Interconnected Codons: Unravelling the Epigenetic Significance of Flanking Sequences in CpG Dyads.

Hypothesizing that CpG codon dyads, formed by consecutive codons containing a cytosine-guanine pair (NNC-GNN), may play a crucial role in gene function, we conducted an extensive analysis to investigate their distribution and conservation within mammalian genes. Our findings reveal that genes characterized by a high density of CpG codon dyads are notably associated with homeobox domains and RNA polymerase II transcription factors. Conversely, genes exhibiting low CpG codon dyad density have links to DNA damage repair and mitosis. Importantly, our study identifies a re-markable increase in expressed genes that harbor CpG during embryonic development, suggesting their potential involvement in gene regulation at these developmental stages. These results under-score the functional significance of CpG codon dyads in DNA methylation and gene expression, fur-ther demonstrating the coevolution of consecutive codons and their contribution to codon usage bias.

Crickets in the spotlight: exploring the impact of light on circadian behavior.

Crickets serve as a well-established model organism in biological research spanning various fields, such as behavior, physiology, neurobiology, and ecology. Cricket circadian behavior was first reported over a century ago and prompted a wealth of studies delving into their chronobiology. Circadian rhythms have been described in relation to fundamental cricket behaviors, encompassing stridulation and locomotion, but also in hormonal secretion and gene expression. Here we review how changes in illumination patterns and light intensity differentially impact the different cricket behaviors as well as circadian gene expression. We further describe the cricket's circadian pacemaker. Ample anatomical manipulations support the location of a major circadian pacemaker in the cricket optic lobes and another in the central brain, possibly interconnected via signaling of the neuropeptide PDF. The cricket circadian machinery comprises a molecular cascade based on two major transcriptional/translational negative feedback loops, deviating somewhat from the canonical model of Drosophila and emphasizing the significance of exploring alternative models. Finally, the nocturnal nature of crickets has provided a unique avenue for investigating the repercussions of artificial light at night on cricket behavior and ecology, underscoring the critical role played by natural light cycles in synchronizing cricket behaviors and populations, further supporting the use of the cricket model in the study of the effects of light on insects. Some gaps in our knowledge and challenges for future studies are discussed.

Epigenetics and seasonal timing in animals: a concise review.

Seasonal adaptation in animals is a complex process that involves genetic, epigenetic, and environmental factors. The present review explores recent studies on epigenetic mechanisms implicated in seasonal adaptation in animals. The review is divided into three main sections, each focusing on a different epigenetic mechanism: DNA methylation, histone modifications, and non-coding RNA. Additionally, the review delves into the current understanding of how these epigenetic factors contribute to the regulation of circadian and seasonal cycles. Understanding these molecular mechanisms provides the first step in deciphering the complex interplay between genetics, epigenetics, and the environment in driving seasonal adaptation in animals. By exploring these mechanisms, a better understanding of how animals adapt to changing environmental conditions can be achieved.

Unveiling Subtle Geographical Clines: Phenotypic Effects and Dynamics of Circadian Clock Gene Polymorphisms.

Our understanding of the gene regulatory network that constitutes the circadian clock has greatly increased in recent decades, notably due to the use of Drosophila as a model system. In contrast, the analysis of natural genetic variation that enables the robust function of the clock under a broad range of environments has developed more slowly. In the current study, we analyzed comprehensive genome sequencing data from wild European populations of Drosophila, which were densely sampled through time and space. We identified hundreds of single nucleotide polymorphisms (SNPs) in nine genes associated with the clock, 276 of which exhibited a latitudinal cline in their allele frequencies. While the effect sizes of these clinal patterns were small, indicating subtle adaptations driven by natural selection, they provided important insights into the genetic dynamics of circadian rhythms in natural populations. We selected nine SNPs in different genes and assessed their impact on circadian and seasonal phenotypes by reconstructing outbred populations fixed for either of the SNP alleles, from inbred DGRP strains. The circadian free-running period of the locomotor activity rhythm was affected by an SNP in doubletime (dbt) and eyes absent (Eya). The SNPs in Clock (Clk), Shaggy (Sgg), period (per), and timeless (tim) affected the acrophase. The alleles of the SNP in Eya conferred different levels of diapause and the chill coma recovery response.

Transcriptional Response of Circadian Clock Genes to an 'Artificial Light at Night' Pulse in the Cricket Gryllus bimaculatus.

