Model Organism In Ecology Research Articles

Fundamental Patterns of Structural Evolution Revealed by Chromosome-Length Genomes of Cactophilic Drosophila.

A thorough understanding of adaptation and speciation requires model organisms with both a history of ecological and phenotypic study as well as a complete set of genomic resources. In particular, high-quality genome assemblies of ecological model organisms are needed to assess the evolution of genome structure and its role in adaptation and speciation. Here, we generate new genomes of cactophilic Drosophila, a crucial model clade for understanding speciation and ecological adaptation in xeric environments. We generated chromosome-level genome assemblies and complete annotations for seven populations across Drosophila mojavensis, Drosophila arizonae, and Drosophila navojoa. We use these data first to establish the most robust phylogeny for this clade to date, and to assess patterns of molecular evolution across the phylogeny, showing concordance with a priori hypotheses regarding adaptive genes in this system. We then show that structural evolution occurs at constant rate across the phylogeny, varies by chromosome, and is correlated with molecular evolution. These results advance the understanding of the D. mojavensis clade by demonstrating core evolutionary genetic patterns and integrating those patterns to generate new gene-level hypotheses regarding adaptation. Our data are presented in a new public database (cactusflybase.arizona.edu), providing one of the most in-depth resources for the analysis of inter- and intraspecific evolutionary genomic data. Furthermore, we anticipate that the patterns of structural evolution identified here will serve as a baseline for future comparative studies to identify the factors that influence the evolution of genome structure across taxa.

Phylogenetic and Comparative Genomics Study of Papilionidae Based on Mitochondrial Genomes.

Most species of Papilionidae are large and beautiful ornamental butterflies. They are recognized as model organisms in ecology, evolutionary biology, genetics, and conservation biology but present numerous unresolved phylogenetic problems. Complete mitochondrial genomes (mitogenomes) have been widely used in phylogenetic studies of butterflies, but mitogenome knowledge within the family Papilionidae is limited, and its phylogeny is far from resolved. In this study, we first report the mitogenome of Byasa confusa from the subfamily Papilioninae of Papilionidae. The mitogenome of B. confusa is 15,135 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and an AT-rich control region (CR), closely mirroring the genomic structure observed in related butterfly species. Comparative analysis of 77 Papilionidae mitogenomes shows gene composition and order to be identical to that of an ancestral insect, and the AT bias, Ka/Ks, and relative synonymous codon usage (RSCU) are all consistent with that of other reported butterfly mitogenomes. We conducted phylogenetic analyses using maximum-likelihood (ML) and Bayesian-inference (BI) methods, with 77 Papilionidae species as ingroups and two species of Nymphalidae and Lycaenidae as outgroups. The phylogenetic analysis indicated that B. confusa were clustered within Byasa. The phylogenetic trees show the monophyly of the subfamily Papilioninae and the tribes Leptocircini, Papilionini, and Troidini. The data supported the following relationships in tribe level on Papilioninae: (((Troidini + Papilionini) + Teinopalpini) + Leptocircini). The divergence time analysis suggests that Papilionidae originated in the late Creataceous. Overall, utilizing the largest number of Papilionidae mitogenomes sequenced to date, with the current first exploration in a phylogenetic analysis on Papilionidae (including four subfamilies), this study comprehensively reveals the mitogenome characteristics and mitogenome-based phylogeny, providing information for further studies on the mitogenome, phylogeny, evolution, and taxonomic revision of the Papilionidae family.

Leaf decomposing fungi influence Saccharomyces paradoxus growth across carbon environments.

Saccharomyces paradoxus is a model organism in ecology and evolution. However, its metabolism in its native habitat remains mysterious: it is frequently found growing on leaf litter, a habitat with few carbon sources that S. paradoxus can metabolize. We hypothesized that leaf-decomposing fungi from the same habitat break down the cellulose in leaf litter extracellularly and release glucose, supporting S. paradoxus growth. We found that facilitation by leaf-decomposing fungi was possible on cellulose and inhibition was common on glucose, suggesting diverse interactions between S. paradoxus and other fungi that have the potential to support S. paradoxus in nature.

Negative Effects of Cyanotoxins and Adaptative Responses of Daphnia.

