Identifizierung von Haplotypen bei Pityogenes chalcographus (Col., Scolytidae) durch SSCP

Pityogenes chalcographus is a widely distributed spruce pest in Eurasia (KNIZEK et al. 2005). In 70ies, E. Führer studied the intraspecific variation of this spruce bark beetle and detected race differentiation among European populations based on crossing experiments (FÜHRER 1977), morphological characters FÜHRER 1978) and allozyme electrophoresis (RITZENGRUBER 1990). In order to verify the hypothesis differentiation, we analysed diverse European P. chalcographus populations using the Cytochrome Oxidase gene (COI) of the mitochondrial DNA. The complete COI gene of 96 individuals was sequenced. In facilitate the screening of the European populations, we applied a PCR-SSCP method. This polyacrylamide electrophoresis technique offers a sensitive but inexpensive, rapid and convenient method for detecting polymorphisms, reducing the amount of samples that require sequencing (SUNNUCKS et al. 2000).Der Kupferstecher Pityogenes chalcographus (Coleoptera, Scolytidae) gehört zu den am weitesten verbreiteten Borkenkäfern Europas. Käferpopulationen aus verschiedenen Gebieten Europas zeigten eine hohe Divergenz hinsichtlich der Nukleotidzusammensetzung des mitochondrialen Cytochrom Oxidase I Gens. Die Unterschiede betragen bis zu 2,26% zwischen einzelnen Herkünften. Die phylogenetische Auswertung der Sequenzen des kompletten Gens (1503bp) ergab einen 6-astigen Baum mit insgesamt 34 Haplotypen. Ausgehend von dieser Struktur wurden Primerpaare entworfen, um jeweils etwa 250 bp lange mutationsreiche Abschnitte des COI Gens zu amplifizieren. Die PCR Produkte wurden mittels Single Strand Conformation Polymorphism (SSCP) untersucht. Zwischen den einzelnen Haplotypen konnten zahlreiche Polymorphismen erkannt werden. Diese eignen sich als schnelles Werkzeug zum screening einzelner Individuen und verringern signifikant die Notwendigkeit klassischer Sequenzierreaktionen

Pityogenes chalcographus (Coleoptera, Scolytinae) at the southernmost borderline of Norway spruce (Picea abies) in Greece

Το φλοιοφάγο έντομο Pityogenes chalcographus L. αποτελεί ένα από τα κύρια βλαπτικά έντομα της υποοικογένειας Scolytinae, που είναι ευρέως εξαπλωμένο στην Ευρώπη. Ο κύριος ξενιστής του εντόμου είναι η ερυθρελάτη (Picea abies) ενώ επίσης μπορεί να αναπτυχθεί επι- τυχώς σε αρκετά ακόμη είδη κωνοφόρων όπως το πεύκο (Pinus sp.), το έλατο (Abies alba), η λάρικα (Larix decidua) και η ψευδοτσούγκα (Pseudotsuga douglasii). Παρά το γεγονός ότι η φυσική εξάπλωση του εντόμου συμπίπτει με αυτή του κύριου ξενιστή του, το P. chalcographus δεν έχει καταγραφεί ούτε και παγιδευτεί στο δάσος ερυθρελάτης στην Ελατιά Δρά- μας, που αποτελεί το νοτιότερο άκρο της φυσικής, αυτόχθονης εξάπλωσης της ερυθρελάτης. Στα πλαίσια της έρευνας εγκαταστάθηκαν στο δάσος της Ελατιάς πέντε φερομονικές παγίδες, οι οποίες περιείχαν την προσελκυστική ουσία chalcogran. Ο συνολικός αριθμός των φλοιοφά- γων εντόμων που συγκεντρώθηκαν μετά το πέρας της περιόδου που οι παγίδες ήταν στο πεδίο, ανήλθε σε αρκετές χιλιάδες ατόμων. Η ερυθρελάτη που αναπτύσσεται στο φυσικό δάσος της Ελατιάς βρίσκεται σε οριακό περιβάλλον όσον αφορά τις συνθήκες ανάπτυξής της, γεγονός που επιτείνεται και από την κλιματική αλλαγή, η οποία γίνεται ακόμη πιο εμφανής σε τέτοια περιβάλλοντα. Η συνδυαστική δράση αυτών των δύο παραγόντων μπορεί να καταστήσει την ερυθρελάτη ιδιαίτερα ευάλωτη στην προσβολή του P. chalcographus, γεγονός που θα μπο- ρούσε εν δυνάμει να θέσει σε κίνδυνο και τα ενδημικά είδη πεύκου της Ελλάδας, καθώς το φλοιοφάγο αυτό έντομο έχει την δυνατότητα ανάπτυξης σε μια πλειάδα ξενιστών.The six toothed bark beetle, Pityogenes chalcographus L. (Coleoptera: Scolytinae) is a widely distributed pest in Europe, infesting mainly Norway spruce (Picea abies) as well as other conifer species such as Pinus sp., Abies alba, Larix deciduas, L. sibirica and Pseudotsuga douglasii. Even though the distribution of this bark beetle coincides that of its main host tree, P. abies, the occurrence of P. chalcographus has never been recorded in the spruce forest of Elatia-Drama, Northern Greece, which is the southernmost area of the natural, autochthonous distribution of P. abies. In this study we installed five pheromone traps baited with chalcogran dispensers in the forest of Elatia. The total number of bark beetles attracted to these traps exceeded several thousands of individuals. Norway spruce trees growing in the natural forest of Elatia demonstrate low vigor, something that can be attributed to the marginal environmental conditions in concert with the effects of climate change. The combination of these factors inhibits the regular growth of spruce, rendering trees more susceptible to the attack of P. chalcographus

