BACKGROUND
in both basic and human health research, there is a pressing need to understand how environmental conditions influence gene functions and, in turn, how individuals and populations cope with changing environments.
invertebrate species have emerged as models for experimental manipulation because of their unique biological attributes, life cycles, large numbers of offspring, easy maintenance, and sophisticated tools for high-throughput biology and open-source informatics  <cit> .
however, the traits observed in laboratories are likely a small subset of the phenotypic variation that is expressed in natural ecosystems.
this may partly explain why  <dig> to 50% or more genes are without experimentally determined functional annotations, even within the best-characterized genomes .
daphnia possess several characteristics that make them valuable for environmental, evolutionary, and developmental genomics research–addressing the added complexity of genome-environment interactions.
daphnia are a ubiquitous, and ecologically important member of freshwater lakes and ponds, and have long been used as a sentinel of the integrity of these aquatic ecosystems.
more recently with the release of the d. pulex genome  <cit> , it now serves as a recognized surrogate model for human health research.
the d. pulex genome possesses more genes than any previously sequenced animal genome , due to a large orphanage of daphnia genes that likely, allows the organism to respond to its environment  <cit> .
in addition to their short generation time, large brood sizes, and ease of laboratory and field manipulation, daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic studies.
at present, however, there are no effective methods for manipulating genes and characterizing gene function, which because of the large gene orphanage limits interspecies extrapolations.
rna interference  is an evolutionarily conserved post-transcriptional gene silencing mechanism, which is triggered by double-stranded rna in a sequence specific manner  <cit> .
since rnai was first reported in the nematode caenorhabditis elegans by fire et al.
<cit> , it has been used as a powerful tool for the analysis of gene function in many organisms such as zebrafish danio rerio <cit> , planarian schmidtea meditteranea <cit> , cnidarian hydra magnipapillata <cit> , fungus neurospora crassa <cit> , fruit-fly drosophila melanogaster <cit>  and mouse mus musculus <cit> .
microinjection is one method of introducing dsrna into cells, and this method has been successfully developed for the daphnid species, d. magna <cit> .
in fact, microinjection techniques enabled the application of not only rnai  <cit> , but also overexpression of foreign genes  <cit>  and the creation of transgenic individuals  <cit> .
establishing these techniques in d. pulex will extend the resources for environmental, evolutionary, and developmental genomics research for this species by providing needed tools to characterize gene function.
distal-less  and its homologs dlx genes, which function as homeodomain transcription factors, play one of the major roles in limb development throughout the animal kingdom  <cit> .
reduction of dll activity caused defects of distal leg segments in arthropods including insects  <cit> , crustaceans , the spider cupiennius salei <cit>  and the spider mite tetranychus urticae <cit> .
because of its conserved role in limb development, defects in the expression of the dll gene produce easily recognizable phenotype and ease of evaluation of its phenotype, which is why this endogenous developmental gene was selected as a target in proof-of-principle rnai in d. pulex.
the goal of this study was to develop a microinjection system for rnai in d. pulex.
culture requirements for isolated embryos were first determined and then microinjection techniques developed for conducting rnai experiments using dll-dsrna.
RESULTS
development of microinjection system
there are two major technical problems for microinjection in daphnid species.
one is the rapid hardening of egg membrane  <cit>  and the other is a considerable difference between the internal and external osmotic pressures of the egg.
the former prevents egg membrane penetration by a needle, and the latter produces leaking of internal contents  when the needle is withdrawn.
kato et al.
<cit>  determined the proper condition for microinjection in d. magna.
eggs were incubated on ice just after ovulation to inhibit the membrane hardening transiently and placed in plastic petri dish with 80 mm sucrose medium dissolved by m <dig> culture medium  to increase external osmotic pressure.
however, these conditions are not suitable for d. pulex possibly because of the difference in egg size and form .
to overcome these hurdles, we first examined culture conditions by varying sucrose concentration and culture media, and determining those conditions that allowed embryos isolated from the brood chamber to develop normally.
survival was greater in m <dig> culture media as compared to dechlorinated freshwater  .
a sucrose concentration of 40 mm on a 2% agar plate yielded the greatest viability, but this concentration of sucrose was not high enough to counter the internal osmolality and, therefore, not sufficient for injection .
taken together, a 2% agar plate covered with 60 mm sucrose dissolved in m <dig> media provided the best conditions for microinjection of d. pulex early embryos, and was employed in subsequent experiments.
these conditions allowed embryos to be injected within  <dig> to 60 min of isolation.
it is critical that injections occur within the first hour following ovulation, because there is no cytokinesis during this period and dsrna can easily diffuse throughout the egg as it remains a single cell  <cit> .
left column represents types of culture plate and top row shows sucrose concentration of culture medium.
sucrose was dissolved in freshwater  or m <dig> culture medium .
dll rnai using microinjection in d. pulex
the dsrna was prepared from a 500 bp region derived from the dll gene , and dsrna derived from a 729-bp region of the escherichia coli male gene  was developed as a negative control.
there is only one dll gene in the d. pulex genome .
the dll-dsrna was synthesized from a region that included the homeodomain.