The plethora of cyanobacterial toxins are an enormous threat to whole ecosystems and humans. Due to eutrophication and increases in lake temperatures from global warming, changes in the distribution of cyanobacterial toxins and selection of few highly toxic species/ strains are likely. Globally, one of the most important grazers that controls cyanobacterial blooms is Daphnia, a freshwater model organism in ecology and (eco)toxicology. Daphnia-cyanobacteria interactions have been studied extensively, often focusing on the interference of filamentous cyanobacteria with Daphnia's filtering apparatus, or on different nutritional constraints (the lack of essential amino acids or lipids) and grazer toxicity. For a long time, this toxicity only referred to microcystins. Currently, the focus shifts toward other deleterious cyanotoxins. Still, less than 10% of the total scientific output deals with cyanotoxins that are not microcystins; although these other cyanotoxins can occur just as frequently and at similar concentrations as microcystins in surface water. This review discusses the effects of different cyanobacterial toxins (hepatotoxins, digestive inhibitors, neurotoxins, and cytotoxins) on Daphnia and provides an elaborate and up-to-date overview of specific responses and adaptations of Daphnia. Furthermore, scenarios of what we can expect for the future of Daphnia-cyanobacteria interactions are described by comprising anthropogenic threats that might further increase toxin stress in Daphnia.

Land snails on islands: building a global inventory

Land snails are one of the most diverse groups of terrestrial animals and are commonly used as model organisms in ecology, biogeography and conservation biology. Despite being poor dispersers, they form crucial components of island faunas and exhibit high percentages of endemism. Insular land snails are also among the most threatened animals on Earth, already having suffered extensive human-caused extinctions. However, current estimates of global insular land snail diversity are based on sporadic records published at the scale of individual islands and/or archipelagos. To tackle this shortfall, we herein present the major features of a global inventory of island snails. We recovered full species lists from existing literature and available species checklists for 727 islands across the globe and collated a database which currently includes the occurrence of 11,139 species, that is approximately 48% of all known land snail species (of which there are an estimated 23,000). Seventy-five percent of the species are single-island endemics, underlining the exceptional nature of islands as global biodiversity hotspots. Overall, our attempt is one of few to examine insular invertebrate diversity at coarser scales and a crucial step to the study of global patterns in island biodiversity.

The genome of the marine monogonont rotifer Brachionus plicatilis: Genome-wide expression profiles of 28 cytochrome P450 genes in response to chlorpyrifos and 2-ethyl-phenanthrene

Brachionus spp. (Rotifera: Monogononta) are globally distributed in aquatic environments and play important roles in the aquatic ecosystem. The marine monogonont rotifer Brachionus plicatilis is considered a suitable model organism for ecology, evolution, and ecotoxicology. In this study, we assembled and characterized the B. plicatilis genome. The total length of the assembled genome was 106.9 Mb and the number of final scaffolds was 716 with an N50 value of 1.15 Mb and a GC content of 26.75%. A total of 20,154 genes were annotated after manual curation. To demonstrate the use of whole genome data, we targeted one of the main detoxifying enzyme of phase I detoxification system and identified in a total of 28 cytochrome P450 s (CYPs). Based on the phylogenetic analysis using the maximum likelihood, 28 B. plicatilis-CYPs were apparently separated into five different clans, namely, 2, 3, 4, mitochondrial (MT), and 46 clans. To better understand the CYPs-mediated xenobiotic detoxification, we measured the mRNA expression levels of 28 B. plicatilis CYPs in response to chlorpyrifos and 2-ethyl-phenanthrene. Most B. plicatilis CYPs were significantly modulated (P < 0.05) in response to chlorpyrifos and 2-ethyl-phenanthrene. In addition, xenobiotic-sensing nuclear receptor (XNR) response element sequences were identified in the 5 kb upstream of promoter regions of 28 CYPs from the genome of B. plicatilis, indicating that these XNR can be associated with detoxification of xenobiotics. Overall, the assembled B. plicatilis genome presented here will be a useful resource for a better understanding the molecular ecotoxicology in the view of molecular mechanisms underlying toxicological responses, particularly on xenobiotic detoxification in this species.