Light is the major signal entraining the circadian clock that regulates physiological and behavioral rhythms in most organisms, including insects. Artificial light at night (ALAN) disrupts the natural light-dark cycle and negatively impacts animals at various levels. We simulated ALAN using dim light stimuli and tested their impact on gene expression in the cricket Gryllus bimaculatus, a model of insect physiology and chronobiology. At night, adult light-dark-regime-raised crickets were exposed for 30 min to a light pulse of 2-40 lx. The relative expression of five circadian-clock-associated genes was compared using qPCR. A dim ALAN pulse elicited tissue-dependent differential expression in some of these genes. The strongest effect was observed in the brain and in the optic lobe, the cricket's circadian pacemaker. The expression of opsin-Long Wave (opLW) was upregulated, as well as cryptochrome1-2 (cry) and period (per). Our findings demonstrate that even a dim ALAN exposure may affect insects at the molecular level, underscoring the impact of ALAN on the circadian clock system.

Photoperiod-Dependent Expression of MicroRNA in Drosophila.

Like many other insects in temperate regions, Drosophila melanogaster exploits the photoperiod shortening that occurs during the autumn as an important cue to trigger a seasonal response. Flies survive the winter by entering a state of reproductive arrest (diapause), which drives the relocation of resources from reproduction to survival. Here, we profiled the expression of microRNA (miRNA) in long and short photoperiods and identified seven differentially expressed miRNAs (dme-mir-2b, dme-mir-11, dme-mir-34, dme-mir-274, dme-mir-184, dme-mir-184*, and dme-mir-285). Misexpression of dme-mir-2b, dme-mir-184, and dme-mir-274 in pigment-dispersing, factor-expressing neurons largely disrupted the normal photoperiodic response, suggesting that these miRNAs play functional roles in photoperiodic timing. We also analyzed the targets of photoperiodic miRNA by both computational predication and by Argonaute-1-mediated immunoprecipitation of long- and short-day RNA samples. Together with global transcriptome profiling, our results expand existing data on other Drosophila species, identifying genes and pathways that are differentially regulated in different photoperiods and reproductive status. Our data suggest that post-transcriptional regulation by miRNA is an important facet of photoperiodic timing.

Nucleotide Variation in Drosophila cryptochrome Is Linked to Circadian Clock Function: An Association Analysis.

Cryptochrome (CRY) is a conserved protein associated with the circadian clock in a broad range of organisms, including plants, insects, and mammals. In Drosophila, cry is a pleiotropic gene that encodes a blue light-dedicated circadian photoreceptor, as well as an electromagnetic field sensor and a geotaxis behavior regulator. We have generated a panel of nearly-isogenic strains that originated from various wild populations and which carry different natural alleles of cry. Sequencing of these alleles revealed substantial polymorphism, the functional role of which was elusive. To link this natural molecular diversity to gene function, we relied on association mapping. Such analysis revealed two major haplogroups consisting of six linked nucleotides associated with circadian phase (haplotypes All1/All2). We also generated a maximum-likelihood gene-tree that uncovered an additional pair of haplogroups (B1/B2). Behavioral analysis of the different haplotypes indicated significant effect on circadian phase and period, as well on the amount of activity and sleep. The data also suggested substantial epistasis between the All and B haplogroups. Intriguingly, circadian photosensitivity, assessed by light-pulse experiments, did not differ between the genotypes. Using CRISPR-mediated transgenic flies, we verified the effect of B1/B2 polymorphism on circadian phase. The transgenic flies also exhibited substantially different levels of cry transcription. We, moreover, analyzed the geographical distribution of the B1/B2 haplotypes, focusing on a 12 bp insertion/deletion polymorphism that differentiates the two haplotypes. Analysis of cry sequences in wild populations across Europe revealed a geographical cline of B1/B2 indel frequency, which correlated with seasonal bioclimatic variables. This spatial distribution of cry polymorphism reinforces the functional importance of these haplotypes in the circadian system and local adaptation.

Adaptation of Drosophila melanogaster to Long Photoperiods of High-Latitude Summers Is Facilitated by the ls-Timeless Allele.

