Sex-specific gene expression in the mosquito Culex pipiens f. molestus in response to artificial light at night

Background Artificial light at night (ALAN) is a typical feature of urban areas and most organisms living in urban or suburban habitats are exposed to low levels of ALAN. Light is one of the most important environmental cues that organisms use to regulate their activities. Studies have begun to quantify the influence of ALAN on the behavior and ecology of organisms, but research on the effects at the molecular level remains limited. Mosquitoes in the Culex pipiens complex (Diptera, Culicidae) are widespread and abundant in urban areas where they are potential disease vectors. It is thus of particular interest to understand how ALAN may influence biologically and ecologically relevant traits. Results We used RNAseq to evaluate the transcriptome response in a Cx. pipiens f. molestus laboratory population that was exposed to near- natural light conditions (light:dark L16:D8 hours, “control”) and ALAN conditions with 3 h of constant low-level light at night (L16 + Llow3:D5 hours, “low-light”). The resulting transcripts were mapped to the reference genome of the closely related Culex quinquefasciatus. Female expression patterns differed significantly between control and treatment conditions at five genes although none showed an absolute fold change greater than two (FC > 2). In contrast, male expression differed at 230 genes (74 with FC > 2). Of these, 216 genes (72 with FC > 2) showed reduced expression in the low-light treatment, most of which were related to gametogenesis, lipid metabolism, and immunity. Of the 14 genes (two with FC > 2) with increased expression, only five had any functional annotation. There was a pronounced sex-bias in gene expression regardless of treatment, with 11,660 genes (51 % of annotated genes; 8694 with FC > 2; 48 % of annotated genes) differentially expressed between males and females, including 14 genes of the circadian clock. Conclusion Our data suggest a stronger response to artificial light by males of Cx. pipiens f. molestus than by females, and that a wide range of physiological pathways may be affected by ALAN at the molecular level. The fact that differences in gene expression appear to be sex-specific may have a strong influence at the population level

Establishing a Large-Scale Field Experiment to Assess the Effects of Artificial Light at Night on Species and Food Webs

Artificial light at night (ALAN) is one of the most obvious hallmarks of human presence in an ecosystem. The rapidly increasing use of artificial light has fundamentally transformed nightscapes throughout most of the globe, although little is known about how ALAN impacts the biodiversity and food webs of illuminated ecosystems. We developed a large-scale experimental infrastructure to study the effects of ALAN on a light-naïve, natural riparian (i.e., terrestrial-aquatic) ecosystem. Twelve street lights (20 m apart) arranged in three rows parallel to an agricultural drainage ditch were installed on each of two sites located in a grassland ecosystem in northern Germany. A range of biotic, abiotic, and photometric data are collected regularly to study the short- and long-term effects of ALAN on behavior, species interactions, physiology, and species composition of communities. Here we describe the infrastructure setup and data collection methods, and characterize the study area including photometric measurements. None of the measured parameters differed significantly between sites in the period before illumination. Results of one short-term experiment, carried out with one site illuminated and the other acting as a control, demonstrate the attraction of ALAN by the immense and immediate increase of insect catches at the lit street lights. The experimental setup provides a unique platform for carrying out interdisciplinary research on sustainable lighting

Microallopatry Caused Strong Diversification in Buthus scorpions (Scorpiones: Buthidae) in the Atlas Mountains (NW Africa)

The immense biodiversity of the Atlas Mountains in North Africa might be the result of high rates of microallopatry caused by mountain barriers surpassing 4000 meters leading to patchy habitat distributions. We test the influence of geographic structures on the phylogenetic patterns among Buthus scorpions using mtDNA sequences. We sampled 91 individuals of the genus Buthus from 51 locations scattered around the Atlas Mountains (Antiatlas, High Atlas, Middle Atlas and Jebel Sahro). We sequenced 452 bp of the Cytochrome Oxidase I gene which proved to be highly variable within and among Buthus species. Our phylogenetic analysis yielded 12 distinct genetic groups one of which comprised three subgroups mostly in accordance with the orographic structure of the mountain systems. Main clades overlap with each other, while subclades are distributed parapatrically. Geographic structures likely acted as long-term barriers among populations causing restriction of gene flow and allowing for strong genetic differentiation. Thus, genetic structure and geographical distribution of genetic (sub)clusters follow the classical theory of allopatric differentiation where distinct groups evolve without range overlap until reproductive isolation and ecological differentiation has built up. Philopatry and low dispersal ability of Buthus scorpions are the likely causes for the observed strong genetic differentiation at this small geographic scale

