Development of an efficient RNA interference method by feeding for the microcrustacean Daphnia
© Schumpert et al. 2015
Received: 22 June 2015
Accepted: 1 October 2015
Published: 7 October 2015
RNA interference (RNAi) is an important molecular tool for analysis of gene function in vivo. Daphnia, a freshwater microcrustacean, is an emerging model organism for studying cellular and molecular processes involved in aging, development, and ecotoxicology especially in the context of environmental variation. However, in spite of the availability of a fully sequenced genome of Daphnia pulex, meaningful mechanistic studies have been hampered by a lack of molecular techniques to alter gene expression. A microinjection method for gene knockdown by RNAi has been described but the need for highly specialized equipment as well as technical expertise limits the wider application of this technique. In addition to being expensive and technically challenging, microinjections can only target genes expressed during embryonic stages, thus making it difficult to achieve effective RNAi in adult organisms.
In our present study we present a bacterial feeding method for RNAi in Daphnia. We used a melanic Daphnia species (Daphnia melanica) that exhibits dark pigmentation to target phenoloxidase, a key enzyme in the biosynthesis of melanin. We demonstrate that our RNAi method results in a striking phenotype and that the phenoloxidase mRNA expression and melanin content, as well as survival following UV insults, are diminished as a result of RNAi.
Overall, our results establish a new method for RNAi in Daphnia that significantly advances further use of Daphnia as a model organism for functional genomics studies. The method we describe is relatively simple and widely applicable for knockdown of a variety of genes in adult organisms.
In order to study gene function in intact organisms, effective techniques to manipulate gene expression to achieve an over-expression or knockdown are essential. RNA interference (RNAi) has revolutionized several fields of biology by making it possible to study loss-of-function effects in various organisms without the time-consuming, laborious genetic manipulations. RNAi is a mechanism in which dsRNA molecules trigger gene silencing in a sequence-specific manner, usually resulting in degradation of the transcript complementary to one strand of the dsRNA ( and the references within). The RNAi pathway is conserved throughout eukaryotes with examples of the RNAi mechanism being used to silence gene expression in numerous model organisms including (but not limiting to) Schizosaccharomyces pombe, Tetrahymena, Drosophila melanogaster, Caenorhabditis elegans, Danio rerio, Xenopus, and Mus musculus . This conserved mechanism of gene silencing has led to exceptional use of reverse genetics methods and has led to a better understanding of molecular pathways at mechanistic levels . Beyond enabling the study of diminished expression of a particular gene leading to advancements in understanding molecular pathways, RNAi has demonstrated a potential for being used in therapeutics for treating human diseases [4, 5].
Daphnia are freshwater microcrustaceans that inhabit inland waters around the world and often are the critical herbivore in aquatic food webs [6, 7]. They have been a major model system in ecology, population genetics, and ecotoxicology for decades due to the ease with which field- and laboratory-based experiments can be conducted. Furthermore, they are cyclic pathenogens, a life cycle that permits genetically diverse natural populations and allows replication of genetically identical individuals through clonal reproduction in the lab . With a fully sequenced genome (D. pulex, ), Daphnia has the potential to be a key model organism in molecular ecology and evolution, and is rapidly emerging as a model organism in non-ecological fields including biology of aging [10–15], and neurobiology [16–20]. Methodology for successful gene knockout using TALEN and CRISPER technologies exists for Daphnia [21, 22]. The U.S. National Institutes of Health list Daphnia as a model organism for biomedical research (http://www.nih.gov/science/models/) citing their extreme phenotypic responses to environmental changes, clonal reproduction, and ecological diversity as advantages in comparison to established biomedical models.
