Dataset Information

Development of a microinjection system for RNA interference in the water flea Daphnia pulex.

ABSTRACT:

Background

The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna, these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments.

Results

We found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds)RNA specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene.

Conclusions

We developed a microinjection system for RNAi studies in D. pulex. These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.

SUBMITTER: Hiruta C

PROVIDER: S-EPMC4228505 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Publications

Development of a microinjection system for RNA interference in the water flea Daphnia pulex.

Hiruta Chizue C Toyota Kenji K Miyakawa Hitoshi H Ogino Yukiko Y Miyagawa Shinichi S Tatarazako Norihisa N Shaw Joseph R JR Iguchi Taisen T

BMC biotechnology 20131105

<h4>Background</h4>The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishm ...[more]

PMID: 24188141

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Project description:Phenotypic plasticity is the ability held in many organisms to produce different phenotypes with a given genome in response to environmental stimuli, such as temperature, nutrition and various biological interactions. It seems likely that environmental signals induce a variety of mechanistic responses that influence ontogenetic processes. Inducible defenses, in which prey animals alter their morphology, behavior and/or other traits to help protect against direct or latent predation threats, are among the most striking examples of phenotypic plasticity. The freshwater microcrustacean Daphnia pulex forms tooth-like defensive structures, "neckteeth," in response to chemical cues or signals, referred to as "kairomones," in this case released from phantom midge larvae, a predator of D. pulex. To identify factors involved in the reception and/or transmission of a kairomone, we used microarray analysis to identify genes up-regulated following a short period of exposure to the midge kairomone. In addition to identifying differentially expressed genes of unknown function, we also found significant up-regulation of genes encoding ionotropic glutamate receptors, which are known to be involved in neurotransmission in many animal species. Specific antagonists of these receptors strongly inhibit the formation of neckteeth in D. pulex, although agonists did not induce neckteeth by themselves, indicating that ionotropic glutamate receptors are necessary but not sufficient for early steps of neckteeth formation in D. pulex. Moreover, using co-exposure of D. pulex to antagonists and juvenile hormone (JH), which physiologically mediates neckteeth formation, we found evidence suggesting that the inhibitory effect of antagonists is not due to direct inhibition of JH synthesis/secretion. Our findings not only provide a candidate molecule required for the inducible defense response in D. pulex, but also will contribute to the understanding of complex mechanisms underlying the recognition of environmental changes, which form the basis of phenotypic plasticity.

Project description:The cladoceran crustacean Daphnia pulex produces female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable external stimuli, it produces male offspring (environmental sex determination: ESD). We recently established an innovative system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be controlled simply by changes in the photoperiod: the long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we demonstrated that de novo methyl farnesoate (MF) synthesis is necessary for male offspring production. These results indicate the key role of innate MF signaling as a conductor between external environmental stimuli and the endogenous male developmental pathway. Despite these findings, the molecular mechanisms underlying up- and downstream signaling of MF have not yet been well elucidated in D. pulex.To elucidate up- and downstream events of MF signaling during sex determination processes, we compared the transcriptomes of daphnids reared under the long-day (female) condition with short-day (male) and MF-treated (male) conditions. We found that genes involved in ionotropic glutamate receptors, known to mediate the vast majority of excitatory neurotransmitting processes in various organisms, were significantly activated in daphnids by the short-day condition but not by MF treatment. Administration of specific agonists and antagonists, especially for the N-methyl-D-aspartic acid (NMDA) receptor, strongly increased or decreased, respectively, the proportion of male-producing mothers. Moreover, we also identified genes responsible for male production (e.g., protein kinase C pathway-related genes). Such genes were generally shared between the short-day reared and MF-treated daphnids.We identified several candidate genes regulating ESD which strongly suggests that these genes may be essential factors for male offspring production as an upstream regulator of MF signaling in D. pulex. This study provides new insight into the fundamental mechanisms underlying how living organisms alter their phenotypes in response to various external environments.

Project description:BackgroundMarine and freshwater zooplankton exhibit daily rhythmic patterns of behavior and physiology which may be regulated directly by the light:dark (LD) cycle and/or a molecular circadian clock. One of the best-studied zooplankton taxa, the freshwater crustacean Daphnia, has a 24 h diel vertical migration (DVM) behavior whereby the organism travels up and down through the water column daily. DVM plays a critical role in resource tracking and the behavioral avoidance of predators and damaging ultraviolet radiation. However, there is little information at the transcriptional level linking the expression patterns of genes to the rhythmic physiology/behavior of Daphnia.ResultsHere we analyzed genome-wide temporal transcriptional patterns from Daphnia pulex collected over a 44 h time period under a 12:12 LD cycle (diel) conditions using a cosine-fitting algorithm. We used a comprehensive network modeling and analysis approach to identify novel co-regulated rhythmic genes that have similar network topological properties and functional annotations as rhythmic genes identified by the cosine-fitting analyses. Furthermore, we used the network approach to predict with high accuracy novel gene-function associations, thus enhancing current functional annotations available for genes in this ecologically relevant model species. Our results reveal that genes in many functional groupings exhibit 24 h rhythms in their expression patterns under diel conditions. We highlight the rhythmic expression of immunity, oxidative detoxification, and sensory process genes. We discuss differences in the chronobiology of D. pulex from other well-characterized terrestrial arthropods.ConclusionsThis research adds to a growing body of literature suggesting the genetic mechanisms governing rhythmicity in crustaceans may be divergent from other arthropod lineages including insects. Lastly, these results highlight the power of using a network analysis approach to identify differential gene expression and provide novel functional annotation.

Dataset Information

Development of a microinjection system for RNA interference in the water flea Daphnia pulex.

Background

Results

Conclusions

Publications

Development of a microinjection system for RNA interference in the water flea Daphnia pulex.

Similar Datasets

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