From whole bodies to single cells: A guide to transcriptomic approaches for ecology and evolutionary biology.

RNA sequencing (RNAseq) methodology has experienced a burst of technological developments in the last decade, which has opened up opportunities for studying the mechanisms of adaptation to environmental factors at both the organismal and cellular level. Selecting the most suitable experimental approach for specific research questions and model systems can, however, be a challenge and researchers in ecology and evolution are commonly faced with the choice of whether to study gene expression variation in whole bodies, specific tissues, and/or single cells. A wide range of sometimes polarised opinions exists over which approach is best. Here, we highlight the advantages and disadvantages of each of these approaches to provide a guide to help researchers make informed decisions and maximise the power of their study. Using illustrative examples of various ecological and evolutionary research questions, we guide the readers through the different RNAseq approaches and help them identify the most suitable design for their own projects.

Validation of biomarkers for neonicotinoid exposure in Folsomia candida under mutual exposure to diethyl maleate.

Neonicotinoid insecticides are harmful to non-target soil invertebrates, which are crucial for sustainable agriculture. Gene expression biomarkers could provide economic and high-throughput metrics of neonicotinoid exposure and toxicity to non-target invertebrates. Thereby, biomarkers can help guide remediation efforts or policy enforcement. Gene expression of Glutathione S-Transferase 3 (GST3) has previously been proposed as a biomarker for the neonicotinoid imidacloprid in the soil ecotoxicological model species Folsomia candida (Collembola). However, it remains unclear how reliably gene expression of neonicotinoid biomarkers, such as GST3, can indicate the exposure to the broader neonicotinoid family under putative GST enzymatic inhibition. In this work, we exposed springtails to two neonicotinoids, thiacloprid and imidacloprid, alongside diethyl maleate (DEM), a known GST metabolic inhibitor that imposes oxidative stress. First, we determined the influence of DEM on neonicotinoid toxicity to springtail fecundity. Second, we surveyed the gene expression of four biomarkers, including GST3, under mutual exposure to neonicotinoids and DEM. We observed no effect of DEM on springtail fecundity. Moreover, the expression of GST3 was only influenced by DEM under mutual exposure with thiacloprid but not with imidacloprid. The results indicate that GST3 is not a robust indicator of neonicotinoid exposure and that probable GST enzymatic inhibition mediates the toxicity of imidacloprid and thiacloprid differentially. Future research should investigate biomarker reliability under shifting metabolic conditions such as provided by DEM exposure.

A Single Nucleotide Variant in the PPARγ-homolog Eip75B Affects Fecundity in Drosophila.

Single nucleotide polymorphisms are the most common type of genetic variation, but how these variants contribute to the adaptation of complex phenotypes is largely unknown. Experimental evolution and genome-wide association studies have demonstrated that variation in the PPARγ-homolog Eip75B has associated with longevity and life-history differences in the fruit fly Drosophila melanogaster. Using RNAi knockdown, we first demonstrate that reduced expression of Eip75B in adult flies affects lifespan, egg-laying rate, and egg volume. We then tested the effects of a naturally occurring SNP within a cis-regulatory domain of Eip75B by applying two complementary approaches: a Mendelian randomization approach using lines of the Drosophila Genetic Reference Panel, and allelic replacement using precise CRISPR/Cas9-induced genome editing. Our experiments reveal that this natural polymorphism has a significant pleiotropic effect on fecundity and egg-to-adult viability, but not on longevity or other life-history traits. Our results provide a rare functional validation at the nucleotide level and identify a natural allelic variant affecting fitness and life-history adaptation.

Combining time-resolved transcriptomics and proteomics data for Adverse Outcome Pathway refinement in ecotoxicology.

Conventional Environmental Risk Assessment (ERA) of pesticide pollution is based on soil concentrations and apical endpoints, such as the reproduction of test organisms, but has traditionally disregarded information along the organismal response cascade leading to an adverse outcome. The Adverse Outcome Pathway (AOP) framework includes response information at any level of biological organization, providing opportunities to use intermediate responses as a predictive read-out for adverse outcomes instead. Transcriptomic and proteomic data can provide thousands of data points on the response to toxic exposure. Combining multiple omics data types is necessary for a comprehensive overview of the response cascade and, therefore, AOP development. However, it is unclear if transcript and protein responses are synchronized in time or time lagged. To understand if analysis of multi-omics data obtained at the same timepoint reveal one synchronized response cascade, we studied time-resolved shifts in gene transcript and protein abundance in the springtail Folsomia candida, a soil ecotoxicological model, after exposure to the neonicotinoid insecticide imidacloprid. We analyzed transcriptome and proteome data every 12 h up to 72 h after onset of exposure. The most pronounced shift in both transcript and protein abundances was observed after 48 h exposure. Moreover, cross-correlation analyses indicate that most genes displayed the highest correlation between transcript and protein abundances without a time-lag. This demonstrates that a combined analysis of transcriptomic and proteomic data from the same time-point can be used for AOP improvement. This data will promote the development of biomarkers for the presence of neonicotinoid insecticides or chemicals with a similar mechanism of action in soils.

