Dimitrios N. Avtzis

Evidence for low-titre infections in insect symbiosis: Wolbachia in the bark beetle Pityogenes chalcographus (Coleoptera, Scolytinae).

Wolbachia are obligatory endosymbiotic alpha-proteobacteria found in many insect species. They are maternally transmitted and often exhibit reproductive phenotypes like cytoplasmic incompatibility. Pityogenes chalcographus is a bark beetle causing severe damage in spruce stands. Its European populations are divided into several mitochondrial clades separated by partial crossing barriers. In this study, we tested a large sample set covering the natural range of the beetle in Europe for the presence of Wolbachia and associations between infection pattern and mitotypes using a highly sensitive nested PCR technique. 35.5% of the individuals were infected with the endosymbiont and two distinct strains were identified. Both strains occur in low titre not accessible by conventional detection methods. The infections are present all over Europe, unlikely to cause the partial crossing barriers in this host and uncoupled from mitochondrial clades. This pattern is indicative for populations evolving towards endosymbiont loss and for repeated intraspecific horizontal transfer of Wolbachia. Alternatively, the low-titre infections found in P. chalcographus are yet another example for Wolbachia that can persist in host species at low densities and frequencies.

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study.

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.

Natural History of the Processionary Moths (Thaumetopoea spp.): New Insights in Relation to Climate Change

It is difficult to find a genus of Lepidoptera showing the high variability of life history traits observed in Thaumetopoea. There are typical summer feeding close to winter feeding species, and in one special case a recent switch has been detected even within one species, the pine processionary moth, indicating that the natural history traits are constantly evolving at a fast rate. There are species adapted to cold conditions of high mountains and high latitude close to truly Mediterranean and sub-desert region species. All species have gregarious behaviour as larva and are protected against vertebrate predators by urticating setae.

Divergent evolutionary histories of two sympatric spruce bark beetle species

Ips typographus and Pityogenes chalcographus are two sympatric Palearctic bark beetle species with wide distribution ranges. As both species are comparable in biology, life history, and habitat, including sharing the same host, Picea abies, they provide excellent models for applying a comparative approach in which to identify common historical patterns of population differentiation and the influence of species‐specific ecological characteristics. We analysed patterns of genetic diversity, genetic structure and demographic history of ten I. typographus and P. chalcographus populations co‐distributed across Europe using both COI and ITS2 markers. Rather than similarities, our results revealed striking differences. Ips typographus was characterised by low genetic diversity, shallow population structure and strong evidence that all extant haplogroups arose via a single Holocene population expansion event. In contrast, genetic variation and structuring were high in P. chalcographus indicating a longer and more complex evolutionary history. This was estimated to be five times older than I. typographus, beginning during the last Pleistocene glacial maximum over 100 000 years ago. Although the expansions of P. chalcographus haplogroups also date to the Holocene or just prior to its onset, we show that these occurred from at least three geographically separated glacial refugia. Overall, these results suggest that the much longer evolutionary history of P. chalcographus greatly influenced the levels of phylogeographic subdivision among lineages and may have led to the evolution of different life‐history traits which in turn have affected genetic structure and resulted in an advantage over the more aggressive I. typographus.

New associations of phoretic mites on Pityokteines curvidens (Coleoptera, Curculionidae, Scolytinae)

The species composition and abundance of phoretic mites of the bark beetle Pityokteines curvidens caught in pheromone traps were investigated in Croatia. The P. curvidens trapping programs have been in an experimental phase in Croatia since 2004 as a possible monitoring and control system. The trapping program also permits the opportunity to sample phoretic mites found associated with the beetles. Beetles were caught using Curviwit pheromones in Theysohn traps placed in the Litorić region of Croatia. A total of 12 mite species were recovered, including Schizostethus simulatrix, Dendrolaelaps quadrisetus, Histiostoma piceae, H. cf. varia, Paraleius leontonychus, Pleuronectocelaeno barbara, Tarsonemus minimax, Trichouropoda lamellosa, Uroobovella ipidis, Schwiebea sp., Phauloppia lucorum and Dolicheremaeus dorni. Five species, Pl. barbara, Schwiebea sp., H. cf. varia, Ph. lucorum and Do. dorni, are identified for the first time in association with P. curvidens. These findings increase the number of mite species known to be phoretic on P. curvidens from 11 to 16. The present study also increases the number of known mite associates of Pityokteines spp. from 14 to 18.