Molekulare Ökologie in der Forstentomologie

Nach der Wiederentdeckung der Mendelschen Regeln Anfang des 20. Jahrhunderts waren es unter anderem Theodosius Dobzhansky und Ernst Mayr, die Evolution erstmals durch die Analyse von Populationen untersuchten. Bis 1980 wurden für diese populationsgenetischen Analysen morphologische, cytologische und enzymelektrophoretische Marker verwendet. Durch die Entdeckung der PCR wurde das Arbeiten mit DNA wesentlich erleichtert. Im Laufe der Zeit setzte sich der Begriff Molekulare Ökologie durch, mit dem man jenen Bereich beschreiben will, der molekulare Methoden der Populationsgenetik und der Genomanalyse mit ökologischen Fragestellungen verbindet. Genetische Marker können vergangene als auch zur Zeit ablaufende Prozesse aufzeigen. Mit Hilfe solcher Marker kann man einerseits Hinweise auf die Refugialgebiete von Organismen während der letzten Eiszeiten erhalten, anderseits Prozesse wie Genfluß, Selektion oder genetische Drift aufzeigen. Bei den meisten der von uns im Folgenden vorgestellten Arbeiten steht das Sequenzieren mitochondrialer Abschnitte im Vordergrund. Die mitochondriale DNA hat durch ihre im Vergleich zu nuklearer DNA erhöhte Mutationsrate und vereinfachte Genstruktur den Vorteil, bei intraspezifischen Fragestellungen gute Resultate zu erzielen.The term Molecular Ecology appeared after the discovery that DNA can be amplified in-vitro by PCR in the late 1980’s. Molecular Ecology can be defined as the scientific field that combines genetic techniques, population genetics and genomics with ecological relevant questions. Since then, many papers have been published in Molecular Ecology. Here we present some examples focusing on Palaearctic scolytid species

EUROPA OSVOJENA U TRENU: PRVI UVID U PRODOR I ŠIRENJE KESTENOVE OSE ŠIŠKARICE (Dryocosmus kuriphilus) U SREDIŠNJOJ EUROPI PUTEM MITOHONDRIJSKE DNK

