Despite increasing recognition of the role of ALAN in ecology and behavior [1–3], our understanding of how it affects organisms at the molecular level remains limited. Here we assessed the effects of artificial light on Cx. pipiens, a widespread and abundant mosquito that is prominent in many urban habitats, by examining changes in gene expression in the entire body. This is the first study to provide data on sex-specific gene expression in adult Culex pipiens f. molestus. The majority of studies on mosquitoes focus on females due to their role as disease vector.
We used Cx. quinquefasciatus genome as a reference to map our data. The only other transcriptomic study of Cx. pipiens f. molestus to date found that the relationship between species was sufficiently close to obtain reliable mapping results . Both species belong to the Cx. pipiens complex. They are known to be able to hybridise and the species status of Cx. quinquefasciatus remains debated [13, 27, 28]. This study, however, focussed on searching for ecotype-specific divergence between genes in samples of mixed sexes .
The experimental design of our study focused on separate analysis of sexes. There was a very strong sex bias in expression, and males in our laboratory population exhibited a much more pronounced response to the treatment mimicking artificial light at night (“low-light”), with many of the affected genes having functional annotation. This study may serve as foundation for future work by providing whole-body transcriptome data for a widespread mosquito and suggests that ALAN can affect a broad range of physiological pathways at the molecular level. Although we are aware that gene expression varies among tissues [8, 16, 17] we chose this approach because our goal was to obtain an overview of processes potentially affected by artificial light at night. This was done at the expense of tissue-specific responses, but we believe that our results provide an important starting point for more detailed studies concerning gene expression in separate tissues, different developmental stages or different light sources and regimes.
The finding that males and females are affected to a different extent fits with the only other published study of sex-specific gene expression in mosquitoes  and suggests possible implications for reproduction biology and, consequently, population-level impacts.
Our experiment was designed to mimic artificial light of the kind generated by street and other outdoor lighting, where the normal transition from natural light (during the day) to darkness at night is altered by an abrupt switching on of light for a period of constant brightness. Veronesi et al.  found that certain light intensity thresholds (e.g. 5 lux for Cx. pipiens) function as cues for commencing or ceasing activity. Although the light regime in the treatment never fell below this threshold, our light regime still provides a cue for anticipation of the onset of darkness by its design to mimic the natural rise and fall of light intensity. In contrast, no cue was provided that allows anticipation of the abrupt switch to “artificial low-light” (300 lux in our experiment, an increase of a factor of ~6). We therefore believe the experiments measured a response to artificial light rather than comparing a short day to a long day. However, our choice to deprive individuals of light cues for 48 h prior to the experiment, and thus prohibiting synchronisation to the ambient light environment, constitutes an acute change relative to the darkness experienced before. As a nocturnal species, Cx. pipiens is naturally exposed to varying, albeit low, light intensities produced by moonlight. Studies have suggested that lunar cycles can influence the activity and biting propensity of mosquitoes [29, 30]. There were no cues regarding the lunar cycle in our laboratory setup. Our results are thus likely to be the response to artificial light at night. However, the presence of moonlight in combination with artificial light may produce a different gene expression profile in natural populations, which remains to be addressed in future studies.
Male response to artificial light at night
The response to artificial light at night in males was primarily detected as reduced expression levels of a number of genes in “low-light” treatment conditions, i.e. in males exposed to artificial light at night instead of the (laboratory-simulated) natural progression from daylight to darkness. These down-regulated genes were mainly related to gametogenesis, immunity and lipid metabolism. There is a scarcity of knowledge about the effects of artificial light on mosquitoes. Under natural conditions, decreasing light triggers activity in nocturnal mosquitoes. Individuals begin the search for food and mates, and males begin to swarm. We may speculate that the expression of genes involved in gametogenesis should increase as light levels decrease, but that artificial light inhibited this here. Our findings are preliminary, and we are not aware of any similar studies, so this remains a hypothesis to be tested in the future. Genes involved in lipid metabolism comprised another important group of genes that were less expressed under ALAN. The last food uptake was 12 h prior to sampling which could mean that the carbohydrate reserves had been used up. It may also mean that males in artificially lit environments were less active. Further work should address whether the observed changes in expression of genes related to lipid metabolism are caused by light-mediated reduced activity or a sign of usage due to starvation. Genes involved in immune response also exhibited lower expression. Some immune genes are known to be rhythmically expressed in An. gambiae [8, 16], and this might also be the case for Cx. pipiens. Despite the evidence for the negative influence of artificial light on immune genes and given their cyclic expression patterns, future studies with a 24-h sampling scheme could provide important insights that enable us to fully understand how these two processes interact.