red fluorescence was used to judge whether microinjection were successful .
results from rnai experiments are summarized in table  <dig>  only 57% of the embryos injected with dll-dsrna developed compared to  <dig> % viability of those injected with of male-dsrna, indicating dll-dsrna produced embryonic lethal phenotypes.
embryonic lethality caused by loss of dll functions was reported in insects  <cit> .
dll
male
sequence-specific gene silencing of d. pulex dll
we injected 1 μg/μl dsrna with a fluorescent dye into each egg and measured the endogenous dll mrna levels using q-pcr at 24 h after injection to determine if dll-dsrna triggers the degradation of endogenous dll mrnas.
the quantity of dll mrna in dll-dsrna-injected embryos was decreased to  <dig> %  of that in male-dsrna-injected control embryos .
these results were generally consistent with the result of d. magna where a  <dig> %  reduction in dll was observed  <cit> .
phenotype of dll rnai in d. pulex
we confirmed the spatial expression pattern of dll in embryo using whole-mount in situ hybridization.
dll was strongly expressed in the distal portion of second antenna, appendages  and labrum, and weakly expressed in first antenna, mandible and first maxilla between  <dig> to 30 h post ovulation .
dll-dsrnai injection of isolated embryos produced defects in the dll-expressing organs  to various degrees.
these defects included a truncation of the second antenna segments  that resembled a phenotype observed in d. magna dll-rnai experiments  <cit> .
the first to fifth thoracic appendages, including each exopodite, were shortened .
in arthropods truncation of the distal portion of the appendages is one of the well-known phenotypes observed in dll mutation/knockdown  <cit> .
in addition, a loss of the ocellus and abdominal setae, and a minimized abdominal claw  were observed in dll-dsrna injected embryos, which are also concordant with d. magna <cit> .
although expression of dll in the ocellus has not been reported in any organisms, dll expression was observed in the prospective ocellus region of d. magna <cit> .
the injection of non-d. pulex male-dsrna did not induce any morphological abnormalities in d. pulex embryos .
these data strongly suggest that the observed phenotypes were specifically generated by dll-dsrna, mediating the disruption of endogenous dll mrnas, in d. pulex.
CONCLUSIONS
we developed a microinjection system for rnai in d. pulex.
the optimum conditions for microinjection were a 2% agar plate with 60 mm sucrose dissolved in m <dig> media.
rnai can be induced in early embryos by injecting dsrna into eggs within 1 hour post-ovulation.
the microinjection system is applicable to not only rnai but also creation of transgenic animals, labeling specific target cells, and so on.
thus this technique will contribute d. pulex to become a more appropriate species as model organism for environmental, evolutionary, and developmental biology.

 in both basic and human health research, there is a pressing need to understand how environmental conditions influence gene functions and, in turn, how individuals and populations cope with changing environments. invertebrate species have emerged as models for experimental manipulation because of their unique biological attributes, life cycles, large numbers of offspring, easy maintenance, and sophisticated tools for high-throughput biology and open-source informatics. however, the traits observed in laboratories are likely a small subset of the phenotypic variation that is expressed in natural ecosystems. this may partly explain why to 50% or more genes are without experimentally determined functional annotations, even within the best-characterized genomes. daphnia possess several characteristics that make them valuable for environmental, evolutionary, and developmental genomics research–addressing the added complexity of genome-environment interactions. daphnia are a ubiquitous, and ecologically important member of freshwater lakes and ponds, and have long been used as a sentinel of the integrity of these aquatic ecosystems. more recently with the release of the d. pulex genome, it now serves as a recognized surrogate model for human health research. the d. pulex genome possesses more genes than any previously sequenced animal genome, due to a large orphanage of daphnia genes that likely, allows the organism to respond to its environment. in addition to their short generation time, large brood sizes, and ease of laboratory and field manipulation, daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic studies. at present, however, there are no effective methods for manipulating genes and characterizing gene function, which because of the large gene orphanage limits interspecies extrapolations. rna interference is an evolutionarily conserved post-transcriptional gene silencing mechanism, which is triggered by double-stranded rna in a sequence specific manner. since rnai was first reported in the nematode caenorhabditis elegans by fire et al., it has been used as a powerful tool for the analysis of gene function in many organisms such as zebrafish danio rerio, planarian schmidtea meditteranea, cnidarian hydra magnipapillata, fungus neurospora crassa, fruit-fly drosophila melanogaster and mouse mus musculus. microinjection is one method of introducing dsrna into cells, and this method has been successfully developed for the daphnid species, d. magna. in fact, microinjection techniques enabled the application of not only rnai, but also overexpression of foreign genes and the creation of transgenic individuals. establishing these techniques in d. pulex will extend the resources for environmental, evolutionary, and developmental genomics research for this species by providing needed tools to characterize gene function. distal-less and its homologs dlx genes, which function as homeodomain transcription factors, play one of the major roles in limb development throughout the animal kingdom. reduction of dll activity caused defects of distal leg segments in arthropods including insects, crustaceans, the spider cupiennius salei and the spider mite tetranychus urticae. because of its conserved role in limb development, defects in the expression of the dll gene produce easily recognizable phenotype and ease of evaluation of its phenotype, which is why this endogenous developmental gene was selected as a target in proof-of-principle rnai in d. pulex. the goal of this study was to develop a microinjection system for rnai in d. pulex. culture requirements for isolated embryos were first determined and then microinjection techniques developed for conducting rnai experiments using dll-dsrna.  