In order to fully realize Daphnia for molecular studies, techniques of experimental genetic manipulation are essential. Currently there is only a single technique described for RNAi in Daphnia [23, 24], which involves microinjecting small dsRNA molecules into the embryos of D. pulex and D. magna. Although this system has allowed for the study of some genes regulating embryonic development, there are several drawbacks and limitations to the microinjection method. The microinjection process is technically challenging, involves specialized equipment, thereby making the protocol expressive, tedious and unlikely to be broadly adopted by researchers interested in Daphnia [3, 25]. Currently, microinjection has been performed successfully only in Daphnia embryos. Therefore, this method limits the number of genes one can target as it is mainly applicable to genes expressed during embryonic development. Thus, there is no reliable RNAi method for studying genes expressed later in Daphnia life span, or for achieving knockdown for a specific duration during the life span.
In C. elegans, the problems associated with microinjection for RNAi were mitigated with the introduction of a new technique that involved feeding the worms with bacteria expressing specific dsRNAs [26–28]. This method for systemic RNAi via feeding has been adapted for multiple organisms including, but not limited to, the house cricket (Acheta domesticus), the lepidopteran pest Spodoptera exigua, the brown apple moth (Epiphyas postwittana), the termite Reticulitermes flavipes, and planarians [29–33]. Since the initial report of the systemic RNAi via feeding method in C. elegans, several different genes have been identified as being essential for systemic RNAi via feeding. One of these essential genes is Sid-1 (systemic RNAi defective), which encodes a transmembrane protein that forms a dsRNA gated channel [34–36]. We searched the recently published Daphnia genome  and found that it contains a Daphnid homologue of Sid-1. We also analyzed the D. pulex genome for the various proteins known to be involved in RNAi  using the online tool PANTHER (Protein Analysis Through Evolutionary Relationships) that allows for identification of various protein homologs based on domain structures and evolution of protein function in various organisms . We determined that the D. pulex genome contains three homologs of Dicer, two homologs of Argonaute, and two homologs of TRBP . Thus, we reasoned that if systemic RNAi via feeding could be achieved for Daphnia, transient gene knockdown experiments would rapidly advance development of this organism as a model system. In our present study, we present a method for efficient RNAi mediated gene knockdown in Daphnia via feeding.
Our study used three different species, D. melanica, D. pulex, and D. pulicaria. Although these taxa have different names, they are part of the same species-complex [38–44], hybridization among them is frequent in the wild [45–47], and experimental crosses do not exhibit reproductive isolation . Therefore, they have limited divergence of their genomes, and can be used in complementary assays of genetic function. They were labeled as distinct species by molecular taxonomists based on mitochondrial divergence [38, 39], though they also have separate ecological niches, with D. melanica specialized for habitats with high UV radiation, and D. pulex and D. pulicaria specialized to small ponds and large lakes with low UV respectively [6, 42, 44, 49]. D. melanica produces melanin as a protective pigment for the high amounts of UV-radiation in its natural habitat [44, 50]. We used the melanin synthesis pathway of D. melanica to develop our RNAi technique, as it provides us with an easily measurable visible phenotype (loss of pigmentation) to assess the effectiveness of gene knockdown. We used D. pulex as a comparison because it is the closest relative to D. melanica that does not exhibit pigmentation .
We selected to target phenoloxidase gene, which encodes an enzyme essential for melanin synthesis in crustaceans, as a target for RNAi [51, 52] based on the easily identifiable phenotype it would produce if RNAi was successful. Our results provide evidence for establishment of an easy, feeding-based RNAi method in Daphnia. We demonstrate reduced phenoloxidase mRNA levels, diminished melanin levels resulting in a dramatic phenotype, and reduced survival in response to UV radiation in Daphnia that are fed on bacteria that express a dsRNA specific for phenoloxidase. This is the first demonstration that systemic RNAi is possible in Daphnia.
