Genetische Charakterisierung und Verbreitung von Stechmücken (Diptera: Culicidae) in Deutschland: Träger humanpathogener Krankheitserreger

Stechmücken (Dipteren: Culicidae) sind weltweit mit über 3500 Arten und mit Ausnahme der arktischen Regionen ubiquitär vertreten. Die medizinische Relevanz dieser Tiergruppe, begründet durch die hämatophage Lebensweise der Weibchen, erschloss sich bereits Ende des 19. Jh. und hat bis heute Bestand. Jedes Jahr sterben rund 600.000 Menschen an den Folgen der Malaria und fast 100 Mio. Menschen infizieren sich mit dem Denguefieber. Zwar beziehen sich diese Zahlen fast ausschließlich auf die Entwicklungsländer, aber im Zuge des Klimawandels und des immer stärkeren Welthandels kommt es auch in Europa und den USA immer wieder zu Ausbrüchen vorher nicht relevanter Krankheiten. So hat sich das West-Nil- Virus seit 1999 in Nordamerika rasant verbreitet. Im Jahr 2013 gab es dort rund 2500 Fälle, von denen 119 zum Tod führten. In Europa traten hingegen Krankheiten wie das Chikungunyafieber (Italien 2007) oder das Denguefieber (Frankreich 2010/2013) auf. Die Gründe für diese Ausbrüche sind vor allem in der Einschleppung neuer Vektorspezies und Krankheitserreger sowie in den veränderten Wirtspräferenzen einheimischer Stechmückenarten zu suchen. Das Wissen um das Vektorpotential der in Deutschland heimischen Stechmücken konnte vor allem durch die seit 2009 initiierten Monitoring-Programme stetig erweitert werden. Auch die Veränderung der heimischen Fauna durch invasive Arten wie Ochlerotatus japonicus japonicus oder Aedes albopictus wird intensiv erforscht. Dennoch ist hinsichtlich der Biologie, Ökologie sowie Genetik vieler Arten noch immer wenig bekannt. Die vorliegende Dissertation, welche auf Basis von vier (ISI-) Einzelpublikationen kumulativ angefertigt wurde, beschäftigte sich mit der Analyse der genetischen Variabilität sowie der Zoogeographie der untersuchten Arten und der Etablierung einer schnellen und kostengünstigen Methode zur Artdiagnostik. Besonderes Augenmerk wurde bei den Analysen auf die beiden heimischen Arten Culex pipiens und Culex torrentium sowie die invasive Art Ochlerotatus japonicus japonicus gelegt. Ziel war es, die noch bestehenden Wissenslücken zu füllen, um zukünftige Monitoring-Programme besser koordinieren sowie Analysen zur Vektorkompetenz und Genetik dieser Arten gezielter durchführen zu können. Es konnte gezeigt werden, dass Cx. pipiens und Cx. torrentium deutliche Unterschiede in ihren Populationsstrukturen aufwiesen welche auf verschiedene evolutive Prozesse hindeuten. Die geringere genetische Variabilität in Cx. pipiens lässt auf positive Selektion durch z.B. Insektizidresistenz im Zuge durchgeführter Bekämpfungsmaßnahmen oder die Infektion mit Wolbachien schließen. Die analysierte Populationsstruktur von Cx. torrentium spricht hingegen für eine geringe Ausbreitung, wodurch der genetische Austausch reduziert wurde und so die untersuchten Populationen genetisch stärker voneinander abwichen. Des Weiteren ließen die Analysen des Cytochrom c Oxidase Untereinheit 1-Fragmentes (cox1) Rückschlüsse auf die Zoogeographie dieser Arten in Deutschland zu - wobei beide Arten über das Untersuchungsgebiet verteilt waren, Cx. torrentium jedoch in den neuen Bundesländern weniger häufig nachgewiesen wurde als in den alten und eine geringere gefangene Individuenzahl aufwies. Basierend auf der ökologischen Nischenmodellierung konnten potentiell neue Verbreitungsgebiete für die Art Ochlerotatus japonicus japonicus identifiziert werden. Als klimatisch besonders günstig zeigten sich dabei Südhessen, das Saarland sowie nördliche Teile Nordrhein-Westfalens. Mit Hilfe der etablierten Methode der direct-PCR wird in Zukunft eine schnellere und kostengünstigere Identifizierung von Stechmücken erfolgen können, welche aufgrund bestimmungsrelevanter Merkmale nicht mehr morphologisch zu identifizieren sind. Um das Wissen über die Stechmücken in Deutschland fortlaufend zu intensivieren, ist sowohl das Weiterführen der Monitoring-Programme als auch die molekularbiologische Aufarbeitung der Proben nötig. Durch die Anwendung neuer Techniken und weiterer molekularer Marker wird es möglich sein, weitere Krankheitserreger sowie genetische Besonderheiten der heimischen Stechmückenfauna nachzuweisen. Aber auch die Überwachung invasiver Stechmückenarten durch die Modellierung potentieller Verbreitungsgebiete und die Anwendung molekularbiologischer Analysemethoden zum Detektieren der Arten und möglicher Krankheitserreger wird ein wichtiger Bestandteil der weiteren Forschung sein