The chestnut gall wasp, Dryocosmus kuriphilus, poses one of the latest additions to the long list of exotic pests that invaded Europe. After its introduction in Italy, chestnut gall wasp expanded rapidly in Europe in a very short period of time. Analysis of a polymorphic mtDNA locus from nine European populations verified the Chinese origin of this invasion. Moreover, the results revealed traces of a severe bottleneck during the phase of introduction that reduced considerably the genetic diversity. It was also shown that the rapid and successful post-introductory expansion was accomplished by a single mtDNA haplotype that has spread in three European countries. The paradox of successful establishment despite the absence of genetic diversity could be attributed to the synergistic effect of several agents. Uniparental propagation and general-purpose genotypes, lack of natural enemies and human-mediated transport seem to have facilitated the invasion and subsequent expansion of D. kuriphilus in Europe.Invazivne vrste smatraju se jednom od najvećih prijetnji bioraznolikosti te su postale i ozbiljan ekonomski problem. Biološkim invazijama pogoduju ljudske aktivnosti koje su značajan vektor pri unosu i širenju novih vrsta. Jedna od nedavno unesenih vrsta kukaca u Europu je kestenova osa šiškarica (Dryocosmus kuriphilus) (Hymenoptera; Cynipidae) koji je globalni štetnik pitomog kestena (Castanea). Napada pupove, čime sprječava rast izbojaka i listova te smanjuje plodonošenje, što konačno može dovesti do smanjenja uroda od čak 70 %.Kako se genetski polimorfizmi akumuliraju tijekom vremena, sadrže vrijedne informacije o povijesti invazije neke vrste, što objašnjava uporabu molekularnih tehnika u istraživanju invazivnih vrsta. Među različitim dostupnim markerima, mitohondrijska DNK (mtDNK) se često koristi u takvim istraživanjima. Usporedbom rezultata ovoga istraživanja s dosadašnjim rezultatima iz Kine, željelo se dokazati ne samo porijeklo izvorne populacije, nego i potencijal širenja (ekspanzije) ovoga štetnika u novom staništu. Ovo istraživanje koristi mtDNK markere za proučavanje genetske varijabilnosti kestenove ose šiškarice, prvi puta nakon njezinog unašanja u Europu.Za potrebe ovog istraživanja uzorkovano je 96 jedinki s 9 različitih lokaliteta u 3 europske zemlje (Slika 1, Tablica 1) u dvije godine (2011. i 2012.). Analiza mitohondrijske DNK napravljena je prema važećim protokolima. Pregledom sekvenci i usporedbom dva lokusa, potvrđena je pojava samo jednog haplotipa u svim populacijama u Italiji, Sloveniji i Hrvatskoj. Taj je haplotip točno odgovarao i jedinom haplotipu pohranjenom u NCBI Genebank, pod pristupnim brojem (Accession Number JF411594) iz Italije, te je bio i istovjetan s haplotipom LZ (Accession Number JF411594) koji je nađen u Kini.Vrlo su blizi i kineski haplotipovi LBX i FS, dok su haplotipovi CC i SY nešto udaljeniji (Tablica 2).Kestenova osa šiškarica unesena je u sjeverozapadnu Italiju iz Kine 2002. godine sa sadnicama pitomog kestena. Kinesko porijeklo ovoga štetnika potvrđeno je i genetskim analizama. Sve talijanske jedinke pokazuju jedan haplotip, koji je istovjetan onom kineskom pohranjenom u NCBI GenBank, pod pristupnim brojem JF411594. To potvrđuje da je osnovna populacija unesena u Europu kineskog podrijetla. Zanimljivo je i da je to i najrasprostranjeniji haplotip u Kini, nađen u nekoliko provincija (Henan, Jiangxi, Hunan and Fujian).Kako je taj haplotip još jednom nađen u Italiji, može se zaključiti da se kestenova osa šiškarica raširila u području istraživanja nakon samo jednog jedinog unosa, iz osnovne populacije ograničene veličine ili niske genetske raznolikosti. Ovo populacijsko usko grlo tijekom prve kolonizacije kestenove ose šiškarice upućuje na samo jedan slučaj unosa s nekoliko jedinki (mala osnivačka populacija). U slučaju višestrukog unosa i/ili velike osnivačke populacije, genetska varijabilnost brzo bi se povećala.Jedan od najučinkovitijih načina savladavanja mana niskih gustoća populacije spojenih s niskom genetskom raznolikosti je partenogeneza. Aseksualni način razmnožavanja je vrlo prilagodljiv, jer dozvoljava malim populacijama da se brzo šire i učinkovito iskorištavaju ograničene resurse. Zbog toga je logično pretpostaviti da je kestenova osa šiškarica, koja se razmnožava partenogenetski, iskoristila tu biološku osobinu kako bi kompenzirala populacijsko usko grlo tijekom unosa i širenja osnovnih populacija u Italiji. Širenje, nakon unosa, neke invazivne vrste određeno je njezinom evolucijskom prilagodljivosti ili plastičnosti u novom području. Evolucijska prilagodljivost olakšava širenje vrste, posebice kada postoji dovoljno genetske varijabilnosti unutar populacije invazivne vrste, kako bi došlo do procesa prirodne selekcije. No to je i paradoks: kako može invazivna vrsta s populacijskim uskim grlom biti uspješna kada su takve vrste vrlo osjetljive na izumiranje? Gubitak genetske raznolikosti tijekom kolonizacije već je primijećena kod invazivnih vrsta, no niti u jednom slučaju nije došlo do prekida takve invazije (daljnjeg širenja). Na temelju rezultata ovoga istraživanja, kestenova osa šiškarica može se uključiti u takve primjere uspješnih invazija bez obzira na nisku genetsku raznolikost prilikom unosa. Za manje od deset godina ovaj se štetnik proširio od mjesta unosa u Italiji na više europskih zemalja (Francuska,Slovenija, Švicarska, Hrvatska, Nizozemska, Austrija, Mađarska) prevalivši udaljenost od 1200 km prema jugu (Sicilija) i 750 km prema istoku (Hrvatska). Na naše iznenađenje, ova ekspanzija uspješno je obavljena samo s jednim haplotipom mtDNK, istovjetnom onome koji je i unesen u Europu. Činjenica da je haplotip koji se proširio na području istraživanja isti kao i onaj najčešći u Kini, nameće hipotezu o univerzalnom genotipu prilagođenom različitim uvjetima okoliša. Populacijsko usko grlo nije utjecalo na sposobnost kestenove ose šiškarice da se udomaći i munjevito širi Europom. Aseksualno razmnožavanje, fenotipska plastičnost zajedno s brzim širenjem uz pomoć čovjeka, nedostatak prirodnih neprijatelja i široka dostupnost biljke domaćina, odredili su smjer i opseg invazije kestenove ose šiškarice u Europi. Primjena molekularnih metoda za objašnjavanje genetske varijabilnosti ovog invazivnog štetnika dala je informacije ne samo o njegovom unosu, nego i širenju. Rezultati ovoga istraživanja mogu se koristiti kako bi se bolje razumjeli mehanizmi kako partenogentski organizmi mogu postati ozbiljni štetnici i bez dovoljne genetske varijabilnosti