Monitoring ecdysteroid activities using genetically encoded reporter gene in Daphnia magna

Ecdysteroid is an important hormone that regulates growth, reproduction, and embryogenesis in arthropods. However, little is known about its role and action mechanism in crustaceans, despite their pivotal role in aquatic ecosystem. Daphnia magna, a freshwater crustacean, is used as a classic model organism in ecology and ecotoxicology. Its ecdysteroid activity has been partially characterized previously. However, the spatio-temporal behavior of ecdysteroid, especially during early embryogenesis, when it is thought to have pivotal roles, is still unclear. Thus, we proposed a genetic modification approach by integrating a reporter gene exhibiting ecdysteroid activity in vivo.We used the clustered regularly interspaced palindromic repeats (CRISPR) genome editing technique, followed by non-homologous end-joining (NHEJ) pathway as the transgenesis method to generate the ecdysteroid reporter transgenic Daphnia. One transgenic Daphnia containing one copy of the ecdysone response element (EcRE)-controlled reporter gene mCherry was successfully obtained and was designated EcRE-mCh.The expression of mCherry was observed during early embryogenesis starting from 12 h after ovulation (hao). The time-lapse imaging during 12–24 hao showed the growing expression of mCherry signal originating from the posterior section of embryo and then migrating toward the anterior section. From 18 hao, the signal was detected around the developing thoracic appendages and localized between the first to third thoracic segments. The establishment of this EcRE-mCh line and its ability to exhibit ecdysteroid activity spatio-temporally might serve as convenient tool to elucidate the roles of ecdysteroid during the early stage of animal development. Moreover, the expression of mCherry in response to the presence of ecdysteroid in water suggests that EcRE-mCh could be used for monitoring ecdysteroid activities in environmental water.

Mitogenome phylogeographic analysis of a planktonic crustacean

Phylogeography places population genetics in an explicitly spatial context, and in doing so attempts to reconstruct the historical and contemporary evolutionary processes acting across a species range through space and time. Here we present the phylogeographical structure of Daphnia magna as determined for full mitochondrial genomes from samples of 60 populations throughout much of the species known range, including Europe, the Middle East, and Asia. Contrary to previous analyses, the present analysis of the mitochondrial genome reveals coarse-grained (continental scale) evidence for spatial structure, and in particular a deep split between Western Eurasia and East Asian D. magna lineages. In contrast to previous analyses with nuclear genetic markers, our mitogenomic analysis reveals much less structure within lineages. We quantify divergence between species using the full mitochondrial genome sequence of a closely related species, D. similis. The distribution of European and Middle Eastern genetic diversity is consistent with a rapid demographic expansion following the end of the most recent ice age about 10,000 years before present. By estimating species wide distributions of dN/dS in mtDNA, we provide evidence that the effectiveness of purifying selection on protein coding genes in the mitochondrial genome of coastal rock pool populations, which have pronounced extinction-colonization dynamics, is reduced compared to larger and more stable non-rock pool populations. The present study adds important insights into the evolutionary history of a widely used model organism in ecology, evolution and ecotoxicology, and highlights the utility of phylogeographic analysis of organellar genomes to understand evolutionary processes.

Worldwide phylogeny of three-spined sticklebacks

Stickleback fishes in the family Gasterosteidae have become model organisms in ecology and evolutionary biology. However, even in the case of the most widely studied species in this family – the three-spined stickleback (Gasterosteus aculeatus) – the worldwide phylogenetic relationships and colonization history of the different populations and lineages remain poorly resolved. Using a large collection of samples covering most parts of the species distribution range, we subjected thousands of single nucleotide polymorphisms to coalescent analyses in order to reconstruct a robust worldwide phylogeny of extant G. aculeatus populations, as well as their ancestral geographic distributions using Statistical-Dispersal Vicariance and Bayesian Binary MCMC analyses. The results suggest that contemporary populations originated from the Pacific Ocean in the Late Pleistocene, and the Atlantic was colonized through the Arctic Ocean by a lineage that diverged from Pacific sticklebacks ca 44.6 Kya. This lineage contains two branches: one that is distributed in the Mediterranean area, from the Iberian Peninsula to the Black Sea (‘Southern European Clade’), and another that is comprised of populations from northern Europe and the east coast of North America (‘Trans-Atlantic Clade’). Hence, the results suggest that the North American East Coast was colonized by trans-Atlantic migration. Coalescence-based divergence time estimates suggest that divergence among major clades is much more recent than previously estimated.