Circadian clocks help animals to be active at the optimal time of the day whereby for most species the daily light-dark cycle is the most important zeitgeber for their circadian clock. In this respect, long arctic summer days are particularly challenging as light is present almost 24 h per day, and continuous light makes the circadian clocks of many animals arrhythmic. This is especially true for the fruit fly, Drosophila melanogaster, which possesses a very light-sensitive clock. The blue-light photoreceptor Cryptochrome (CRY) and the clock protein Timeless (TIM) are the light-sensitive components of the circadian clock and are responsible for constant light-induced arrhythmicity even at very low light intensities. Nevertheless, D. melanogaster was able to spread from its tropical origin and invade northern latitudes. Here, we tested whether a natural polymorphism at the timeless (tim) locus, s-tim and ls-tim, helped adaptation to very long photoperiods. The recently evolved natural allele, ls-tim, encodes a longer, less light sensitive form of TIM (L-TIM) in addition to the shorter (S-TIM) form, the only form encoded by the ancient s-tim allele. ls-tim has evolved in southeastern Italy and slowly spreads to higher latitudes. L-TIM is known to interact less efficiently with CRY as compared with S-TIM. Here, we studied the locomotor activity patterns of ~40 wild s-tim and ls-tim isofemale lines caught at different latitudes under simulated high-latitude summer light conditions (continuous light or long photoperiods with 20-h daily light). We found that the ls-tim lines were significantly more rhythmic under continuous light than the s-tim lines. Importantly, the ls-tim lines can delay their evening activity under long photoperiods, a behavioral adaptation that appears to be optimal under high-latitude conditions. Our observations suggest that the functional gain associated with ls-tim may drive the northern spread of this allele by directional selection.

Metagenomic analysis reveals the signature of gut microbiota associated with human chronotypes.

Patterns of diurnal activity differ substantially between individuals, with early risers and late sleepers being examples of opposite chronotypes. Growing evidence suggests that the late chronotype significantly impacts the risk of developing mood disorders, obesity, diabetes, and other chronic diseases. Despite the vast potential of utilizing chronotype information for precision medicine, those factors that shape chronotypes remain poorly understood. Here, we assessed whether the various chronotypes are associated with different gut microbiome compositions. Using metagenomic sequencing analysis, we established a distinct signature associated with chronotype based on two bacterial genera, Alistipes (elevated in "larks") and Lachnospira (elevated in "owls"). We identified three metabolic pathways associated with the early chronotype, and linked distinct dietary patterns with different chronotypes. Our work demonstrates an association between the gut microbiome and chronotype and may represent the first step towards developing dietary interventions aimed at ameliorating the deleterious health correlates of the late chronotype.

Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

The genetic basis of diurnal preference in Drosophila melanogaster.

BACKGROUND: Most animals restrict their activity to a specific part of the day, being diurnal, nocturnal or crepuscular. The genetic basis underlying diurnal preference is largely unknown. Under laboratory conditions, Drosophila melanogaster is crepuscular, showing a bi-modal activity profile. However, a survey of strains derived from wild populations indicated that high variability among individuals exists, including flies that are nocturnal. RESULTS: Using a highly diverse population, we performed an artificial selection experiment, selecting flies with extreme diurnal or nocturnal preference. After 10 generations, we obtained highly diurnal and nocturnal strains. We used whole-genome expression analysis to identify differentially expressed genes in diurnal, nocturnal and crepuscular (control) flies. Other than one circadian clock gene (pdp1), most differentially expressed genes were associated with either clock output (pdf, to) or input (Rh3, Rh2, msn). This finding was congruent with behavioural experiments indicating that both light masking and the circadian pacemaker are involved in driving nocturnality. CONCLUSIONS: Our study demonstrates that genetic variation segregating in wild populations contributes to substantial variation in diurnal preference. We identified candidate genes associated with diurnality/nocturnality, while data emerging from our expression analysis and behavioural experiments suggest that both clock and clock-independent pathways are involved in shaping diurnal preference. The diurnal and nocturnal selection strains provide us with a unique opportunity to understand the genetic architecture of diurnal preference.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Validating the Demethylating Effects of 5-aza-2'-deoxycytidine in Insects Requires a Whole-Genome Approach.

We previously demonstrated that treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) alters the offspring sex ratios produced by females of the parasitoid wasp Nasonia vitripennis. Females allocate offspring sex ratio in line with local mate competition theory, producing more or less female-biased sex ratios as the number of other females laying eggs on a patch varies, thereby reducing competition among their sons for mates. Interestingly, treatment with 5-aza-dC did not ablate the facultative sex allocation response. Instead, sex ratios became less female biased, a shift in the direction of the optimum sex ratio for paternally inherited alleles according to genomic conflict theory. This was the first (albeit indirect) experimental evidence for genomic conflict over sex allocation. In their comment, Ellers and colleagues assayed the effects of 5-aza-dC on DNA methylation in 10 Nasonia genes, finding no evidence of demethylation in these 10 genes, from which they conclude that 5-aza-dC has no demethylating capability in N. vitripennis. Quantifying the efficacy of 5-aza-dC in terms of demethylation is indeed crucial to in-depth interpretation of studies using 5-aza-dC to link phenotypes to epigenetic regulation. Here we outline the mode of action of 5-aza-dC and demonstrate that determining the efficacy of 5-aza-dC in insect systems requires a whole-genome approach.

