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Seminal fluid proteins (SFPs) play vital roles for optimizing reproductive success in diverse animals. Underlining their significance, SFP production and transfer are highly plastic, e.g., depending on the presence of rivals or mating status of partners. However, surprisingly little is known about replenishing SFPs after mating. This is especially relevant in species that mate multiple times, as they continuously produce and use SFPs throughout their reproductive life. Here we examined the expression pattern of SFP genes after mating in the great pond snail, Lymnaea stagnalis. Our results show that two out of the six SFP genes investigated here were upregulated 1 week after mating. Surprisingly, most SFP genes did not change their expression immediately after mating. Even after 1 week, when supposedly seminal fluid is fully replenished, the expression of SFP genes is rather high. In addition, the difference with previous studies hints at the possibility that SFP production after mating is plastic and depends on the mating history of female-acting snails. Our results shed light on unexplored aspects of SFP production, thereby expanding the understanding of reproductive strategies in animals.

Urbanisation is rapidly altering ecosystems, leading to profound biodiversity loss. To mitigate these effects, we need a better understanding of how urbanisation impacts dispersal and reproduction. Two contrasting population demographic models have been proposed that predict that urbanisation either promotes (facilitation model) or constrains (fragmentation model) gene flow and genetic diversity. Which of these models prevails likely depends on the strength of selection on specific phenotypic traits that influence dispersal, survival, or reproduction. Here, we a priori examined the genomic impact of urbanisation on the Neotropical túngara frog (Engystomops pustulosus), a species known to adapt its reproductive traits to urban selective pressures. Using whole-genome resequencing for multiple urban and forest populations we examined genomic diversity, population connectivity and demographic history. Contrary to both the fragmentation and facilitation models, urban populations did not exhibit substantial changes in genomic diversity or differentiation compared with forest populations, and genomic variation was best explained by geographic distance rather than environmental factors. Adopting an a posteriori approach, we additionally found both urban and forest populations to have undergone population declines. The timing of these declines appears to coincide with extensive human activity around the Panama Canal during the last few centuries rather than recent urbanisation. Our study highlights the long-lasting legacy of past anthropogenic disturbances in the genome and the importance of considering the historical context in urban evolution studies as anthropogenic effects may be extensive and impact nonurban areas on both recent and older timescales.

Common-garden experiments with populations sampled along natural thermal gradients help to reveal local adaptation, disentangle environmental and genetic effects, and ultimately predict, by analogy, future biotic responses to climate change. In this regard, geothermal habitats are useful model systems as they exhibit dramatic changes in soil temperature. The springtail Protaphorura pseudovanderdrifti has apparently coped with such local geothermal warming in Iceland, as this species occurs along a more than half-century-old geothermal gradient in a grassland and persists along a newly emerged temperature gradient in a previously non-geothermal planted spruce forest. We measured thermal reaction norms for development and walking speed and acute cold shock tolerance of P. pseudovanderdrifti originating from the grassland and forest geothermal gradients. Temperature-dependent juvenile development showed little variation among subpopulations from the recently warmed forest, probably due to insufficient evolutionary time, but springtails from the warmed grassland plots had significantly steeper reaction norms than their counterparts from the corresponding unwarmed plot. In contrast, cold tolerance and locomotory activity showed no conclusive clinal pattern despite significant within-habitat variation. There appeared to be significant differences between habitats, as springtails from the forest had more temperature-sensitive developmental rate and locomotory activity, walked faster, and exhibited more variable cold tolerance than grassland springtails did. The planting of a forest, therefore, seems to have exerted a stronger effect on the thermal phenotype of P. pseudovanderdrifti than the emergence of a geothermal gradient. Thus, habitat properties may be no less important in shaping thermal reaction norms than the mean temperature. These local-scale findings suggest that, in addition to warming per se, global transformation of communities may drive the evolution of t