Einblicke aus Genetik, Physiologie, Verhalten und Populationsstruktur

Summary The term urbanisation describes, on the one hand, the process of outward expansion of cities and on the other hand a changed behaviour of rural populations, i.e. functional urbanisation. Especially the physical expansion of cities creates increasing pressure on the surrounding environments and its plant and animal communities. To date, there are 32 Megacities (>10 million inhabitants) around the globe, 11 of them with more than 20 million inhabitants. As a habitat, cities are quite challenging as they are highly dynamic, crowded, noisy, polluted and extremely patchy in terms of comparable habitat types or local temperature. One important feature accompanying the majority of human settlements is artificial light at night. In fact, its presence became as natural to us as the moon and stars. However, light itself is a stimulus to above-ground species that regulates timekeeping, daily activity and seasonal behaviours. Consequently, light could potentially impact a given organism on an array of levels, for example genetically or behaviourally, but also over different time scales, for instance daily or seasonally. In order to assess the effects of artificial light at night I analysed different levels, i.e. gene expression, activity, fecundity and population structure, in one species, the common house mosquito Culex pipiens. In the first chapter of my thesis I examined the transcriptome, which is the entirety of the transcribed genes at a given moment in time, of male and female mosquitoes after exposure to different light regimes (1. “control”: light:16h and dark:8h; 2. “twilight” light:16+3h and dark:5h). I found that gene expression between males and females differed by more than 50%, independent of the light treatment. This underlines the importance of studying both sexes separately, rather than pooling the data, in order to avoid under- or overestimation of the effect of artificial light arising by opposing or additive patterns of sex-specific gene expression. The extended light treatment elicited a reduction in the expression of genes related to gametogenesis, immune response and lipid metabolism in males but not in females. I then specifically analysed the gene expression of 5 genes (period, timeless, clock, cycle and cryptochrome-2) comprising most of the central circadian clock using qRT-PCR. The photoreceptor CRYPTOCHROME-1, another clock component, picks up blue light and feeds the information, via a reaction cascade, into the clock, which results in daily fine-tuning. Therefore, information obtained from ambient light levels should lead to a shift in expression levels in the extended light treatment (light:dark: 16+3:8h). I indeed found a down-regulation of the period and the clock gene expression in the light treatment. When analysing this for each sex separately, I found the same expression pattern in males. However, in females expression of the period gene was down-regulated while the cycle gene expression was up-regulated. There was a clear difference in expression, even though I did not find evidence for an overall shift in the clock timing compared to the baseline group. The circadian clock regulates a number of downstream processes, daily activity being one of them. A change in timekeeping should thus be reflected in daily locomotor activity. This was tested using a Locomotor Activity Monitor, which records activity as the number of crossings of infra red beams in a given time interval. Males and females displayed very different movement patterns, with males being less active than females. The extended light period significantly reduced activity in both sexes throughout the day, not only at the time of application. In constant darkness, the individuals resumed a similar activity pattern, i.e. active phases, indicating that the clock rhythm (~24h) was not shifted, even though the magnitude of activity was reduced. To understand if less activity in the light treatment had implications for the population I analysed female fecundity as the number and size of eggs produced per population. Less active individuals may have less time for feeding and thus the nutritional status may not be optimal. Optimising reproductive output could then be achieved by producing either fewer eggs of the same size (as in optimal conditions) or the same number but smaller eggs. I found fewer and smaller eggs per raft in the light treatment, indicating that light may have effects on the population level as well. The next step was to evaluate whether the presence of artificial light leads to a detectable differentiation between natural urban and rural populations. A microsatellite analysis was conducted to estimate the degree of differentiation along an urban-rural gradient. Unexpectedly, there was no differentiation. Instead there was a high connectivity between local populations ensuring gene flow and thus a high genetic variability in a panmictic population across the study area. In this thesis, I was able to show that artificial light at night does influence mosquitoes on several levels. Light can be a stressor potentially influencing adult individual fitness. Additionally, the fitness of the offspring might be negatively impacted. On the other hand, the high amount of genetic variability and potential exchange of rural and urban local populations ensures a large gene pool and a high adaptive potential. To date, this is the only work addressing the impact of artificial light at night on several levels. It therefore presents important information on potential influences of light pollution. Furthermore, valuable insights on mosquito ecology, especially in males, have been gained, which may be of great interest for estimating the success