Differential gene expression is not required for facultative sex allocation: a transcriptome analysis of brain tissue in the parasitoid wasp Nasonia vitripennis.

Whole-transcriptome technologies have been widely used in behavioural genetics to identify genes associated with the performance of a behaviour and provide clues to its mechanistic basis. Here, we consider the genetic basis of sex allocation behaviour in the parasitoid wasp Nasonia vitripennis. Female Nasonia facultatively vary their offspring sex ratio in line with Hamilton's theory of local mate competition (LMC). A single female or 'foundress' laying eggs on a patch will lay just enough sons to fertilize her daughters. As the number of 'foundresses' laying eggs on a patch increases (and LMC declines), females produce increasingly male-biased sex ratios. Phenotypic studies have revealed the cues females use to estimate the level of LMC their sons will experience, but our understanding of the genetics underlying sex allocation is limited. Here, we exposed females to three foundress number conditions, i.e. three LMC conditions, and allowed them to oviposit. mRNA was extracted from only the heads of these females to target the brain tissue. The subsequent RNA-seq experiment confirmed that differential gene expression is not associated with the response to sex allocation cues and that we must instead turn to the underlying neuroscience to reveal the underpinnings of this impressive behavioural plasticity.

Interspecific studies of circadian genes period and timeless in Drosophila.

The level of rescue of clock function in genetically arrhythmic Drosophila melanogaster hosts using interspecific clock gene transformation was used to study the putative intermolecular coevolution between interacting clock proteins. Among them PER and TIM are the two important negative regulators of the circadian clock feedback loop. We transformed either the D. pseudoobscura per or tim transgenes into the corresponding arrhythmic D. melanogaster mutant (per01 or tim01) and observed >50% rhythmicity but the period of activity rhythm was either longer (D. pseudoobscura-per) or shorter than 24 h (D. pseudoobscura-tim) compared to controls. By introducing both transgenes simultaneously into double mutants, we observed that the period of the activity rhythm was rescued by the pair of hemizygous transgenes (~24 h). These flies also showed a more optimal level of temperature compensation for the period. Under LD 12:12 these flies have a D. pseudoobscura like activity profile with the absence of morning anticipation as well as a very prominent earlier evening peak of activity rhythm. These observation are consistent with the view that TIM and PER form a heterospecific coevolved module at least for the circadian period of activity rhythms. However the strength of rhythmicity was reduced by having both transgenes present, so while evidence for a coevolution between PER and TIM is observed for some characters it is not for others.

Inverse European Latitudinal Cline at the timeless Locus of Drosophila melanogaster Reveals Selection on a Clock Gene: Population Genetics of ls-tim.

The spread of adaptive genetic variants in populations is a cornerstone of evolutionary theory but with relatively few biologically well-understood examples. Previous work on the ls-tim variant of timeless, which encodes the light-sensitive circadian regulator in Drosophila melanogaster, suggests that it may have originated in southeastern Italy. Flies characterized by the new allele show photoperiod-related phenotypes likely to be adaptive in seasonal environments. ls-tim may be spreading from its point of origin in Italy by directional selection, but there are alternative explanations for its observed clinal geographical distribution, including balancing selection and demography. From population analyses of ls-tim frequencies collected on the eastern side of the Iberian Peninsula, we show that ls-tim frequencies are inverted compared with those in Italy. This pattern is consistent with a scenario of directional selection rather than latitude-associated balancing selection. Neutrality tests further reveal the signature of directional selection at the ls-tim site, which is reduced a few kb pairs either side of ls-tim. A reanalysis of allele frequencies from a large number of microsatellite loci do not demonstrate any frequent ls-tim-like spatial patterns, so a general demographic effect or population expansion from southeastern Italy cannot readily explain current ls-tim frequencies. Finally, a revised estimate of the age of ls-tim allele using linkage disequilibrium and coalescent-based approaches reveals that it may be only 300 to 3000 years old, perhaps explaining why it has not yet gone to fixation. ls-tim thus provides a rare temporal snapshot of a new allele that has come under selection before it reaches equilibrium.

Mapping Quantitative Trait Loci Underlying Circadian Light Sensitivity in Drosophila.