The use of DNA demethylating agents has been popular in epigenetic studies. Recently, Cook and colleagues, in a 2015 American Naturalist article, claimed an effect of 5-aza-2'-deoxycytidine (5-aza-dC) on the sex ratio of a parasitoid wasp without verifying its effect on DNA methylation. We repeated the 5-aza-dC feeding treatment to test its effectiveness. We used bisulfite amplicon sequencing of 10 genes that either were heavily methylated, previously showed a response to 5-aza-dC, or were suggested to regulate fatty acid synthesis epigenetically, and we demonstrate that wasps fed 5-aza-dC did not show reduced DNA methylation at these loci. Therefore, the conclusion that demethylation shifts sex ratios upward needs reconsideration.

Parasitoid insects are important model systems for a multitude of biological research topics and widely used as biological control agents against insect pests. While the parasitoid lifestyle has evolved numerous times in different insect groups, research has focused almost exclusively on Hymenoptera from the Parasitica clade. The genomes of several members of this group have been sequenced, but no genomic resources are available from any of the other, independent evolutionary origins of the parasitoid lifestyle. Our aim here was to develop genomic resources for three parasitoid insects outside the Parasitica. We present draft genome assemblies for Goniozus legneri, a parasitoid Hymenopteran more closely related to the non-parasitoid wasps and bees than to the Parasitica wasps, the Coleopteran parasitoid Aleochara bilineata and the Dipteran parasitoid Paykullia maculata. The genome assemblies are fragmented, but complete in terms of gene content. We also provide preliminary structural annotations. We anticipate that these genomic resources will be valuable for testing the generality of findings obtained from Parasitica wasps in future comparative studies.

Background: Folsomia candida is a model in soil biology, belonging to the family of Isotomidae, subclass Collembola. It reproduces parthenogenetically in the presence of Wolbachia, and exhibits remarkable physiological adaptations to stress. To better understand these features and adaptations to life in the soil, we studied its genome in the context of its parthenogenetic lifestyle. Results: We applied Pacific Bioscience sequencing and assembly to generate a reference genome for F. candida of 221.7 Mbp, comprising only 162 scaffolds. The complete genome of its endosymbiont Wolbachia, was also assembled and turned out to be the largest strain identified so far. Substantial gene family expansions and lineage-specific gene clusters were linked to stress response. A large number of genes (809) were acquired by horizontal gene transfer. A substantial fraction of these genes are involved in lignocellulose degradation. Also, the presence of genes involved in antibiotic biosynthesis was confirmed. Intra-genomic rearrangements of collinear gene clusters were observed, of which 11 were organized as palindromes. The Hox gene cluster of F. candida showed major rearrangements compared to arthropod consensus cluster, resulting in a disorganized cluster. Conclusions: The expansion of stress response gene families suggests that stress defense was important to facilitate colonization of soils. The large number of HGT genes related to lignocellulose degradation could be beneficial to unlock carbohydrate sources in soil, especially those contained in decaying plant and fungal organic matter. Intra- as well as inter-scaffold duplications of gene clusters may be a consequence of its parthenogenetic lifestyle. This high quality genome will be instrumental for evolutionary biologists investigating deep phylogenetic lineages among arthropods and will provide the basis for a more mechanistic understanding in soil ecology and ecotoxicology.

Phenotypic regression of morphological, behavioral, or physiological traits can evolve when reduced trait expression has neutral or beneficial effects on overall performance. Studies on the evolution of phenotypic degradation in animals have concentrated mostly on the evaluation of resulting phenotypes, whereas much less research has been dedicated to uncovering the molecular mechanisms that underlie phenotypic regression. The majority of parasitoids (i.e., insects that develop on or inside other arthropods), do not accumulate lipid reserves during their free-living adult life-stage and represent an excellent system to study phenotypic regression in animals. Here, we study transcriptional patterns associated with lack of lipogenesis in the parasitic wasp Nasonia vitripennis. We first confirmed that N. vitripennis does not synthesize lipids by showing a reduction in lipid reserves despite ingestion of dietary sugar, and a lack of incorporation of isotopic labels into lipid reserves when fed deuterated sugar solution. Second, we investigated transcriptional responses of 28 genes involved in lipid and sugar metabolism in short- and long-term sugar-fed females relative to starved females of N. vitripennis. Sugar feeding did not induce transcription of fatty acid synthase (fas) or other key genes involved in the lipid biosynthesis pathway. Furthermore, several genes involved in carbohydrate metabolism had a lower transcription in fed than in starved females. Our results reveal that N. vitripennis gene transcription in response to dietary sugar deviates markedly from patterns typically observed in other organisms. This study is the first to identify differential gene transcription associated with lack of lipogenesis in parasitoids and provides new insights into the molecular mechanism that underlies phenotypic regression of this trait.