of control programs.Urbanisierung beschreibt die physische Expansion von Städten einerseits, zum Anderen aber auch das zunehmend verstädterte Verhalten von Bewohnern kleinerer, ruraler Ortschaften. Dieser Vorgang wird als funktionelle Urbanisierung bezeichnet. Insbesondere die Ausdehnung von Städten sorgt für steigenden Druck auf Flora und Fauna der umgebenden Landschaft. So gibt es bis heute 32 Megastädte (> 10 Millionen Einwohner), 11 davon haben bereits mehr als 20 Millionen Einwohner. Als Habitat sind Städte eine Herausforderung da sie hoch-dynamisch, überfüllt, laut und verschmutzt sind. Vergleichbare Habitat Typen sind sehr ungleichmäßig über Stadt verteilt, vor allem auch in Bezug auf lokal vorherrschende Temperaturen. Ein weiteres Merkmal urbaner Lebensräume ist künstliches Licht in der Nacht. Tatsächlich ist künstliches Licht für uns so alltäglich geworden wie Mond und Sterne. Nichtsdestotrotz, Licht als solches ist ein wichtiger Stimulus für überirdische Lebewesen, der Zeiterfassung sowie tägliche und saisonale Aktivität reguliert. Dies verdeutlicht, das Licht einen Organismus möglicherweise auf einer ganzen Reihe von Ebenen beeinflussen könnte, zum Beispiel genetisch oder verhaltensbiologisch, aber auch über Zeiträume hinweg, etwa im Tages- oder Jahresverlauf. Um den Einfluss künstlichen Lichts bewerten zu können, analysierte ich unterschiedliche Ebenen innerhalb einer Stechmückenart, der gemeinen Hausmücke (Culex pipiens). Im ersten Teil dieser Arbeit untersuchte ich das Transkriptom, die Gesamtheit der Gentranskripte zu einem bestimmten Zeitpunkt, von männlichen und weiblichen Stechmücken nachdem sie unterschiedlichen Lichtregimes (1. „control“ Licht: 16 Std. und dunkel:8 Std.; 2. „twilight“ Licht: 16+3 Std. und Dunkel: 8 Std.) ausgesetzt waren. Mehr als 50% der Gene wurden allein beim Vergleich von Männchen mit Weibchen unterschiedlich exprimiert. Ein Befund, der die Wichtigkeit unterstreicht, beide Geschlechter getrennt zu untersuchen, anstatt die Genexpression aller Tiere gemeinsam zu betrachten. Nur so lässt sich vermeiden, dass aufgrund von gegensätzlichen oder additiven geschlechtsspezifischen Genexpressionsmustern die Auswirkungen künstlichen Lichtes unter- oder überschätzt werden. Des Weiteren verursachte die verlängerte Lichtperiode eine Reduktion der Expression von Genen, die in der Gametogenese, der Immunreaktion und im Fettstoffwechsel involviert sind in Männchen, jedoch nicht in Weibchen. Danach untersuchte ich mittels qRT-PCR speziell die Expression von 5 Genen (period, timeless, clock, cycle und cryptochrome-2) der zentralen, circadianen Uhr. Der Fotorezeptor CRYPTOCHROME-1 nimmt Photonen blauer Wellenlängen auf und setzt so eine Reaktionskaskade in Gang. Auf diesem Weg gelangen Informationen zur Tageszeit zum zentralen Uhr-Mechanismus, was für dessen Feineinstellung sorgt. Eine Veränderung des Lichtregimes, wie im durchgeführten Versuch, sollte also einer Veränderung der Genexpression führen. Tatsächlich fand eine Herunterregulierung der Genexpression von period und clock bei verlängerter Hellphase statt. Bei der Untersuchung nach Geschlechtern getrennt fand sich das gleiche Expressionsmuster bei Männchen. Bei den Weibchen wurde das period- Gen ebenfalls herunter- das cycle-Gen jedoch heraufreguliert. Obwohl es insgesamt klare Unterschiede in der Genexpression zwischen Mücken beider Lichtregimes gab, konnte ich keinen Hinweis auf eine allgemeine Verschiebung des circadianen Rhythmus finden. Die innere Uhr reguliert die Funktion einiger nachgeschalteter Prozesse, tägliche Aktivität ist einer davon. Unterschiede in der Zeiterfassung sollten daher in divergierenden täglichen Bewegungsmustern resultieren. Dies wurde mit Hilfe von einem Locomotor Activity Monitor getestet. Dieses Gerät zählt die Anzahl der Durchtritte durch Infrarot Schranken in einem gegebenen Zeitintervall. Männchen und Weibchen zeigten sehr unterschiedliche Aktivitätsmuster, die Männchen waren generell weniger aktiv. Bei verlängerter Hellphase war die Aktivität beider Geschlechter über den ganzen Tag hinweg signifikant reduziert, nicht nur in der Phase, in der Licht appliziert wurde. In ständiger Dunkelheit zeigten die Individuen beider Gruppen ähnliche Aktivitätsmuster, d. H. ähnlich lange Phasen der Aktivität. Dies deutet darauf hin, dass der Rhythmus der Uhr (ca. 24h) nicht verändert war, obwohl das Ausmaß der Aktivität reduziert war. Um zu verstehen, ob reduzierte Aktivität Auswirkungen für die gesamte Population hat untersuchte ich die Fekundität der Weibchen anhand der Anzahl und Größe der Eier pro Population in beiden Lichtregime-Gruppen. Weniger aktive Individuen könnten weniger Zeit zur Nahrungsaufnahme haben und somit einen schlechteren Ernährungszustand. Im Falle einer optimierten Nachkommen-Produktion könnte das wiederum entweder zu weniger aber gleich großen Eiern pro Eischiffchen führen, oder aber zu kleineren Eiern, die aber ähnlich zahlreich sind wie bei gutem Ernährungszustand. Ich fand heraus, dass im Lichtversuch die Anzahl der Eier pro Schiffchen geringer war. Gleichzeitig waren die Eier aber ebenfalls kleiner. Dies deutet darauf hin, dass künstliches Licht auch Auswirkungen auf Populationsebene haben könnte. Somit war der nächste Schritt zu evaluieren, ob die Präsenz künstlichen Lichts zu detektierbarer Differenzierung zwischen natürlichen urbanen und ruralen Populationen führt. Mittels einer Mikrosatelliten-Analyse wurde der Grad der Differenzierung entlang eines urban-rural Gradienten bestimmt. Entgegen den Erwartungen konnte keine Differenzierung gefunden werden. Stattdessen scheint eine hohe Konnektivität zwischen lokalen Populationen zu bestehen, die dann Genfluss und somit eine hohe genetische Variabilität in einer panmiktischen Population über das ganze Untersuchungsgebiet sichert