Natural alleles at the Doa locus underpin evolutionary changes in Drosophila lifespan and fecundity.

'Evolve and resequence' (E&R) studies in Drosophila melanogaster have identified many candidate loci underlying the evolution of ageing and life history, but experiments that validate the effects of such candidates remain rare. In a recent E&R study we have identified several alleles of the LAMMER kinase Darkener of apricot (Doa) as candidates for evolutionary changes in lifespan and fecundity. Here, we use two complementary approaches to confirm a functional role of Doa in life-history evolution. First, we used transgenic RNAi to study the effects of Doa at the whole-gene level. Ubiquitous silencing of expression in adult flies reduced both lifespan and fecundity, indicating pleiotropic effects. Second, to characterize segregating variation at Doa, we examined four candidate single nucleotide polymorphisms (SNPs; Doa-1, -2, -3, -4) using a genetic association approach. Three candidate SNPs had effects that were qualitatively consistent with expectations based on our E&R study: Doa-2 pleiotropically affected both lifespan and late-life fecundity; Doa-1 affected lifespan (but not fecundity); and Doa-4 affected late-life fecundity (but not lifespan). Finally, the last candidate allele (Doa-3) also affected lifespan, but in the opposite direction from predicted.

Biomarker development for neonicotinoid exposure in soil under interaction with the synergist piperonyl butoxide in Folsomia candida.

Pesticide toxicity is typically assessed by exposing model organisms to individual compounds and measuring effects on survival and reproduction. These tests are time-consuming, labor-intensive, and do not accurately capture the effect of pesticide mixtures. Moreover, it is unfeasible to screen the nearly infinite combinations of mixtures for synergistic effects on model organisms. Therefore, reliable molecular indicators of pesticide exposure have to be identified, i.e., biomarkers. These biomarkers can form the basis of rapid and economical screening procedures to assess the toxicity of pesticides even under synergistic interaction with other pollutants. In this study, we screened the expression patterns of eight genes for suitability as a biomarker for neonicotinoid exposure in the soil ecotoxicological model Folsomia candida (springtails). Springtails were exposed to the neonicotinoids imidacloprid and thiacloprid either alone or with various levels of piperonyl butoxide (PBO), which inhibits cytochrome P450 enzymes (CYPs): a common point of synergistic interaction between neonicotinoid and other pesticides. First, we confirmed PBO as a potency enhancer for neonicotinoid toxicity to springtail fecundity, and then used it as a tool to confirm biomarker robustness. We identified two genes that are reliably indicative for neonicotinoid exposure even under metabolic inhibition of CYPs by PBO, nicotinic acetylcholine receptor-subunit alpha 1 (nAchR) and sodium-coupled monocarboxylate transporter (SMCT). These results can form the basis for developing high-throughput screening procedures for neonicotinoid exposure in varying mixture compositions.

Distinct genomic signals of lifespan and life history evolution in response to postponed reproduction and larval diet in Drosophila.

Reproduction and diet are two major factors controlling the physiology of aging and life history, but how they interact to affect the evolution of longevity is unknown. Moreover, although studies of large-effect mutants suggest an important role of nutrient sensing pathways in regulating aging, the genetic basis of evolutionary changes in lifespan remains poorly understood. To address these questions, we analyzed the genomes of experimentally evolved Drosophila melanogaster populations subjected to a factorial combination of two selection regimes: reproductive age (early versus postponed), and diet during the larval stage ("low," "control," "high"), resulting in six treatment combinations with four replicate populations each. Selection on reproductive age consistently affected lifespan, with flies from the postponed reproduction regime having evolved a longer lifespan. In contrast, larval diet affected lifespan only in early-reproducing populations: flies adapted to the "low" diet lived longer than those adapted to control diet. Here, we find genomic evidence for strong independent evolutionary responses to either selection regime, as well as loci that diverged in response to both regimes, thus representing genomic interactions between the two. Overall, we find that the genomic basis of longevity is largely independent of dietary adaptation. Differentiated loci were not enriched for "canonical" longevity genes, suggesting that naturally occurring genic targets of selection for longevity differ qualitatively from variants found in mutant screens. Comparing our candidate loci to those from other "evolve and resequence" studies of longevity demonstrated significant overlap among independent experiments. This suggests that the evolution of longevity, despite its presumed complex and polygenic nature, might be to some extent convergent and predictable.