Daphnia pulex was isolated from waterbodies in southwest Michigan in 2008 and have since been cultured in the lab. Daphnia melanica were isolated from high altitude alpine lakes in the Sierra Nevada region in eastern California. The isolate used was known as “Sierra”, and ND5 mitochondrial gene sequencing confirmed the D. melanica species identity [50, 53]. D. pulex species identity was determined by ldh allozyme characterization (designated “slow-slow”) as described previously [54, 55]. D. pulex was maintained at a temperature of 20° C with a photoperiod of 12:12 light:dark in a Percival growth chamber. D. melanica were maintained at a temperature of 15° C with a photoperiod of 16:8 light:dark. All Daphnia were maintained at a concentration of 3 to 5 animals per 250 ml beaker in 100 ml of filtered lake water until experimentation. Lake water was obtained from public access Lake Murray in central South Carolina and was filtered (1 μm) before use. Young newborn Daphnia were transferred to a new beaker with fresh water on alternate days. D. pulex cultures were fed every day with vitamin-supplemented algae Ankistrodesmus falcatus at a concentration of 20,000 cells/ml. D. melanica cultures were fed 20,000 cells/ml of Ankistrodesmus falcatus on alternate days.
Vectors and feeding system
BL21(DE3): ompT hsdSB (rB-mB-) gal dcm (Novagen) - a strain deficient in lon and ompT proteases and used for efficient production of recombinant proteins.
HT115 (DE3) (W3110, rnc14::DTn10 (Addgene, ) - a strain deficient in RNase III and used for efficient production of dsRNAs.
E. coli cells that are (DE3) contain an integrated T7 RNA polymerase ORF under the control of a LacUV5 promoter the cultures can be induced with 2 mM IPTG to produce T7 RNA polymerase. This leads to the production of dsRNA from the PCR product cloned between the two T7 promoters. We also used the plasmid construct L4417, which contains the 5′ half (750 bp) of the GFP ORF cloned between the two T7 promoters (to be used as a negative control in the experiments as it does not have a natural target RNA in Daphnia). All the recombinant plasmids that we generated would produce a dsRNA product of about 800 bp in the bacterial host that expresses T7 RNA polymerase. Both L4440 and L4417 were obtained from Addgene (L4440 Plasmid #: 1654, L4417 Plasmid #1649- both gifts from Dr. A. Fire to Addgene).
Target genes and plasmid constructs
In our current study, we targeted phenoloxidase transcripts for degradation by RNAi. The PCR primers were designed based on D. pulex phenoloxidase (NCBI_GNO_8300047) sequence. We generated several constructs using L4440 plasmid vector to generate dsRNA corresponding to different regions of our target transcripts (Fig. 1c and d). Note for each construct the PCR product was cloned into pGEMT Easy (Promega) and the sequence was confirmed. The following primers were used to generate the PCR products using either the genomic DNA or cDNA as a template. Appropriate restriction enzyme sites were engineered into the 5′ends of both PCR primers for sub-cloning from pGEMT Easy to L4440. Primers PO245 and GAPDH were used only in qPCR tests of gene expression levels.
P1: (gDNA template) Forward: CACCATGTCAGATTTGCAGC
P2: (gDNA template) Forward: AATTCTTGCCGATCAAGGTG
P3: (cDNA template) Forward: GCGTGGCAGGTTATTTTCAT
PO245 (qPCR): Forward: CCATTCAGTCCTAAACCGGA
GFP: Forward: GCCCGAAGGTTATGTACAGG
GAPDH: Forward: TTATCACCTCCTCAACTTC
The following abbreviations denote which targeting vector was in the bacteria administered as feed to Daphnia: EV: Empty Vector L4440, GFP: GFP Control (L4417), P1: 5′ half of phenoloxidase gene amplified from genomic DNA, P2: 3′ half of phenoloxidase gene amplified from genomic DNA, P3: internal region of phenoloxidase mRNA amplified from cDNA, NM: Nonmelanic D. pulex (Clone: RW20), served as a control.