Eretmochelys imbricata shells present a dynamic substrate for a facilitative epibiont relationship between macrofauna richness and nematode diversity, structure and function

Although nematodes are the most abundant metazoans in marine environments and present an important biological and ecological model organism to assess marine ecosystem processes and functions, there are in fact very few studies that use nematodes to investigate ecological communities and relationships on “mobile” ecosystems. Arguably one of the most mobile or dynamic marine ecosystems is a sea turtle carapace, hosting tens to hundreds or even thousands of epibiotic organisms; and as the turtle breeds, feeds and migrates, provides an ecosystem that is continuously exposed to changes and potential colonizers. In this study we investigated the nematode communities associated with 19 Hawksbill sea turtle carapaces (Eretmochelys imbricata), and compared nematode structural (composition, richness and diversity) and functional (trophic types and gender/life stages) community parameters with those of other comparable epibiotic substrates (macrophytes, natural and artificial hard substrates) to see whether turtle carapaces are hotspots of nematode diversity and function among substrates suitable for epifauna. We also addressed potential epibiotic macrofauna-nematode interactions by looking at the relationships between macrofauna richness and nematode richness, diversity and community composition. Results suggest that the macrofauna play a bioconstructing role, creating several microenvironments, and thereby enhancing the richness and diversity of the associated nematode assemblages. This was supported by a direct and positive relationship between macrofauna and nematode richness, and implies a genera enrichment process across size classes and phyla. All heterotrophic nematode feeding guilds were recovered from the carapaces, with dominance of predators/omnivores and epistrate feeders. Nematode juveniles dominated in terms of abundance, and a female/male ratio of 1.11 was observed. Nematode richness and diversity were higher than found on other substrates, but feeding guild, gender and life stage structure did not differ compared to nematode communities from all other epibiotic substrates. As a result, we argue that turtle carapaces can be seen as hotspots for nematode biodiversity compared to other epibiotic substrates, but this is not reflected in the function of the nematode community. This study is the first to investigate in detail sea turtle carapace nematode communities, their richness, diversity, trophic and life cycle structure, and potential interactions with their co-epibionts, the macrofauna.

Functional genomics in the wild: a case study with paper wasps shows challenges and prospects for RNA interference in ecological systems.

RNA interference (RNAi) is a useful tool to assess gene function by knocking down expression of a target gene and has been used successfully in domestic and laboratory organisms. However, the use of RNAi for functional genomics has not fully extended into ecological model organisms in natural environments. Assessment of gene function in the wild is important because gene function can be environmentally and context dependent. Here, we present a case study using RNAi to assess gene function in wild paper wasps Polistes metricus, to test roles for two candidate genes (NADH dehydrogenase (NADHdh) and retinoid and fatty acid binding protein (RfaBp)) in the development of reproductive castes. Previous studies have shown that these genes are upregulated in larvae that become queens compared to workers, but this pattern was reversed in the laboratory, making field-based studies necessary. We orally administered dsRNA to larvae in field colonies and found evidence of a short-term knockdown followed by a compensatory rebound in expression for RfaBp. We also observed the predicted worker-like decrease in lipid stores in NADHdh dsRNA treated wasps, suggesting a possible role for NADHdh in caste development. We discuss our results in the context of challenges for using RNAi for functional genomics in ecological model organisms in the field.

Multigene phylogenetic analysis redefines dung beetles relationships and classification (Coleoptera: Scarabaeidae: Scarabaeinae).

BackgroundDung beetles (subfamily Scarabaeinae) are popular model organisms in ecology and developmental biology, and for the last two decades they have experienced a systematics renaissance with the adoption of modern phylogenetic approaches. Within this period 16 key phylogenies and numerous additional studies with limited scope have been published, but higher-level relationships of this pivotal group of beetles remain contentious and current classifications contain many unnatural groupings. The present study provides a robust phylogenetic framework and a revised classification of dung beetles.ResultsWe assembled the so far largest molecular dataset for dung beetles using sequences of 8 gene regions and 547 terminals including the outgroup taxa. This dataset was analyzed using Bayesian, maximum likelihood and parsimony approaches. In order to test the sensitivity of results to different analytical treatments, we evaluated alternative partitioning schemes based on secondary structure, domains and codon position. We assessed substitution models adequacy using Bayesian framework and used these results to exclude partitions where substitution models did not adequately depict the processes that generated the data. We show that exclusion of partitions that failed the model adequacy evaluation has a potential to improve phylogenetic inference, but efficient implementation of this approach on large datasets is problematic and awaits development of new computationally advanced software. In the class Insecta it is uncommon for the results of molecular phylogenetic analysis to lead to substantial changes in classification. However, the results presented here are congruent with recent morphological studies and support the largest change in dung beetle systematics for the last 50 years. Here we propose the revision of the concepts for the tribes Deltochilini (Canthonini), Dichotomiini and Coprini; additionally, we redefine the tribe Sisyphini. We provide and illustrate synapomorphies and diagnostic characters supporting the new concepts to facilitate diagnosability of the redefined tribes. As a result of the proposed changes a large number of genera previously assigned to these tribes are now left outside the redefined tribes and are treated as incertae sedis.ConclusionsThe present study redefines dung beetles classification and gives new insight into their phylogeny. It has broad implications for the systematics as well as for various ecological and evolutionary analyses in dung beetles.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0822-x) contains supplementary material, which is available to authorized users.