Adaptation to developmental diet influences the response to selection on age at reproduction in the fruit fly.

Experimental evolution (EE) is a powerful tool for addressing how environmental factors influence life-history evolution. While in nature different selection pressures experienced across the lifespan shape life histories, EE studies typically apply selection pressures one at a time. Here, we assess the consequences of adaptation to three different developmental diets in combination with classical selection for early or late reproduction in the fruit fly Drosophila melanogaster. We find that the response to each selection pressure is similar to that observed when they are applied independently, but the overall magnitude of the response depends on the selection regime experienced in the other life stage. For example, adaptation to increased age at reproduction increased lifespan across all diets; however, the extent of the increase was dependent on the dietary selection regime. Similarly, adaptation to a lower calorie developmental diet led to faster development and decreased adult weight, but the magnitude of the response was dependent on the age-at-reproduction selection regime. Given that multiple selection pressures are prevalent in nature, our findings suggest that trade-offs should be considered not only among traits within an organism, but also among adaptive responses to different-sometimes conflicting-selection pressures, including across life stages.

Selection for associative learning of color stimuli reveals correlated evolution of this learning ability across multiple stimuli and rewards.

We are only starting to understand how variation in cognitive ability can result from local adaptations to environmental conditions. A major question in this regard is to what extent selection on cognitive ability in a specific context affects that ability in general through correlated evolution. To address this question, we performed artificial selection on visual associative learning in female Nasonia vitripennis wasps. Using appetitive conditioning in which a visual stimulus was offered in association with a host reward, the ability to learn visual associations was enhanced within 10 generations of selection. To test for correlated evolution affecting this form of learning, the ability to readily form learned associations in females was also tested using an olfactory instead of a visual stimulus in the appetitive conditioning. Additionally, we assessed whether the improved associative learning ability was expressed across sexes by color-conditioning males with a mating reward. Both females and males from the selected lines consistently demonstrated an increased associative learning ability compared to the control lines, independent of learning context or conditioned stimulus. No difference in relative volume of brain neuropils was detected between the selected and control lines.

Amino acid modulation of lifespan and reproduction in Drosophila.

Manipulating amino acid (AA) intake in Drosophila can profoundly affect lifespan and reproduction. Remarkably, AA manipulation can uncouple the commonly observed trade-off between these traits. This finding seems to challenge the idea that this trade-off is due to competitive resource allocation, but here we argue that this view might be too simplistic. We also discuss the mechanisms of the AA response, mediated by the IIS/TOR and GCN2 pathways. Elucidating how these pathways respond to specific AA will likely yield important insights into how AA modulate the reproduction-lifespan relationship. The Drosophila model offers powerful genetic tools, combined with options for precise diet manipulation, to address these fundamental questions.

Differentially expressed genes linked to natural variation in long-term memory formation in Cotesia parasitic wasps.

Even though learning and memory are universal traits in the Animal Kingdom, closely related species reveal substantial variation in learning rate and memory dynamics. To determine the genetic background of this natural variation, we studied two congeneric parasitic wasp species, Cotesia glomerata and C. rubecula, which lay their eggs in caterpillars of the large and small cabbage white butterfly. A successful egg laying event serves as an unconditioned stimulus (US) in a classical conditioning paradigm, where plant odors become associated with the encounter of a suitable host caterpillar. Depending on the host species, the number of conditioning trials and the parasitic wasp species, three different types of transcription-dependent long-term memory (LTM) and one type of transcription-independent, anesthesia-resistant memory (ARM) can be distinguished. To identify transcripts underlying these differences in memory formation, we isolated mRNA from parasitic wasp heads at three different time points between induction and consolidation of each of the four memory types, and for each sample three biological replicates, where after strand-specific paired-end 100 bp deep sequencing. Transcriptomes were assembled de novo and differential expression was determined for each memory type and time point after conditioning, compared to unconditioned wasps. Most differentially expressed (DE) genes and antisense transcripts were only DE in one of the LTM types. Among the DE genes that were DE in two or more LTM types, were many protein kinases and phosphatases, small GTPases, receptors and ion channels. Some genes were DE in opposing directions between any of the LTM memory types and ARM, suggesting that ARM in Cotesia requires the transcription of genes inhibiting LTM or vice versa. We discuss our findings in the context of neuronal functioning, including RNA splicing and transport, epigenetic regulation, neurotransmitter/peptide synthesis and antisense transcription. In conclusion, these brain transcriptomes provide candidate genes that may be involved in the observed natural variation in LTM in closely related Cotesia parasitic wasp species.