Novel morphological and genetic markers for the discrimination of three European Pityokteines (Coleoptera: Curculionidae: Scolytinae) species

Background and Purpose: The three palearctic species Pityokteines spinidens, P. curvidens and P. vorontzowi are main pests on Abies species and their impact on Abies stands is increasing. As the three scolytid species, particularly females, are difficult to distinguish, this study aimed to find additional morphological characters for identification. Further, part of the mitochondrial COI gene was sequenced to develop a significant barcode marker for future use. Material and Methods: All three bark beetle species were collected from logs in Croatia (Litori} and Trako{}an), in order to quantify the number of strial and interstrial punctures. Insect DNA was extracted and PCR products were purified, directly sequenced, aligned and analyzed by MP analysis and Bayesian analysis. Results and Conclusion: The number of punctures in the first and second interstriae between the elytral base and the sutural tubercle proved to be a valuable tool for the differentiation of P. spinidens from P. curvidens and P. vorontzowi. This morphological feature was consistent with the number of punctures which varied for the first and the second interstriae in P. spinidens compared to P. curvidens and P. vorontzowi. The mitochondrial COI gene provided another means in the discrimination of Pityokteines species, revealing that P. curvidens and P. vorontzowi are sister species

Intraspecific divergence between Grapholitha molesta (Tortricidae, Lepidoptera) populations from Italy and Greece in relation to temperature.