Parasite diversity of European Myotis species with special emphasis on Myotis myotis (Microchiroptera, Vespertilionidae) from a typical nursery roost

Background: Bats belong to one of the most species-rich orders within the Mammalia. They show a worldwide distribution, a high degree of ecological diversification as well as a high diversity of associated parasites and pathogens. Despite their prominent and unique role, the knowledge of their parasite-host-relationships as well as the mechanisms of co-evolutionary processes are, partly due to strict conservation regulations, scarce. Methods: Juvenile specimens of the greater mouse-eared bat (Myotis myotis) from a roosting colony in Gladenbach (Hesse, Germany) were examined for their metazoan endo-and ectoparasite infections and pathogens. Morphometric data were recorded and the individuals were checked for Lyssavirus-specific antigen using a direct immunofluorescence test. For unambiguous species identification, the bats were analysed by cyt-b sequence comparison. Results: Myotis myotis were parasitized by the six insect and arachnid ectoparasite species, i.e. Ixodes ricinus, Ischnopsyllus octactenus, Ichoronyssus scutatus, Steatonyssus periblepharus, Spinturnix myoti and Cimex dissimilis. Additionally, the nematode Molinostrongylus alatus and the cestode Vampirolepis balsaci were recorded. Each bat was parasitized by at least four species. The parasites showed partially extreme rates of infection, never recorded before, with more than 1,440 parasites per single host. Ichoronyssus scutatus, Steatonyssus periblepharus, Vampirolepis balsaci and Molinostrongylus alatus are recorded for the first time in Germany. A checklist for Europe is presented containing records of 98 parasite species of 14 Myotis species. Conclusions: The Myotis myotis from Gladenbach (Hesse, Germany) were parasitized by a diverse parasite fauna with high infestation rates. We assume that in juvenile Myotis the number of parasites is generally higher than in adults due to only later acquired immune competence and behavioural adaptations. Our results revealed new insights into parasite fauna of M. myotis and European bats in general. The finding of endoparasitic cyclophyllidean cestodes that have a two-host lifecycle is, considering the stationary behaviour of the juvenile bats, rather unusual and suggests a non-predatory transmission mechanism (e.g. via autoinfection). A new insight gained from the collated literature was that the European wide composition of the Myotis parasite fauna is dominated by a few specific taxonomic groups in Europe

Population structure and distribution patterns of the sibling mosquito apecies Culex pipiens and Culex torrentium (Diptera: Culicidae) reveal different evolutionary paths

Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species Culex pipiens and Culex torrentium. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima's D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for Cx. pipiens, widely distributed within Germany and a more homogeneous pattern for Cx. torrentium. The low genetic differences within Cx. pipiens could be a result of an infection with Wolbachia which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (FST) ranged significantly from moderate to very great between populations of Cx. pipiens and Cx. torrentium. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations (Cx. pipiens [88.38%]; Cx. torrentium [66.54%]). Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species' genetic variation. Overall, the results confirm that Cx. pipiens and Cx. torrentium underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction

Population Structure and Distribution Patterns of the Sibling Mosquito Species <i>Culex pipiens</i> and <i>Culex torrentium</i> (Diptera: Culicidae) Reveal Different Evolutionary Paths