Learning-induced gene expression in the heads of two Nasonia species that differ in long-term memory formation.

BACKGROUND: Cellular processes underlying memory formation are evolutionary conserved, but natural variation in memory dynamics between animal species or populations is common. The genetic basis of this fascinating phenomenon is poorly understood. Closely related species of Nasonia parasitic wasps differ in long-term memory (LTM) formation: N. vitripennis will form transcription-dependent LTM after a single conditioning trial, whereas the closely-related species N. giraulti will not. Genes that were differentially expressed (DE) after conditioning in N. vitripennis, but not in N. giraulti, were identified as candidate genes that may regulate LTM formation. RESULTS: RNA was collected from heads of both species before and immediately, 4 or 24 hours after conditioning, with 3 replicates per time point. It was sequenced strand-specifically, which allows distinguishing sense from antisense transcripts and improves the quality of expression analyses. We determined conditioning-induced DE compared to naïve controls for both species. These expression patterns were then analysed with GO enrichment analyses for each species and time point, which demonstrated an enrichment of signalling-related genes immediately after conditioning in N. vitripennis only. Analyses of known LTM genes and genes with an opposing expression pattern between the two species revealed additional candidate genes for the difference in LTM formation. These include genes from various signalling cascades, including several members of the Ras and PI3 kinase signalling pathways, and glutamate receptors. Interestingly, several other known LTM genes were exclusively differentially expressed in N. giraulti, which may indicate an LTM-inhibitory mechanism. Among the DE transcripts were also antisense transcripts. Furthermore, antisense transcripts aligning to a number of known memory genes were detected, which may have a role in regulating these genes. CONCLUSION: This study is the first to describe and compare expression patterns of both protein-coding and antisense transcripts, at different time points after conditioning, of two closely related animal species that differ in LTM formation. Several candidate genes that may regulate differences in LTM have been identified. This transcriptome analysis is a valuable resource for future in-depth studies to elucidate the role of candidate genes and antisense transcription in natural variation in LTM formation.

Natural variation in long-term memory formation among Nasonia parasitic wasp species.

Closely related species of parasitic wasps can differ substantially in memory dynamics. In this study we demonstrate differences in the number of conditioning trials required to form long-term memory between the closely related parasitic wasp species Nasonia vitripennis and Nasonia giraulti (Hymenoptera: Pteromalidae). A single conditioning trial, in which a female wasp associates an odour with the reward of finding a host, results in the formation of transcription-dependent long-term memory in N. vitripennis, whereas N. giraulti requires spaced training to do so. Memory formation does not depend on the type of reward: oviposition, which was hypothesized to be a 'larger' reward results in similar memory retention as host feeding in both Nasonia species. There are several genetic and genomic tools available for Nasonia species to identify genetic mechanisms that underlie the observed variation in the number of trials required to form long-term memory.

Natural variation in learning rate and memory dynamics in parasitoid wasps: opportunities for converging ecology and neuroscience.

Although the neural and genetic pathways underlying learning and memory formation seem strikingly similar among species of distant animal phyla, several more subtle inter- and intraspecific differences become evident from studies on model organisms. The true significance of such variation can only be understood when integrating this with information on the ecological relevance. Here, we argue that parasitoid wasps provide an excellent opportunity for multi-disciplinary studies that integrate ultimate and proximate approaches. These insects display interspecific variation in learning rate and memory dynamics that reflects natural variation in a daunting foraging task that largely determines their fitness: finding the inconspicuous hosts to which they will assign their offspring to develop. We review bioassays used for oviposition learning, the ecological factors that are considered to underlie the observed differences in learning rate and memory dynamics, and the opportunities for convergence of ecology and neuroscience that are offered by using parasitoid wasps as model species. We advocate that variation in learning and memory traits has evolved to suit an insect's lifestyle within its ecological niche.