City-Dwellers and Country Folks: Lack of Population Differentiation Along an Urban-Rural Gradient in the Mosquito Culex pipiens (Diptera: Culicidae)

Mosquitoes (Diptera, Culicidae) occur in natural, urban, and peri-urban areas throughout the globe. Although the characteristics of urban and peri-urban habitats differ from those of natural habitats in many ways (e.g., fragmentation, pollution, noise, and light), few studies have examined the population connectivity of mosquitoes in urban areas. To obtain an overview of the species composition, we sampled mosquitoes from 23 sites in and around the city of Berlin, Germany. Of 23 species, five occurred in urban, 10 in peri-urban, and 20 in rural areas. Culex pipiens Linnaeus (Diptera: Culicidae) was the most common species collected (75% of all individuals) and occurred in all habitats. Hence this species was selected to be analysed at 10 microsatellite markers. There was no significant differentiation (FST = 0.016, P = 0.9) or isolation by distance (P = 0.06) among Cx. pipiens populations along an urban-rural gradient. The only significant differences detected were between Cx. pipiens and a laboratory population of Cx. pipiens f. molestus (pairwise FST = 0.114-0.148, P ≤ 0.001 in all comparisons), suggesting that the markers chosen were suitable for the identification of population differentiation. Our results indicate that Cx. pipiens gene flow is widespread within and among urban, peri-urban, and rural areas and that urban habitat does not necessarily impede or enhance gene flow among these populations

City-dwellers and country folks: lack of population differentiation along an urban-rural gradient in the mosquito culex pipiens (Diptera: culicidae)

Mosquitoes (Diptera, Culicidae) occur in natural, urban, and peri-urban areas throughout the globe. Although the characteristics of urban and peri-urban habitats differ from those of natural habitats in many ways (e.g., fragmentation, pollution, noise, and light), few studies have examined the population connectivity of mosquitoes in urban areas. To obtain an overview of the species composition, we sampled mosquitoes from 23 sites in and around the city of Berlin, Germany. Of 23 species, five occurred in urban, 10 in peri-urban, and 20 in rural areas. Culex pipiens Linnaeus (Diptera: Culicidae) was the most common species collected (75% of all individuals) and occurred in all habitats. Hence this species was selected to be analysed at 10 microsatellite markers. There was no significant differentiation (FST = 0.016, P = 0.9) or isolation by distance (P = 0.06) among Cx. pipiens populations along an urban-rural gradient. The only significant differences detected were between Cx. pipiens and a laboratory population of Cx. pipiens f. molestus (pairwise FST = 0.114-0.148, P ≤ 0.001 in all comparisons), suggesting that the markers chosen were suitable for the identification of population differentiation. Our results indicate that Cx. pipiens gene flow is widespread within and among urban, peri-urban, and rural areas and that urban habitat does not necessarily impede or enhance gene flow among these populations