<div><p>Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species <i>Culex pipiens</i> and <i>Culex torrentium</i>. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima's D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for <i>Cx. pipiens</i>, widely distributed within Germany and a more homogeneous pattern for <i>Cx. torrentium</i>. The low genetic differences within <i>Cx. pipiens</i> could be a result of an infection with <i>Wolbachia</i> which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (F_ST) ranged significantly from moderate to very great between populations of <i>Cx. pipiens</i> and <i>Cx. torrentium</i>. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations (<i>Cx. pipiens</i> [88.38%]; <i>Cx. torrentium</i> [66.54%]). Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species' genetic variation. Overall, the results confirm that <i>Cx. pipiens</i> and <i>Cx. torrentium</i> underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction.</p></div

Sampling localities in Germany with abbreviations and number of sequences and detected haplotypes at each locality.

<p>Sampling localities in Germany with abbreviations and number of sequences and detected haplotypes at each locality.</p

Sampling localities of <i>Culex pipiens</i> across Germany with significant different population pairwise F_ST values.

<p>Significant different pairwise F_ST values between populations are indicated using different line colors. Significant F_ST values were grouped into the four following categories: very great population differentiation (red lines), great population differentiation (yellow lines), moderate population differentiation (green lines) and low population differentiation (purple lines) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102158#pone.0102158-Balloux1" target="_blank">[67]</a>. Pictured are all sampling points listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102158#pone-0102158-t004" target="_blank">Table 4</a> with a summary of their haplotypes. Map was created with ArcMap 10.1.</p

Sampling localities of <i>Culex torrentium</i> across Germany with significant different population pairwise F_ST values.

<p>Significant F_ST values were grouped into the four following categories: very great population differentiation (red lines), great population differentiation (yellow lines), moderate population differentiation (green lines) and low population differentiation (purple lines) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102158#pone.0102158-Balloux1" target="_blank">[67]</a>. Pictured are all sampling points listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102158#pone-0102158-t005" target="_blank">Table 5</a> with a summary of their haplotypes. There were no significant moderate or low F_ST values. Map was created with ArcMap 10.1.</p

AMOVA group structure of <i>Culex pipiens</i> and <i>Culex torrentium</i>.

<p>Group structures are based on pairwise F_ST's of <i>Culex pipiens</i> and <i>Culex torrentium</i>.</p

Haplotype networks of <i>Culex pipiens</i> and <i>Culex torrentium</i> for the cox1 gene segment calculated using statistical parsimony as implemented in TCS 1.21.

<p>The squares stand for the most probable ancestral haplotypes, the circle for all other haplotypes. The Numbers are equal to the haplotypes of each species. Each line represents a single mutation while small white dots symbolize hypothetical missing haplotypes. The size of the circles and the square is proportional to the number of the occurring haplotypes. The number of individuals can be derived from the scale which is given in the figure. Different colors represent the different geographical sampling localities. The colored area is proportional to the occurrence at the respective site.</p

Distribution of <i>Culex torrentium</i> (white) and <i>Culex pipiens</i> (grey) in Germany (A) and the Hessian Rhine-Main area (B).

<p>Small circles in Figure 1A (excluding the circles for FFM, BV, AS and GR) indicate that only one of the two species was detected at this specific locality. Pie charts indicate the ratio of the two detected species at this locality. The sizes of the pie chart and the circles do not relate to the number of investigated individuals (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102158#pone-0102158-t001" target="_blank">Table 1</a>). A: Overview of the sampling localities across Germany. Abbreviations: AS = Altenstadt, BV = Bad Vilbel, MF = Berlin-Marienfelde, BI = Bielefeld, BL = Bad Lippspringe, DB = Duisburg, DK = Dresden-Klotzsche, EW = Eberswalde, FFM = Frankfurt/Main (four different localities: Bornheim (FB), Bockenheim (KS), Sachsenhausen (FS) and Ostend (FZ)), FT = Fuldatal, GR = Gründau-Rothenbergen, HU = Husum, KL = Klein Linden, LE = Lebus, LL = Langenlehsten, MG = Mönchengladbach, MÜ = Müncheberg, RI = Rietschen, ST = Stralsund and WI = Wismar. B: Detailed view of the Rhine-Main area with Höchst a.d.N. (A1), Eichen (AS2), Heldenbergen (AS3), Klein Linden. Map was created with ArcMap 10.1.</p