RNAi feeding protocol
Ten Daphnia aged 4–5 weeks selected for experimentation were placed in 100 ml of filtered lakewater in a 250 ml beaker. E. coli strain BL21(DE3) bacteria transformed with the plasmid of choice (L4440 with one of the inserts indicated in Fig. 1) were grown overnight in Luria Broth (LB) with 2 mM IPTG to induce the expression of T7 RNA polymerase and the dsRNA corresponding to the cloned PCR product. The OD600 of the overnight cultures was measured, and bacteria from 2.8 OD600 units of overnight culture were pelleted (usually about 1 ml). The pelleted bacteria were resuspended in 1 ml of filtered lake water and dispensed directly into the beakers, which contained Daphnia, thereby diluting 1 ml of resuspended bacteria in 100 ml of filtered lake water. This corresponds to a final OD600 of 0.028 or about 2.4 × 107 E. coli cells in 100 ml. This same procedure was repeated for 10 days, with the Daphnia also being fed algae, Ankistrodesmus falcatus, at a concentration of 20,000 cells/ml each day for D. pulex and and on alternate days for D. melanica. The water being changed every other day for D. melanica or every day for D. pulex. New bacterial culture in fresh LB was prepared for feeding on each feeding day, and fresh algae (20,000 cells/ml) added after addition of bacteria to water, thus Daphnia were always fed with a mixture of algae and bacteria. During RNAi feeding regimen, the same photoperiods as stated under Daphnia cultures section were maintained for each species.
UV treatment of D. melanica
For the experiments involving the knockdown of phenoloxidase in D. melanica, on the tenth and eleventh days of the bacterial feeding regimen, Daphnia were exposed to UV radiation by using a transilluminator with 312 nm UVB emission. Daphnia in 250 ml beakers (in 100 ml of water) were placed on the transilluminator. Beakers were arranged such that individual Daphnia were an average 10 cm from the UV source. Daphnia were exposed to UV for 5 min, and then returned to the Percival chamber. On the day 12 after beginning of bacterial feeding, the Daphnia were sacrificed and assayed for visual phenotypes and harvested to assay melanin content or isolate total RNA. This UV treatment allowed for a rigorous assessment of the RNAi targeting phenoloxidase via feeding method. D. melanica, if stressed, can stop production of melanin synthesis and this may result in false positive scoring of the loss of pigmentation phenotype since the presence of bacteria in water may induce some level of stress. By exposing Daphnia to UV radiation, the synthesis of melanin can be induced, over-riding any probable down regulation due to stress.
The assay was performed as per Hebert and Emery . The body length of Daphnia to be analyzed via the Melanin Assay for their melanin content was measured and then Daphnia were placed in 50 μl of 5 M NaOH and incubated at 40 °C for 4 days. The melanin content of the resulting solution was determined by measuring optical density at 420 nm with a plate reader. We performed a melanin standard curve using commercially available bovine melanin. By generating a standard curve, we were able to convert our OD420 values into micrograms of melanin per millimeter of Daphnia [53, 58]. We also measured the melanin content of a nonmelanic Daphnia species as a negative control.
UV sensitivity and survival assay
We tested Daphnia’s ability to survive UV exposure following the loss of pigmentation in response to feeding on bacteria expressing the phenoloxidase dsRNA. Daphnia in control and treatment groups were fed bacteria as described above for 10 days. On the eleventh day, Daphnia were subjected to UV radiation (10 min of UV radiation on the transillimuminator). We subjected the Daphnia a second time to this UV dosage on day 12 and on the thirteenth day examined the viability in control and treatment groups.
Reverse transcriptase (RT)-PCR
Total RNA was isolated using RNAzol B reagent (TelTest) from 6 to 10 Daphnia following a bacterial RNAi feeding regimen for 10 days. Prior to RNA isolation, for the Daphnia fed on bacteria expressing any of the described dsRNAs, the entire gut was removed from each individual to avoid contamination from the bacteria in the gut that contain the dsRNA. Daphnia were collected in a microcentrifuge tube, rinsed once with 1 ml of PBS, and were homogenized in 0.8 ml of RNAzol B. Total RNA was isolated as per the supplied protocol. cDNA was synthesized using random hexamer primers, 1 μg total RNA, 10–20 units M-MuLV reverse transcriptase, 500 μM dNTPs, 40 units RNase Inhibitor RNasin (Promega) in appropriate reaction buffer. For each PCR reaction, 2 μl (1/10th of total) cDNA was used with 50 pmoles each of the forward and reverse primers designed to amplify phenoloxidase PCR product using the Promega GoTaq PCR kit. The following conditions were used for PCR: 95° C for 5 min (initial denaturation), denaturation at 95° C for 30 s, annealing at 58° C for 30 s, extension at 72° C for 30 s for 27 cycles in order to stay within linear range of amplification. The linear range was determined by varying cycle numbers and performing a densitometric analysis of the amplified product. PCR products were separated on a 1 % agarose gel.
