Project description:BackgroundThe planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data.ResultsIn this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (Tbp) syntaxin 16 (Stx16), X-box binding protein 1 (Xbp1) and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP), was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH) were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are found in the D. pulex genome sequence. When applying aTub for expression normalization Xbp1 and Tbp are falsely reported as significantly upregulated.ConclusionsOur results suggest that the genes Xbp1, Tbp, CAPON and Stx16 are suitable reference genes for accurate normalization in qRT-PCR studies using Chaoborus-induced D. pulex specimens. Furthermore, our study underscores the importance of verifying the expression stability of putative reference genes for normalization of expression levels.

Project description:Across evolutionary time, nearly all animal species have harnessed photoperiod to initiate processes that ultimately influence seasonal behavior and life history traits. In the freshwater cladoceran Daphnia magna, the effect of photoperiod on various life history traits has generally been investigated in conjunction with additional environmental stimuli. In the present study, we sought to untangling responses directly attributable to photoperiod in D. magna and identify the molecular processes underlying resultant behavioral and life history responses using functional analysis of global transcriptomic expression. D. magna were exposed to five different photoperiods immediately post-hatch for 21d where a standard long-day photoperiod of 16 hours light and 8 hours dark (16L:8D) served as the control relative to 4L:20D, 8L:16D, 12L:12L, and 20L:4D photoperiods. Entrainment to short-day photo-periods (4L:20D, 8L:16D, and 12L:12L) resulted in significantly increased light-avoidance behaviors relative to the control photoperiod where young Daphnia (7d old) displayed the most pronounced avoidance responses. Correspondingly, functional transcriptomics identified differential transcriptional expression of genes involved in glutamate signaling, which is critical in arthropod light-avoidance responses, as well as period circadian protein and proteins coding F-box/LRR-repeat domains, all of which contribute to establishing circadian rhythms in arthropods. Short-day photoperiods also induced increased metabolic rates which corresponded with broad-scale changes in transcriptional expression across multiple systems-level energy metabolism pathways. The most striking observation was increased male production across short-day photoperiods (4L:20D, 8L:16D, and 12L:12D). Transcriptional expression consistent with multiple putative mechanisms of male production were observed including expression suggestive of increased glutamate signaling; a response observed to induce male production in D. pulex via photo-period sensitive mechanisms. Overall, the results demonstrate the importance of photoperiod on behavior and life history trajectories in D. magna where we have now established multiple putative mechanistic pathways underlying several critical responses.

Dataset Information

Light intensity controls anti-predator defences in Daphnia: the suppression of life-history changes.

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Light intensity controls anti-predator defences in Daphnia: the suppression of life-history changes.

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