Genetic changes contributing to phenotypic and phenological differences within or between species have been identified for a handful of traits, but the relationship between alleles underlying intraspecific polymorphism and interspecific divergence is largely unknown. Particularly for economically important insects macroscopic diagnosis and assessment of species boundaries and related population dynamics is often problematic because of the limited morphological and/or biological traits. In this work we attempt to compare temperature driven physiological responses of Grapholita molesta populations from different areas along with an initial phylogenetic analysis. To evaluate region-specific moth phenology patterns, we used the data sets of flights during two successive growth seasons (2011 and 2012). Population sizes of G. molesta moth phenology depended strongly on the particular location of research and model performance that were applied to predict population dynamics was less effective in predicting the related data sets. In addition, several individual from two populations (Italy and Greece) were screened with two mtDNA loci (761 bp and 627 bp in length) revealing the levels of intraspecific divergence within this species between different geographic regions. Beside that, however, DNA barcoding revealed that at least two species other than G. molesta were also attracted by the G. molesta lures. It was thus made evident that DNA-based approaches can shed more light into variation in phenology observed among populations of the same species. With that, more work is on the way, including a comprehensive population study and a rigorous sampling of many more geographical regions to offer a more robust explanation of intraspecific divergence

Shallow Genetic Structure among the European Populations of the Six-Toothed Bark Beetle Ips sexdentatus (Coleoptera, Curculionidae, Scolytinae)

The six-toothed bark beetle, Ips sexdentatus, is one of the most abundant scolytid species of the central and southern European countries. It mostly feeds on Pinus sp., whereas during population outbreaks it can also attack Picea sp. In spite of its broad distribution, its phylogeography has never been studied before. To do that, we employed an mtDNA marker on 489 individuals that covered most of its native range in Europe. Geographic distribution of the 86 haplotypes showed that at least three glacial refugia have played a significant role in shaping the currently observed pattern of genetic divergence in Europe, without excluding the contribution of minor refugial areas that acted in a similar manner. The revealed shallow structure can be considered an artifact of factors that reduced intraspecific diversity, at the same time favoring gene flow. As such, biological traits of the species itself (flying ability and host preference) and even human-mediated transport of wood seem to be the most prevailing and probable reasons that gave rise to the observed pattern.Austrian Science Fund FWF (project number P26749-B25)

Pine Pitch Canker and Insects: Regional Risks, Environmental Regulation, and Practical Management Options

Producción CientíficaPine pitch canker (PPC), caused by the pathogenic fungus Fusarium circinatum (Nirenberg and O’ Donnell), is a serious threat to pine forests globally. The recent introduction of the pathogen to Southern Europe and its spread in Mediterranean region is alarming considering the immense ecological and economic importance of pines in the region. Pines in forests and nurseries can be infected, resulting in severe growth losses and mortality. The pathogen is known to spread in plants for planting and in seeds, and results from recent studies have indicated that F. circinatum may also spread through phoretic associations with certain insects. With this review, we aim to expand the current understanding of the risk of insect-mediated spread of PPC in different parts of Europe. Through the joint action of a multinational researcher team, we collate the existing information about the insect species spectrum in different biogeographic conditions and scrutinize the potential of these insects to transmit F. circinatum spores in forests and nurseries. We also discuss the impact of environmental factors and forest management in this context. We present evidence for the existence of a high diversity of insects with potential to weaken pines and disseminate PPC in Europe, including several common beetle species. In many parts of Europe, temperatures are projected to rise, which may promote the activity of several insect species, supporting multivoltinism and thus, further amplifying the risk of insect-mediated dissemination of PPC. Integrated pest management (IPM) solutions that comply with forest management practices need to be developed to reduce this risk. We recommend careful monitoring of insect populations as the basis for successful IPM. Improved understanding of environmental control of the interaction between insects, the pathogen, and host trees is needed in order to support development of bio-rational strategies to safeguard European pine trees and forests against F. circinatum in future.European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH)Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project AGL2015-69370-R)Portuguese Foundation for Science and Technology (contract IF/00471/2013/CP1203/CT0001)Russian Foundation for Basic Research (grant 17-04-01486)Saint Petersburg State Polytechnical University (project 2019-0420