Real time PCR
We first determined the efficiency of the real time PCR reactions with serial dilutions of all cDNAs. Every reaction was performed in triplicate in a total volume of 20 μl. This included 4 μl cDNA, 250nM phenoloxidase or GAPDH primers, and SensiFast Supermix (BioLine). GAPDH was used for normalization. Phenoloxidase and GFP primers were validated by running serial dilutions with a template of known quality and the efficiency of the reactions were determined to be greater than 98 %. GAPDH primers were previously validated . In addition, we also ensured that the GAPDH mRNA levels did not change during our treatments prior to performing experimental qRT-PCR for analyzing the phenoloxidase mRNA levels. In all samples analyzed (EV, GFP, P1, P2, P3 and D. pulex), the GAPDH threshold cycle (Ct) value was determined to be 24.5 +/− 0.10. This initial standardization was important because GAPDH mRNA levels were reported to change under certain treatment conditions in Daphnia . All reactions were run on a BioRad CFX96 Real Time System C1000 Thermal cycler machine with the following conditions: 95° C for 30 s, 95° C for 5 s, 58° C for 5 s for phenoloxidase and 54 C for 5 s for GFP (the last three steps repeated for 50 cycles), 65° C for 5 s, and then 95° C for 5 s. We analyzed our data using the Bio-Rad CFX Manager Software with the 2-ΔΔCt method. Note that three independent RNA isolations were used from three independent groups of Daphnia to serve as biological replicates.
To determine statistical significance, a two tailed Student’s T-test assuming equal variance or chi square analysis was performed. Each figure legend denotes p values as set forth by brackets and special F characters. Note that our alpha level was p = 0.05.
Results and discussion
Selection of target
In order to establish an RNAi method for Daphnia via feeding, we selected target genes that would result in easily identifiable visible phenotypes. A deficiency of phenoloxidase enzyme would result in a reduction of melanin pigment, thus producing a visible loss of pigmentation in D. melanica. As shown in Fig. 1a, we selected to target phenoloxidase gene based on its involvement in several essential steps in the melanin synthesis pathway [52, 60]. In order to test dsRNAs corresponding to three different regions of phenoloxidase gene for their effectiveness, we used three PCR primer pairs to amplify the indicated regions for sub-cloning into the plasmid vector L4440. As is shown in Fig. 1b, two regions from the phenoloxidase gene (with primers binding in introns of the gene) corresponding to the 5′ region (P1) and 3′ region (P2) were selected for PCR amplification. Another primer pair was used with cDNA as a template (therefore no intronic regions would be present), which corresponds to a central region of the phenoloxidase transcript (named P3, Fig. 1b). A different primer pair was used for real time PCR in order to measure changes in phenoloxidase transcript levels (termed PO245, Fig. 1b) after RNAi feeding regimen.
Optimization of the RNAi feeding protocol
We selected the concentration of bacteria in feed as well as the duration of the RNAi feeding regimen based on our initial experiments to determine the effective dose that would result in the least lethality and a quick phenotypic response (loss of pigmentation in D. melanica). As seen in Table 1, we selected the bacterial dose that resulted in a phenotypic change in 10 days with the least lethality (which was 2.4 x 107 bacterial cells/day/100 ml lake water for 10 days). Note that although this concentration and duration was optimal for targeting phenoloxidase in D. melanica, the optimal concentration and duration may have to be determined for specific species and clones of Daphnia as well as for different target genes using the RNAi via feeding system. The method used for this initial optimization would be similar to one we outline here.