It is well known that some genes exhibit cyclic expression over time . Our initial analysis using ‘treatment’ and ‘timepoint’ resulted in only two differentially expressed genes over time and in response to treatment (in males). Neither of, these genes were detected as differentially expressed when the factor ‘timepoint’ was removed from the model, thus our results using only ‘treatment’ as a factor present a robust estimate for the gene expression level response to the low-light treatment, although this comes at the expense of detecting temporal differences that may occur in the response to light treatment. Future experiments using extended time series sampling under different light regimes may provide more insight into temporal changes in response to ALAN.
Of the genes more highly expressed in the low-light condition in males, the majority encoded conserved hypothetical proteins, i.e. have no clearly defined function. Only the one of the two genes with FC > 2 was annotated, namely as 4-coumarate-CoA ligase 1 (Additional file 1: Table S1). To date, the function of this gene is not known in mosquitoes.
Female response to artificial light at night
It was striking that the different light regimes did not induce detectable changes in gene expression in female Cx. pipiens. One biological explanation is that females in both treatments were inseminated and this may have played a role. Male accessory gland secretion is a powerful modulator of female behaviour and activity , which might potentially render females insensitive to light at night. This could have implications for biting propensity, as accessory gland secretion can trigger ovulation and oviposition behaviour. An evaluation of the effect of light at night at different life stages (e.g., virgin, inseminated, or after oviposition) would provide important insights. However, females often mate soon after emergence  and thus it is reasonable to assume that the vast majority of adult females in nature are inseminated at a given point in time.
Half of all genes were differentially expressed in males and females, indicating a strong sex-specific pattern of expression regardless of the light treatment. Sex-biased differences in gene expression are known to occur in a number of species and sexual dimorphism (in morphology, behaviour and physiology) is believed to be a main driver of this . Our findings were similar to those of a recent study of the mosquito An. gambiae that reported 72 % of genes to show sex-biased differential expression in whole bodies . In Drosophila, approximately 50 % of genes are sex-biased . By determining ortholog relationships between the sex-biased genes from our study of Cx. pipiens and those in An. gambiae  we found a larger overlap of sex-biased genes in males compared to females. This pattern could be explained by greater conservation of male-biased gene expression between Anopheles and Culex. Alternatively, this could reflect differences in ecology of females from these two species. An. gambiae is an obligate blood feeder whereas Cx pipiens f. molestus displays facultative autogeny (a blood meal is not essential). Furthermore, the present study sampled individuals reared on a sugar diet whereas An. gambiae data were derived from blood-engorged females and sugar-fed males , potentially contributing to differences in female-biased gene expression.
Among the genes that were differentially expressed in males and females were the circadian clock genes. Although clock-gene expression varies among tissues and our approach was a whole-body sampling, some notable findings suggest areas for more research on the effect of artificial light on clock genes. Male-biased genes related to the clock were generally much more highly expressed compared to females, in which genes were only slightly (although significantly) up-regulated. This suggests that the internal circadian clock system may either be influenced differently in males and females or is inherently different between the sexes, as is the case in Anopheles gambiae . To our knowledge it has not been tested whether this sex-specific circadian rhythm is the case in Culex pipiens. The highly expressed male-biased genes included photolyase. This is a domain of the cryptochrome-1 protein which contains two light-harvesting cofactors and is mainly responsible for DNA repair after UV- and blue-light exposure . timeout/timeless-2 is a paralogue of circadian clock gene timeless and is involved in chromosome stability and light entrainment . disc overgrown protein kinase, in Drosophila also referred to as doubletime. It phosphorylates the period protein and thus contributes to circadian rhythmicity . All of these genes are involved in the perception of light and relate directly to circadian clock function, suggesting a greater potential for the low-light treatment to influence males compared to females, in agreement with our overall findings. Further underlining this potential sex-specific influence is the fact that we found the gene cryptochrome-1 to be more highly expressed as a response to ALAN. The presence of light clearly changed the expression of the gene of paramount importance in synchronisation of the circadian clock to the environment and clock-controlled processes.