Strong phylogeographic co-structure between the anther-smut fungus and its white campion host.

Although congruence between host and pathogen phylogenies has been extensively investigated, the congruence between host and pathogen genetic structures at the within-species level has received little attention. Using an unprecedented and comprehensive collection of associated plant-pathogen samples, we investigated the degree of congruence between the genetic structures across Europe of two evolutionary and ecological model organisms, the anther-smut pathogen Microbotryum lychnidis-dioicae and its host plant Silene latifolia. We demonstrated a significant and particularly strong level of host-pathogen co-structure, with three main genetic clusters displaying highly similar spatial ranges in Western Europe, Eastern Europe and Italy, respectively. Correcting for the geographical component of genetic variation, significant correlations were still found between the genetic distances of anther-smut and host populations. Inoculation experiments suggested plant local adaptation, at the cluster level, for resistance to pathogens. These findings indicate that the pathogen remained isolated in the same fragmented southern refugia as its host plant during the last glaciation, and that little long-distance dispersal has occurred since the recolonization of Europe for either the plant or the pathogen, despite their known ability to travel across continents. This, together with the inoculation results, suggests that coevolutionary and competitive processes may be drivers of host-pathogen co-structure.

An ecological model organism flies into the genomics era.

Despite the very rapid 'genomicization' of the field of Molecular Ecology in recent years, there have been relatively few annotated whole-genome assemblies of nonmodel organisms published. Instead, molecular ecologists have more frequently utilized next-generation sequencing technologies to develop genome-wide markers or to generate transcriptome data. Whole-genome assemblies are more expensive and require considerable computational resources and bioinformatic expertise. However, the availability of an annotated genome offers exciting opportunities to address fundamental questions in ecology and evolution that are difficult to address with moderate sets of markers or by transcriptome sequencing. Such questions include elucidating the roles of natural and sexual selection in shaping diversity, determining the roles of regulatory and protein-coding change in the evolution of traits, and determining the genomic architecture of sex-specific trait variation. Arguably, these questions are most tractable--and most interesting--in well-characterized species for which there is already some knowledge of natural and sexual selection, and of the traits that are most likely to link to fitness. In this issue, Mueller et al. (2016) present the assembly and annotation of the genome of the blue tit (Cyanistes caeruleus), a model ecological species. In addition, by sequencing the transcriptome of male and female blue tits, the authors identify and annotate sex-biased gene expression and conclude that noncoding RNA genes are likely to play a significant role in sex-biased expression. By making their assembly and annotation publically available and accessible via a genome browser, Mueller et al. (2016) offer exciting possibilities for further research into the genomic basis of adaptation, and investigation of the roles of natural and sexual selection, in this well-studied ecological model species.

Accumulation and transformation of nanomaterials in ecological model organisms investigated by using synchrotron radiation techniques

Application of SR techniques to study the accumulation and transformation of engineered nanomaterials in different model organisms of terrestrial, aquatic and atmospheric systems.