Phenotypic regression of morphological, behavioral, or physiological traits can evolve when reduced trait expression has neutral or beneficial effects on overall performance. Studies on the evolution of phenotypic degradation in animals have concentrated mostly on the evaluation of resulting phenotypes, whereas much less research has been dedicated to uncovering the molecular mechanisms that underlie phenotypic regression. The majority of parasitoids (i.e., insects that develop on or inside other arthropods), do not accumulate lipid reserves during their free-living adult life-stage and represent an excellent system to study phenotypic regression in animals. Here, we study transcriptional patterns associated with lack of lipogenesis in the parasitic wasp Nasonia vitripennis.We first confirmed that N. vitripennis does not synthesize lipids by showing a reduction in lipid reserves despite ingestionofdietary sugar,anda lack of incorporationof isotopic labels into lipid reserveswhenfeddeuteratedsugar solution. Second, we investigated transcriptional responses of 28 genes involved in lipid and sugar metabolism in short- and long-term sugar-fed females relative to starved females of N. vitripennis. Sugar feeding did not induce transcription of fatty acid synthase (fas) or other key genes involved in the lipid biosynthesis pathway. Furthermore, several genes involved in carbohydrate metabolism had a lower transcription in fed than in starved females. Our results reveal that N. vitripennis gene transcription in response to dietary sugar deviates markedly from patterns typically observed in other organisms. This study is the first to identify differential gene transcription associated with lack of lipogenesis in parasitoids and provides newinsights into themolecularmechanism that underlies phenotypic regression of this trait. © 2012 The Author(s).

BACKGROUND: Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation.RESULTS: Eight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable.CONCLUSION: We present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements.

BACKGROUND: Metallothionein (mt) transcription is elevated in heavy metal tolerant field populations of Orchesella cincta (Collembola). This suggests that natural selection acts on transcriptional regulation of mt in springtails at sites where cadmium (Cd) levels in soil reach toxic values This study investigates the nature and the evolutionary origin of polymorphisms in the metallothionein promoter (pmt) and their functional significance for mt expression.RESULTS: We sequenced approximately 1600 bp upstream the mt coding region by genome walking. Nine pmt alleles were discovered in NW-European populations. They differ in the number of some indels, consensus transcription factor binding sites and core promoter elements. Extensive recombination events between some of the alleles can be inferred from the alignment. A deviation from neutral expectations was detected in a cadmium tolerant population, pointing towards balancing selection on some promoter stretches. Luciferase constructs were made from the most abundant alleles, and responses to Cd, paraquat (oxidative stress inducer) and moulting hormone were studied in cell lines. By using paraquat we were able to dissect the effect of oxidative stress from the Cd specific effect, and extensive differences in mt induction levels between these two stressors were observed.CONCLUSION: The pmt alleles evolved by a number of recombination events, and exhibited differential inducibilities by Cd, paraquat and molting hormone. In a tolerant population from a metal contaminated site, promoter allele frequencies differed significantly from a reference site and nucleotide polymorphisms in some promoter stretches deviated from neutral expectations, revealing a signature of balancing selection. Our results suggest that the structural differences in the Orchesella cincta metallothionein promoter alleles contribute to the metallothionein -over-expresser phenotype in cadmium tolerant populations.