Despite the significant advance in our understanding of the molecular basis of light entrainment of the circadian clock in Drosophila, the underlying genetic architecture is still largely unknown. The aim of this study was to identify loci associated with variation in circadian photosensitivity, which are important for the evolution of this trait. We have used complementary approaches that combined quantitative trait loci (QTL) mapping, complementation testing, and transcriptome profiling to dissect this variation. We identified a major QTL on chromosome 2, which was subsequently fine mapped using deficiency complementation mapping into 2 smaller regions spanning 139 genes, some of which are known to be involved in functions that have been previously implicated in light entrainment. Two genes implicated with the clock and located within that interval, timeless and cycle, failed to complement the QTL, indicating that alleles of these genes contribute to the variation in light response. Specifically, we find that the timeless s/ ls polymorphism that has been previously shown to constitute a latitudinal cline in Europe is also segregating in our recombinant inbred lines and is contributing to the phenotypic variation in light sensitivity. We also profiled gene expression in 2 recombinant inbred strains that differ significantly in their photosensitivity and identified a total of 368 transcripts that showed differential expression (false discovery rate < 0.1). Of 131 transcripts that showed a significant recombinant inbred line by treatment interaction (i.e., putative expression QTL), 4 are located within QTL2.

Is diapause an ancient adaptation in Drosophila?

D. melanogaster enters a state of reproductive arrest when exposed to low temperatures (12°C) and shorter photoperiods. A number of studies have suggested that diapause has recently evolved in European D. melanogaster populations, that it is not present in the sibling species D. simulans, that it is non-photoperiodic in American D. melanogaster populations, and that it spontaneously terminates after 6-8weeks. We have studied the overwintering phenotype under different conditions and observe that American, European and, surprisingly, African D. melanogaster populations can show photoperiodic diapause, as can European, but not African D. simulans. Surprisingly other Drosophila species from pan-tropical regions can also show significant levels of photoperiodic diapause. We observe that spontaneous termination of diapause after a few weeks can be largely avoided with a more realistic winter simulation for D. melanogaster, but not D. simulans. Examining metabolite accumulation during diapause reveals that the shallow diapause of D. melanogaster has similar features to that of other more robustly-diapausing species. Our results suggest that diapause may be an ancient character that emerged in the tropics to resist unfavourable seasonal conditions and which has been enhanced during D. melanogaster's colonisation of temperate regions. Our results also highlight how different methodologies to quantify diapause can lead to apparently conflicting results that we believe can now largely be resolved.

Geographical analysis of diapause inducibility in European Drosophila melanogaster populations.

Seasonal overwintering in insects represents an adaptation to stressful environments and in European Drosophila melanogaster females, low temperatures and short photoperiods can induce an ovarian diapause. Diapause may represent a recent (<15Ky) adaptation to the colonisation of temperate Europe by D. melanogaster from tropical sub-Saharan Africa, because African D. melanogaster and the sibling species D. simulans, have been reported to fail to undergo diapause. Over the past few centuries, D. melanogaster have also invaded North America and Australia, and eastern populations on both continents show a predictable latitudinal cline in diapause induction. In Europe however, a new diapause-enhancing timeless allele, ls-tim, is observed at high levels in southern Italy (∼80%), where it appears to have arisen and has spread throughout the continent with a frequency of ∼20% in Scandinavia. Given the phenotype of ls-tim and its geographical distribution, we might predict that it would work against any latitudinal cline in diapause induction within Europe. Indeed we reveal that any latitudinal cline for diapause in Europe is very weak, as predicted by ls-tim frequencies. In contrast, we determine ls-tim frequencies in North America and observe that they would be expected to strengthen the latitudinal pattern of diapause. Our results reveal how a newly arisen mutation, can, via the stochastic nature of where it initially arose, blur an otherwise adaptive geographical pattern.

Neural and non-neural contributions to sexual dimorphism of mid-day sleep in Drosophila melanogaster: a pilot study.

Many of the characteristics associated with mammalian sleep are also observed in Drosophila melanogaster Meigen, making the fruit fly a powerful model organism for studying the genetics of this important process. Included among the similarities is the presence of sexual dimorphic sleep patterns, which, in flies, are manifested as increased mid-day sleep ('siesta') in males compared with females. In the present study, targeted mis-expression of the genes transformer (tra) and tra2 is used to either feminize or masculinize specific neural and non-neural tissues in the fly. Feminization of male D. melanogaster using three different GAL4 drivers that are expressed in the mushroom bodies induces a female-like reduced siesta, whereas the masculinization of females using these drivers triggers the male-like increased siesta. A similar reversal of sex-specific sleep is also observed by mis-expressing tra in the fat body, which is a key tissue in energy metabolism and hormone secretion. In addition, the daily expression levels of takeout, an important circadian clock output gene, are sexually dimorphic. Taken together, these experiments suggest that sleep sexual dimorphism in D. melanogaster is driven by multiple neural and non-neural circuits, within and outside the brain.