Artificial Light at Night Influences Clock-Gene Expression, Activity, and Fecundity in the Mosquito Culex pipiens f. molestus

Light is an important environmental cue, and exposure to artificial light at night (ALAN) may disrupt organismal physiology and behavior. We investigated whether ALAN led to changes in clock-gene expression, diel activity patterns, and fecundity in laboratory populations of the mosquito Culex pipiens f. molestus (Diptera, Culicidae), a species that occurs in urban areas and is thus regularly exposed to ALAN. Populations were kept under 16 h:8h light:dark cycles or were subjected to an additional 3.5 h of light (100–300 lx) in the evenings. ALAN induced significant changes in expression in all genes studied, either alone (period) or as an interaction with time (timeless, cryptochrome2, Clock, cycle). Changes were sex-specific: period was down-regulated in both sexes, cycle was up-regulated in females, and Clock was down-regulated in males. ALAN-exposed mosquitoes were less active during the extra-light phase, but exposed females were more active later in the night. ALAN-exposed females also produced smaller and fewer eggs. Our findings indicate a sex-specific impact of ALAN on the physiology and behavior of Culex pipiens f. molestus and that changes in clock-gene expression, activity, and fecundity may be linked

Additional file 4: Table S4. of Sex-specific gene expression in the mosquito Culex pipiens f. molestus in response to artificial light at night

Spreadsheet listing all differentially expressed gene s between male and female mosquitoes. GeneID = gene ID; base Mean = the mean of the normalized counts for all samples;log2FoldChange = Log 2 fold change in expression low-light relative to control; lfcMLE = unshrunken maximum likelihood estimates of Log2 fold change in expression; lfcSE = Standard Error of log2FoldChange; Stat = Wald statisic; pvalue; padj = p value adjusted after Benjamini-Hochberg; Annotation = functional annotation of the gene; Domain(s) = Interpro domain annotations. Genes with an absolute fold change of two are shown in bold. (XLS 2665 kb

Additional file 2: Table S2. of Sex-specific gene expression in the mosquito Culex pipiens f. molestus in response to artificial light at night

Spreadsheet listing all differentially expressed genes in females exposed to the ALAN treatment based on adjusted p-values. Gene = gene ID; base Mean = the mean of the normalized counts for all samples; log2FoldChange = Log 2 fold change in expression low-light relative to control; lfcMLE = unshrunken maximum likelihood estimates of Log2 fold change in expression; lfcSE = Standard Error of log2FoldChange; Stat = Wald statisic; pvalue; padj = p value adjusted after Benjamini-Hochberg; Annotation = functional annotation of the gene; Domain(s) = Interpro domain annotations. (XLS 19 kb

Multiple introductions and overwintering shape the progressive invasion of Aedes albopictus beyond the Alps

Abstract Aedes albopictus originates from Southeast Asia and is considered one of the most invasive species globally. This mosquito is a nuisance and a disease vector of significant public health relevance. In Europe, Ae. albopictus is firmly established and widespread south of the Alps, a mountain range that forms a formidable biogeographic barrier to many organisms. Recent reports of Ae. albopictus north of the Alps raise questions of (1) the origins of its recent invasion, and (2) if this mosquito has established overwintering populations north of the Alps. To answer these questions, we analyzed population genomic data from >4000 genome‐wide SNPs obtained through double‐digest restriction site‐associated DNA sequencing. We collected SNP data from specimens from six sites in Switzerland, north and south of the Alps, and analyzed them together with specimens from other 33 European sites, five from the Americas, and five from its Asian native range. At a global level, we detected four genetic clusters with specimens from Indonesia, Brazil, and Japan as the most differentiated, whereas specimens from Europe, Hong Kong, and USA largely overlapped. Across the Alps, we detected a weak genetic structure and high levels of genetic admixture, supporting a scenario of rapid and human‐aided dispersal along transportation routes. While the genetic pattern suggests frequent re‐introductions into Switzerland from Italian sources, the recovery of a pair of full siblings in two consecutive years in Strasbourg, France, suggests the presence of an overwintering population north of the Alps. The suggestion of overwintering populations of Ae. albopictus north of the Alps and the expansion patterns identified points to an increased risk of further northward expansion and the need for increased surveillance of mosquito populations in Northern Europe