Mitochondrial phylogenies in the light of pseudogenes and Wolbachia: re-assessment of a bark beetle dataset

Abstract Phylogenetic studies based on mtDNA become increasingly questioned because of potential pitfalls due to mitochondrial pseudogenes and mitochondrial selective sweeps. While the inclusion of nuclear markers should preferentially be considered for future studies, there is no need to abandon mtDNA as long as tests for the known mtDNA artefacts are performed. In this study we presentadditionaldata and test previous phylogeographical studies of Pityogenes chalcographus. We did not detect nuclear copies (numts) of the previously used mitochondrial markers by performing a combined long range/nested PCR of the COI gene and by an in silico analysis of the COI sequence data. This confirms the robustness of our previous phylogenetic study of Pityogenes chalcographus. Results of an in-situ hybridization of Wolbachia in Pityogenes chalcographus confirm the presence of this endosysmbiont in this species. However, we did not detect a correlation between infection status, geographical region and mtDNA haplotypes. The hybridisation data also support a previous hypothesis that infections do not result from parasitoids or parasitic nematodes, insect surface or laboratory contaminations and are hence a true infection of Pityogenes chalcographus. We conclude that the deep structure found in mitochondrial populations of Pityogenes chalcographus indeed represents the evolutionary history of European populations.

Lessons from a Beetle and an Ant: Coping with Taxon-Dependent Differences in Microsatellite Development Success

Microsatellites are powerful markers often isolated de novo for species yet to be investigated. Enriched genomic libraries are usually used for isolation purposes. We critically evaluate the outcome of an enrichment-based protocol applied to two insect species (the ant Lasius austriacus and the beetle Pityogenes chalcographus) which yielded contrasting numbers of suitable loci. Our findings of differences in microsatellite isolation are consistent with the available data on differences in genomic characteristics across these taxa. In the beetle repeated isolation of identical motifs, difficulties in primer development, and multibanded products caused loss of most candidate clones. We identified critical steps during marker development.

Climate Warming and Past and Present Distribution of the Processionary Moths (Thaumetopoea spp.) in Europe, Asia Minor and North Africa

Pine processionary moth, Thaumetopea pityocampa, is a model insect indicator of global warming, the northwards and upwards range expansion of this Mediterranean species being directly associated with the recent warming up. The knowledge about the drivers of moth expansion is synthesized. A first standardized mapping of the northern expansion edge, from Western Europe to Turkey, is presented, then detailed for 20 countries of Europe, Asia Minor and North Africa, including future trends. Additional data about the responses of the other Thaumetopoea species are given. Finally, the chapter points out the importance of the man-mediated introductions in the expansion process.

What is Next in Bark Beetle Phylogeography

Bark beetle species within the scolytid genera Dendroctonus, Ips, Pityogenes and Tomicus are known to cause extensive ecological and economical damage in spruce and pine forests during epidemic outbreaks all around the world. Dendroctonus ponderosae poses the most recent example having destroyed almost 100,000 km2 of conifer forests in North America. The success and effectiveness of scolytid species lies mostly in strategies developed over the course of time. Among these, a complex system of semiochemicals promotes the communication and aggregation on the spot of infestation facilitating an en masse attack against a host tree’s defenses; or an association with fungi that evolved either in the form of nutrition (ambrosia fungi) or even by reducing the resistance of host trees (blue-stain fungi). Although often specific to a tree genus or species, some bark beetles are polyphagous and have the ability to switch on to new hosts and extend their host range (i.e., between conifer genera such as Pityogenes chalcographus or even from conifer to deciduous trees as Polygraphus grandiclava). A combination of these capabilities in concert with life history or ecological traits explains why bark beetles are considered interesting subjects in evolutionary studies. Several bark beetle species appear in phylogeographic investigations, in an effort to improve our understanding of their ecology, epidemiology and evolution. In this paper investigations that unveil the phylogeographic history of bark beetles are reviewed. A close association between refugial areas and postglacial migration routes that insects and host trees have followed in the last 15,000 BP has been suggested in many studies. Finally, a future perspective of how next generation sequencing will influence the resolution of phylogeographic patterns in the coming years is presented. Utilization of such novel techniques will provide a more detailed insight into the genome of scolytids facilitating at the same time the application of neutral and non-neutral markers. The latter markers in particular promise to enhance the study of eco-physiological reaction types like the so-called pioneer beetles or obligate diapausing individuals.

Geographic distribution of chestnut feeding insects in Greece

Greece has been traditionally considered as a chestnut-producing country; yet, chestnut production has experienced a dramatic decline in the last decades, mainly due to chestnut blight Cryphonectria parasitica and chestnut feeding pests. Though chestnut blight is partially under control, little attention has been granted on chestnut feeding pests in Greece. Two species are described as major pests, i.e., Cydia splendana and Curculio elephas, without any information regarding their distribution. This study aimed to identify species diversity that feed on chestnuts trees using genetic markers and further to obtain insight into their distribution. Besides C. splendana and C. elephas, three additional species were determined namely Cydia fagiglandana, Curculio glandium as well as a Curculio sp. that has never been genetically determined before. The distribution of these species was inhomogeneous in Greece. These results demonstrate clearly the need for a carefully planned pest management approach adapted to the local mixture of chestnut feeding pests of each area rather than one general approach.