Timing matters: Sensitivity of Daphnia magna dormant eggs to fenoxycarb exposure depends on embryonic developmental stage

Although Daphnia magna is a key species in many lentic freshwater ecosystems and is commonly used as model organism in ecology and ecotoxicology, very little is known about the effects of chemicals on their dormant life stages. Dormant eggs (ephippia) are produced when environmental conditions deteriorate, and Daphnia switch from clonal to sexual reproduction. Ephippia produced over different growing seasons can accumulate in the sediment of ponds and lakes, where they can be exposed to pesticides and other (anthropogenic) stressors. In the present study, we have investigated the effects of pesticide exposure on dormant eggs at different embryonic developmental stages and evaluated the degree of protection against pollution provided by the ephippial case. We therefore conducted a hatching experiment in which decapsulated and encapsulated dormant eggs were exposed to an insect growth regulator (fenoxycarb) at different stages during their development, both before and after activation of the eggs. In addition, we developed an analytical method to measure fenoxycarb concentrations in the dormant eggs. Fenoxycarb negatively affected development and hatching success and changed the timing of hatching in activated and in dormant eggs. Hatching characteristics as well as fenoxycarb concentrations inside the eggs differed significantly between exposure treatments. Final stages of embryonic development were most sensitive to pesticide exposure and had the highest tissue concentrations of fenoxycarb. Tissue concentrations did not differ significantly between decapsulated and encapsulated eggs, suggesting that the ephippial case offers limited or no direct protection against pesticide exposure. With this study we provide new evidence showing that pesticides can bioconcentrate in and affect D. magna dormant eggs. The severity of the effects on developing embryos depends on the timing of pesticide exposure. Our results stress the importance of considering the full life-cycle of model organisms used in ecotoxicological studies, since these are ultimately aimed at assessing risks of chemical exposure on natural aquatic ecosystems.

Underground evolution: New roots for the old tree of lumbricid earthworms

Earthworms belonging to the family Lumbricidae are extremely abundant in terrestrial temperate regions. They affect soil properties and nutrient cycling, thus shaping plant community composition and aboveground food webs. Some lumbricids are also model organisms in ecology and toxicology. Despite the intense research efforts dedicated to lumbricids over the last 130years, the evolutionary relationships and taxonomic classification of these organisms are still subject to great debate. Resolution of their systematics is hampered by the structural simplicity of the earthworm body plan and the existence of cryptic species. We sampled 160 earthworm specimens belonging to 84 lumbricid species (28 genera) and 22 Lumbricoidea outgroups, sequenced two nuclear genes, four mitochondrial genes and seven mitochondrial tRNAs and examined 22 morphological characters. We then applied a combination of phylogenetic methods to generate the first robust genus-level phylogeny of the Lumbricidae. Our results show that the current Lumbricidae classification and the underlying hypotheses of character evolution must be revised. Our chronogram suggests that lumbricids emerged in the Lower Cretaceous in the holarctic region and that their diversification has been driven by tectonic processes (e.g. Laurasia split) and geographical isolation. Our chronogram and character reconstruction analysis reveal that spermathecae number does not follow a gradual pattern of reduction and that parthenogenesis arose from sexual relatives multiple times in the group; the same analysis also indicates that both epigeic and anecic earthworms evolved from endogeic ancestors. These findings emphasize the strong and multiple changes to which morphological and ecological characters are subjected, challenging the hypothesis of character stasis in Lumbricidae.

The complete mitochondrial genome of Parage aegeria (Insecta: Lepidoptera: Papilionidae)

Pararge aegeria is a palearctic butterfly used as a model organism in ecology, behavior, evolution and development, with significant geographically-correlated morphological variation and two close relatives in Macaronesia. In this study, I have determined the complete mitogenome sequence of P. aegeria. The genome is 15,240 bp long, and has the typical gene organization of other lepidopteran mitogenomes. Salient features include the IMQ order of tRNA genes, a sizable (52 bp) spacer between trnQ and nad2, a CGA start codon in cox1 and the ATAGA(T)19 motif in the control region. Interestingly, this region also contains four copies of the imperfect palindrome TAAATATWTATAWATATTTA.

De novo transcriptome of Ischnura elegans provides insights into sensory biology, colour and vision genes.

