Janine Mariën

Genetic structure in Orchesella cincta (Collembola): strong subdivision of European populations inferred from mtDNA and AFLP markers.

Population genetic structure is determined both by current processes and historical events. Current processes include gene flow, which is largely influenced by the migration capacity of a species. Historical events are, for example, glaciation periods, which have had a major impact on the distribution of many species. Species with a low capacity or tendency to move about or disperse often exhibit clear spatial genetic structures, whereas mobile species mostly show less spatial genetic differentiation. In this paper we report on the genetic structure of a small, wingless arthropod species (Orchesella cincta: Collembola) in Europe. For this purpose we used mtDNA COII sequences and AFLP markers. We show that large genetic differences exist between populations of O. cincta, as expected from O. cinctas winglessness and sedentary lifestyle. Despite the fact that most variability was observed within populations (59%), a highly significant amount of AFLP variation (25%) was observed between populations from northwestern Europe, central Europe and Italy. This suggests that gene flow among regions is extremely low, which is additionally supported by the lack of shared mtDNA alleles between regions. Based on the genetic variation and sequence differences observed we conclude that the subdivision occurred long before the last glaciation periods. Although the populations still interbreed in the lab, we assume that in the long term the genetic isolation of these regions may lead to speciation processes.

Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)

BackgroundGenomic 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.ResultsEight 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.ConclusionWe 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.

Adaptive differences in gene expression associated with heavy metal tolerance in the soil arthropod Orchesella cincta

Field‐selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola) populations occurring at metal‐contaminated mining sites. This tolerance correlated with heritable increase in metal excretion efficiency, less pronounced cadmium (Cd)‐induced growth reduction and overexpression of the metallothionein gene. We applied transcriptomics to determine differential gene expression caused by this abiotic stress in reference and Cd‐tolerant populations. Many cDNAs responded to Cd exposure in the reference population. Significantly fewer clones were Cd responsive in tolerant animals. Analysis of variance revealed transcripts that interact between Cd exposure and population. Hierarchical cluster analysis of these clones identified two major groups. The first one contained cDNAs that were up‐regulated by Cd in the reference culture but non‐responsive or down‐regulated in tolerant animals. This cluster was also characterized by elevated constitutive expression in the tolerant population. Gene ontology analysis revealed that these cDNAs were involved in structural integrity of the cuticle, anti‐microbial defence, calcium channel‐blocking, sulphur assimilation and chromatin remodelling. The second group consisted of cDNAs down‐regulated in reference animals but not responding or slightly up‐regulated in tolerant animals. Their functions involved carbohydrate metabolic processes, Ca2+‐dependent stress signalling, redox state, proteolysis and digestion. The reference population showed a strong signature of stress‐induced genome‐wide perturbation of gene expression, whereas the tolerant animals maintained normal gene expression upon Cd exposure. We confirmed the micro‐evolutionary processes occurring in soil arthropod populations and suggest a major contribution of gene regulation to the evolution of a stress‐adapted phenotype.

Collembase: a repository for springtail genomics and soil quality assessment.

BackgroundEnvironmental quality assessment is traditionally based on responses of reproduction and survival of indicator organisms. For soil assessment the springtail Folsomia candida (Collembola) is an accepted standard test organism. We argue that environmental quality assessment using gene expression profiles of indicator organisms exposed to test substrates is more sensitive, more toxicant specific and significantly faster than current risk assessment methods. To apply this species as a genomic model for soil quality testing we conducted an EST sequencing project and developed an online database.DescriptionCollembase is a web-accessible database comprising springtail (F. candida) genomic data. Presently, the database contains information on 8686 ESTs that are assembled into 5952 unique gene objects. Of those gene objects ~40% showed homology to other protein sequences available in GenBank (blastx analysis; non-redundant (nr) database; expect-value < 10-5). Software was applied to infer protein sequences. The putative peptides, which had an average length of 115 amino-acids (ranging between 23 and 440) were annotated with Gene Ontology (GO) terms. In total 1025 peptides (~17% of the gene objects) were assigned at least one GO term (expect-value < 10-25). Within Collembase searches can be conducted based on BLAST and GO annotation, cluster name or using a BLAST server. The system furthermore enables easy sequence retrieval for functional genomic and Quantitative-PCR experiments. Sequences are submitted to GenBank (Accession numbers: EV473060 – EV481745).ConclusionCollembase http://www.collembase.org is a resource of sequence data on the springtail F. candida. The information within the database will be linked to a custom made microarray, based on the Agilent platform, which can be applied for soil quality testing. In addition, Collembase supplies information that is valuable for related scientific disciplines such as molecular ecology, ecogenomics, molecular evolution and phylogenetics.