development of microinjection system there are two major technical problems for microinjection in daphnid species. one is the rapid hardening of egg membrane and the other is a considerable difference between the internal and external osmotic pressures of the egg. the former prevents egg membrane penetration by a needle, and the latter produces leaking of internal contents when the needle is withdrawn. kato et al. determined the proper condition for microinjection in d. magna. eggs were incubated on ice just after ovulation to inhibit the membrane hardening transiently and placed in plastic petri dish with 80 mm sucrose medium dissolved by m culture medium to increase external osmotic pressure. however, these conditions are not suitable for d. pulex possibly because of the difference in egg size and form. to overcome these hurdles, we first examined culture conditions by varying sucrose concentration and culture media, and determining those conditions that allowed embryos isolated from the brood chamber to develop normally. survival was greater in m culture media as compared to dechlorinated freshwater. a sucrose concentration of 40 mm on a 2% agar plate yielded the greatest viability, but this concentration of sucrose was not high enough to counter the internal osmolality and, therefore, not sufficient for injection. taken together, a 2% agar plate covered with 60 mm sucrose dissolved in m media provided the best conditions for microinjection of d. pulex early embryos, and was employed in subsequent experiments. these conditions allowed embryos to be injected within to 60 min of isolation. it is critical that injections occur within the first hour following ovulation, because there is no cytokinesis during this period and dsrna can easily diffuse throughout the egg as it remains a single cell. left column represents types of culture plate and top row shows sucrose concentration of culture medium. sucrose was dissolved in freshwater or m culture medium. dll rnai using microinjection in d. pulex the dsrna was prepared from a 500 bp region derived from the dll gene, and dsrna derived from a 729-bp region of the escherichia coli male gene was developed as a negative control. there is only one dll gene in the d. pulex genome. the dll-dsrna was synthesized from a region that included the homeodomain. red fluorescence was used to judge whether microinjection were successful. results from rnai experiments are summarized in table  only 57% of the embryos injected with dll-dsrna developed compared to % viability of those injected with of male-dsrna, indicating dll-dsrna produced embryonic lethal phenotypes. embryonic lethality caused by loss of dll functions was reported in insects. dll male sequence-specific gene silencing of d. pulex dll we injected 1 μg/μl dsrna with a fluorescent dye into each egg and measured the endogenous dll mrna levels using q-pcr at 24 h after injection to determine if dll-dsrna triggers the degradation of endogenous dll mrnas. the quantity of dll mrna in dll-dsrna-injected embryos was decreased to % of that in male-dsrna-injected control embryos. these results were generally consistent with the result of d. magna where a % reduction in dll was observed. phenotype of dll rnai in d. pulex we confirmed the spatial expression pattern of dll in embryo using whole-mount in situ hybridization. dll was strongly expressed in the distal portion of second antenna, appendages and labrum, and weakly expressed in first antenna, mandible and first maxilla between to 30 h post ovulation. dll-dsrnai injection of isolated embryos produced defects in the dll-expressing organs to various degrees. these defects included a truncation of the second antenna segments that resembled a phenotype observed in d. magna dll-rnai experiments. the first to fifth thoracic appendages, including each exopodite, were shortened. in arthropods truncation of the distal portion of the appendages is one of the well-known phenotypes observed in dll mutation/knockdown. in addition, a loss of the ocellus and abdominal setae, and a minimized abdominal claw were observed in dll-dsrna injected embryos, which are also concordant with d. magna. although expression of dll in the ocellus has not been reported in any organisms, dll expression was observed in the prospective ocellus region of d. magna. the injection of non-d. pulex male-dsrna did not induce any morphological abnormalities in d. pulex embryos. these data strongly suggest that the observed phenotypes were specifically generated by dll-dsrna, mediating the disruption of endogenous dll mrnas, in d. pulex.  we developed a microinjection system for rnai in d. pulex. the optimum conditions for microinjection were a 2% agar plate with 60 mm sucrose dissolved in m media. rnai can be induced in early embryos by injecting dsrna into eggs within 1 hour post-ovulation. the microinjection system is applicable to not only rnai but also creation of transgenic animals, labeling specific target cells, and so on. thus this technique will contribute d. pulex to become a more appropriate species as model organism for environmental, evolutionary, and developmental biology. 
we found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mm sucrose dissolved in m media, providing optimal conditions for microinjections. these techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics. although previous investigations developed an rna interference 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. the ubiquitous, freshwater microcrustacean daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. then, we injected double-stranded rna specific to the distal-less gene, which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. 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. we developed a microinjection system for rnai studies in d. pulex.
 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 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.  we found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mm sucrose dissolved in m media, providing optimal conditions for microinjections. then, we injected double-stranded rna specific to the distal-less gene, 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.  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. daphnia pulexmicroinjectionrna interferencedistal-lessparthenogenetic egg 