BackgroundThere is growing interest in odonates (damselflies and dragonflies) as model organisms in ecology and evolutionary biology but the development of genomic resources has been slow. So far only one draft genome (Ladona fulva) and one transcriptome assembly (Enallagma hageni) have been published. Odonates have some of the most advanced visual systems among insects and several species are colour polymorphic, and genomic and transcriptomic data would allow studying the genomic architecture of these interesting traits and make detailed comparative studies between related species possible. Here, we present a comprehensive de novo transcriptome assembly for the blue-tailed damselfly Ischnura elegans (Odonata: Coenagrionidae) built from short-read RNA-seq data. The transcriptome analysis in this paper provides a first step towards identifying genes and pathways underlying the visual and colour systems in this insect group.ResultsIllumina RNA sequencing performed on tissues from the head, thorax and abdomen generated 428,744,100 paired-ends reads amounting to 110 Gb of sequence data, which was assembled de novo with Trinity. A transcriptome was produced after filtering and quality checking yielding a final set of 60,232 high quality transcripts for analysis. CEGMA software identified 247 out of 248 ultra-conserved core proteins as ‘complete’ in the transcriptome assembly, yielding a completeness of 99.6%. BLASTX and InterProScan annotated 55% of the assembled transcripts and showed that the three tissue types differed both qualitatively and quantitatively in I. elegans. Differential expression identified 8,625 transcripts to be differentially expressed in head, thorax and abdomen. Targeted analyses of vision and colour functional pathways identified the presence of four different opsin types and three pigmentation pathways. We also identified transcripts involved in temperature sensitivity, thermoregulation and olfaction. All these traits and their associated transcripts are of considerable ecological and evolutionary interest for this and other insect orders.ConclusionsOur work presents a comprehensive transcriptome resource for the ancient insect order Odonata and provides insight into their biology and physiology. The transcriptomic resource can provide a foundation for future investigations into this diverse group, including the evolution of colour, vision, olfaction and thermal adaptation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-808) contains supplementary material, which is available to authorized users.

071 Towards the eternal life for Daphnia magna clones

Daphnia magna is an important model organism for ecology, evolutionary biology and ecotoxicology. One of the main advantages of using Daphnia in research lies in its mode of reproduction. The life cycle of Daphnia is characterized by an alternation of sexual and asexual reproduction, called cyclic parthenogenesis, which enables researchers to work with clonal lines and provides an unlimited amount of genetically identical individuals for experimentation. This way of reproduction is used in case of a fast population increase during the favorable conditions of spring and summer. When females sense stimuli of detrimental conditions they start to produce males and switch to sexual reproduction. Fertilized eggs are deposited in an ephippium and which in turn is deposited in the water to form an egg bank, where they stay viable for a long period of time. The dormant eggs are able to cope with severe freezing temperatures and drought. It is even possible to freeze them in liquid nitrogen without the use of cryoprotectants and revive them without significant loss of viability; however this is not possible for the asexual eggs. To date, maintenance of those clonal lines can only be achieved through continuous culturing. This is a labor intensive process and it is not without risk of losing important lines because of contamination or disease. Cryopreservation could provide us with a welcome alternative to safely store genetically well characterized lines. In this study, we tested important steps towards developing a cryopreservation protocol for the asexual eggs of D. magna. Based on similar work with the tropical nematode Radophilis similis and related literature, a set up was designed to establish a vitrification protocol comprising four steps. Before the actual freezing, eggs must be prepared to resist the freezing procedure by reducing their water content as much as possible and for these steps we evaluated mortality and other parameters associated with survival. In the first experiment the eggs were loaded with the penetrating cryoprotectant glycerol. We used glycerol because previous studies have shown that the amount of glycerol significantly differs between sexual and asexual eggs of D. magna . After exposure to 10% glycerol for 30 min 54% of the eggs survived and besides this resulted in a 20% volume reduction and a glycerol uptake of 4.30 μg/ml. Next, a vitrification solution was developed consisting of a mixture of the cryoprotectants glycerol, ethylene glycol and trehalose. Trehalose was selected since we found that sexual eggs, which are highly resistant to various stressors, contain significantly higher levels of this compound and secondly it is known that sugars can have beneficial effects on the properties of EG-based solutions. The vitrification capacities of the solutions were tested by screening presence/absence of ice crystal during rapid freezing and thawing. Subsequently, the toxicity of the vitrifying solutions at 0 °C towards asexual eggs of the water flea was tested. The vitrifying mixture with the lowest possible solute concentration and the lowest toxicity consists of 20% glycerol, 25% ethylene glycol and 0.5M trehalose. Cryoprotection is followed by ultra-rapid freezing in liquid nitrogen and thawing. For this the method of droplet vitrification is applied. Source of funding: IWT Vlaanderen. Conflict of interest: None declared. adinda.putman@bio.kuleuven.be