established model species in ecotoxicology, ecology and evolution. Several new genomics tools are presently under development for this species; among them, a linkage map is a first requirement for estimating the genetic background of phenotypic traits in quantitative trait loci (QTL) studies and is also very useful in assembling the genome. It also enables comparative studies between D. magna and D. pulex, for which a linkage map already exists. RESULTS: Here we describe the first genetic linkage map of D. magna. We generated 214 F2 (intercross) clonal lines as the foundation of the linkage analysis. The linkage map itself is based on 109 microsatellite markers, which produced ten major linkage groups ranging in size from 31.1 cM to 288.5 cM. The total size of this linkage map extends to 1211.6 Kosambi cM, and the average interval for the markers within linkage groups is 15.1 cM. The F2 clones can be used to map QTLs for traits that differ between the parental clones. We successfully mapped the location of two loci with infertility alleles, one inherited from the paternal clone (Iinb1) and the other from the maternal clone (Xinb3). CONCLUSIONS: The D. magna linkage map presented here provides extensive coverage of the genome and a given density of markers that enable us to detect QTLs of moderate to strong effects. It is similar in size to the linkage map of D. pulex.

Signals of demographic expansion in Drosophila virilis.

BACKGROUND: The pattern of genetic variation within and among populations of a species is strongly affected by its phylogeographic history. Analyses based on putatively neutral markers provide data from which past events, such as population expansions and colonizations, can be inferred. Drosophila virilis is a cosmopolitan species belonging to the virilis group, where divergence times between different phylads go back to the early Miocene. We analysed mitochondrial DNA sequence variation among 35 Drosophila virilis strains covering the species' range in order to detect demographic events that could be used to understand the present characteristics of the species, as well as its differences from other members of the group. RESULTS: Drosophila virilis showed very low nucleotide diversity with haplotypes distributed in a star-like network, consistent with a recent world-wide exponential expansion possibly associated either with domestication or post-glacial colonization. All analyses point towards a rapid population expansion. Coalescence models support this interpretation. The central haplotype in the network, which could be interpreted as ancestral, is widely distributed and gives no information about the geographical origin of the population expansion. The species showed no geographic structure in the distribution of mitochondrial haplotypes, in contrast to results of a recent microsatellite-based analysis. CONCLUSION: The lack of geographic structure and the star-like topology depicted by the D. virilis haplotypes indicate a pattern of global demographic expansion, probably related to human movements, although this interpretation cannot be distinguished from a selective sweep in the mitochondrial DNA until nuclear sequence data become available. The particular behavioural traits of this species, including weak species-discrimination and intraspecific mate choice exercised by the females, can be understood from this perspective.

Variation in male courtship song traits in Drosophila virilis: the effects of selection and drift on song divergence at the intraspecific level.

Genetic and phenotypic divergence of Drosophila virilis laboratory strains originating from different parts of the species range were studied with the aid of microsatellite markers and by analysing male courtship songs. The strains from America, Europe, continental Asia and Japan showed moderate geographic clustering both at the genetic level and in several traits of the male song. The genetic distances and the song divergence of the strains did not show significant association, which suggests that the songs have not diverged solely as a side-effect of genetic divergence. Comparison of the songs of the laboratory strains to those of freshly collected strains showed that pulse characters of the song are quite sensitive to culture conditions. While laboratory rearing of the flies had no effect on the number of pulses in a pulse train or the pulse train length, the tendency of the sound pulses to become longer during laboratory maintenance could explain the lack of geographic variation in pulse length and inter pulse interval. Sensitivity of songs to culturing conditions should be taken in account in studies on song divergence.

Microsatellite-based species identification method for Drosophila virilis group species.

Species of the D. virilis group are widely used in evolutionary research, but the individuals of different species are difficult to distinguish from each other morphologically. We constructed a fast and easy microsatellite-based identification method for the species of the group occurring sympatrically in northern Europe. The neighbor joining tree based on 14 microsatellite loci also gave a good resolution of the species divergence pattern in the whole group.

Localization of hrpA-induced Pseudomonas syringae pv. tomato DC3000 in infected tomato leaves.

SUMMARY Pseudomonas syringae pv. tomato is the causative agent of bacterial speck of tomato. The key virulence determinant of P. syringae is the hrp gene cluster, which encodes a type III secretion system. The type III system is used by a wide variety of pathogenic bacteria for transporting virulence proteins from the bacteria directly into the eukaryotic host cell. Hrp pilus, which is composed of HrpA pilin subunits, is an indispensable component of the type III secretion system in P. syringae. Here we have determined the spatial and temporal expression pattern of hrpA of P. syringae DC3000 in intact leaves, using a HrpA-GFP protein fusion and confocal microscopy. The hrpA gene was strongly and rapidly induced inside the leaf tissues after infiltration of the bacteria. After spray-inoculation, hrpA-induced bacteria were detected endophytically 72 h post-inoculation, and 96 h after spray-inoculation, disease symptoms appeared and GFP-expressing bacteria were observed at symptom sites, both endo- and epiphytically. Live/dead staining of the bacteria showed that Pst DC3000 does not survive well on leaf surfaces. Apoplastic populations were apparently bursting on to the leaf surface through stomata. Kinetics of population sizes of wild-type DC3000 and hrpA(-) showed significant differences, initially endophytically and only later epiphytically. Our results suggest that the Hrp pilus is first induced in the apoplast and apparently functions mainly inside the leaf tissues. These results suggest that P. syringae DC3000 mainly multiplies endophytically.