Revealing pancrustacean relationships: Phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers

BackgroundIn recent years, several new hypotheses on phylogenetic relations among arthropods have been proposed on the basis of DNA sequences. One of the challenged hypotheses is the monophyly of hexapods. This discussion originated from analyses based on mitochondrial DNA datasets that, due to an unusual positioning of Collembola, suggested that the hexapod body plan evolved at least twice. Here, we re-evaluate the position of Collembola using ribosomal protein gene sequences.ResultsIn total 48 ribosomal proteins were obtained for the collembolan Folsomia candida. These 48 sequences were aligned with sequence data on 35 other ecdysozoans. Each ribosomal protein gene was available for 25% to 86% of the taxa. However, the total sequence information was unequally distributed over the taxa and ranged between 4% and 100%. A concatenated dataset was constructed (5034 inferred amino acids in length), of which ~66% of the positions were filled. Phylogenetic tree reconstructions, using Maximum Likelihood, Maximum Parsimony, and Bayesian methods, resulted in a topology that supports monophyly of Hexapoda.ConclusionAlthough ribosomal proteins in general may not evolve independently, they once more appear highly valuable for phylogenetic reconstruction. Our analyses clearly suggest that Hexapoda is monophyletic. This underpins the inconsistency between nuclear and mitochondrial datasets when analyzing pancrustacean relationships. Caution is needed when applying mitochondrial markers in deep phylogeny.

Recombinational micro-evolution of functionally different metallothionein promoter alleles from Orchesella cincta

BackgroundMetallothionein (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.ResultsWe 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.ConclusionThe 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.

A Functional Isopenicillin N Synthase in an Animal Genome

Horizontal transfer of genes is widespread among prokaryotes, but is less common between microorganisms and animals. Here, we present evidence for the presence of a gene encoding functional isopenicillin N synthase, an enzyme in the β-lactam antibiotics biosynthesis pathway, in the genome of the soil-living collembolan species, Folsomia candida (FcIPNS). At present, this gene is only known from bacteria and fungi, as is the capacity to produce β-lactam antibiotics. The FcIPNS gene was located on two genomic contigs, was physically linked to a predicted insect ATP-binding cassette transporter gene, and contained three introns each flanked by eukaryotic splicing recognition sites (GT/AG). Homology searches revealed no similarity between these introns and the FcIPNS regions of bacteria or fungi. All amino acids conserved across bacteria and fungi were also conserved in F. candida. Recombinant FcIPNS was able to convert its substrate amino δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine into isopenicillin N, providing strong evidence that FcIPNS is functional. Phylogenetic analysis clustered FcIPNS outside the bacterial IPNS clade, and also outside the fungal IPNS clade, suggesting an ancient gene transfer followed by divergence in the F. candida genome. In conclusion, the data suggest that the soil-living collembolan F. candida has assimilated the capacity for antibacterial activity by horizontal gene transfer, which may be an important adaptive trait in the microbe-dominated soil ecosystem.

Influence of adaptive evolution of cadmium tolerance on neutral and functional genetic variation in Orchesella cincta

Adaptation to environmental toxicants, such as metals, can affect population genetic diversity, both at neutral and selectable loci. At the transcriptional level, evolution of metal tolerance is possible due to the existence of polymorphisms in the cis-regulatory sequences of stress-responsive genes such as the metallothionein gene (mt). This study investigated the influence of cadmium adaptation on genetic diversity of soil-living Orchesella cincta (Collembola) populations in neutral (microsatellites and AFLP) and in functional (mt promoter) markers. Also, the influence of cis- and trans-acting factors on increased tolerance was addressed. No reduced genetic diversity was observed in two tolerant populations compared to five sensitive populations, either in neutral or in selectable markers. Extensive migration along with a large population size may explain the high genetic diversity measured. The metal-tolerant phenotype seems to be mostly influenced by genetic factors acting in cis on mt gene expression. The results suggest that certain promoter genotypes, which are found mainly or exclusively in Cd-tolerant populations, contribute to higher constitutive mt gene expression in individuals from these populations. However, more studies are needed to clearly unravel the influence of cis/trans-regulatory evolution in tolerant populations.

Coping with living in the soil: the genome of the parthenogenetic springtail Folsomia candida

BackgroundFolsomia 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.ResultsWe 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.ConclusionsThe 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.

Narcotic mechanisms of acute toxicity of chlorinated anilines in Folsomia candida (Collembola) revealed by gene expression analysis

In order to clarify the mechanisms of reproductive toxicity in a QSAR approach, the transcriptional signatures upon the 2 day exposure to the 28 days EC₅₀ of a series of increasingly chlorinated aniline compounds and 1,2,3,4-tetrachlorobenzene were measured in Folsomia candida. In general, the transcriptional patterns associated with all compounds revealed toxicity at the cellular membranes and hence components of narcosis type I, but a principal component analysis revealed a deviating response by the pentachloroaniline and 2,3,5,6-tetrachloroaniline exposure. Moreover the expression of a subset of mainly biotransformation related genes showed a significant relationship with the logK(ow,) which stresses the presence of narcosis type I. This was confirmed by GO term enrichment at the level of cellular component. Genes involved in the three phases of xenobiotic biotransformation exhibited strict compound specific transcription patterns, which may reflect biotransformation processes in F. candida. Additional toxic mechanisms were especially observed for the 2,3,5,6-tetrachloroaniline, which possible works as an uncoupler or inhibitor of electron transport systems, which is revealed by the up-regulation of genes that encode different members of the electron transport chain. The aniline and 2,3,4-trichloroaniline exposure caused the induction of genes in the ROS defense system. Additional toxicity mechanisms were less clear, but they include the attack of microbial pathogens for the six other compounds and for 2,3,5,6-tetrachloroaniline an effect on mitochondrial protein folding.