GFP dsRNA can be detected in Daphnia after being fed on bacteria expressing GFP dsRNA
Phenotypic change in D. melanica fed on bacteria expressing phenoloxidase dsRNA
During the course of the phenoloxidase RNAi feeding regimen, some progeny organisms born to treated mothers (about 10–15 %) exhibited clear carapaces, while others in the same clutch were melanic. Due to the unpredictability of the clear offspring being born over the course of the experiment, we did not analyze steady state levels of phenoloxidase mRNA in the progeny with clear carapaces. Once removed from the RNAi feeding regimen, the offspring with clear carapaces always returned to normal melanic pigmentation within the next 4–6 days. It is worth a note that the survival as well as the total offspring number was not affected by the RNAi feeding regimen.
Phenoloxidase transcript levels are diminished in Daphnia fed on bacteria expressing Phenoloxidase dsRNA
Melanin levels are diminished in Daphnia fed on bacteria expressing phenoloxidase dsRNA
Daphnia fed on bacteria expressing phenoloxidase dsRNA are sensitized to UV radiation
Delivery of dsRNA by feeding is a convenient and reliable RNAi method
In order to knock down specific gene expression, several methods for the delivery of dsRNA or siRNA have been used in recent years. Among these, RNAi via direct injection of a dsRNA solution is a simple method that works in larger insects due to the easy protocol. It is effective in knocking down expression of target genes in invertebrates such as the cricket G. bimaculatus, the mosquito Aedes aegypti , the German cockroach Blattella germanica , and the silkworm larvae Bombyx mori . For a small freshwater microcrustacean such as Daphnia, it is difficult to achieve RNAi via injection in adult organisms as injection may result in high mortality since Daphnia will lose viability rapidly if not kept immersed in cool water. Thus, delivering dsRNA by feeding offers several advantages over direct injection of dsRNA method, as it is less labor-intensive, less expensive, and is also applicable for screening a large number of essential Daphnia genes because of its simplicity. Our study also suggests for the first time that RNAi in Daphnia is systemic, and is the first report of phenoloxidase knockdown using RNAi which not only produced significant reduction in mRNA and melanin levels but it also resulted in a marked increase in lethality in response to UV exposure.
It is worth noting that melanin synthesis was restored in D. melanica at about 7 days after bacterial feeding was discontinued, thereby demonstrating that the RNAi was transient and thus this method holds a potential to test the effect of gene knockdown at selected time points during the life span. This may be a very useful feature, especially for aging and longevity research to study the contribution of specific genes in an age-dependent manner. As there was no apparent effect of RNAi bacterial feeding regimen on reproduction in any species we examined, the technique would be widely applicable to most genes. In this regard, it is worth noting that we attempted a knockdown of distal-less and eyeless by the maternal feeding method. A knockdown of distal-less produced no viable progeny, which could be a sign of embryonic lethality specifically due to achieving efficient RNAi for distal-less. Drosophila distal-less null mutants die as embryos due to defects in development of sensory organs . The knockdown of eyeless was effective and produced a deformed eye phenotype in the progeny as early as in the first clutch after 4–5 days of RNAi feeding regimen. However the organisms exhibiting deformed eyes did not survive for more than a few hours and no RNA could be isolated for analysis of eyeless mRNA levels. Thus, although the preliminary data suggests that targeting mRNAs expressed in developing embryos via feeding the mothers is possible, further experiments are essential to establish the experimental conditions and validation of RNAi effects.
Thus, we present a fast and effective method to achieve gene-specific knockdown in adult organisms as well as developing Daphnia embryos that holds a tremendous potential to become a mainstream method in various types of biological studies that use Daphnia as a model organism.
We describe a new method to achieve gene specific knockdown by RNAi in Daphnia via feeding. By using E. coli cells that express gene-specific dsRNAs as a food additive for adult Daphnia, we can achieve an efficient RNAi for genes that are expressed in adult tissues. This method provides a powerful tool for genetic manipulation of this important model organism for environmental, evolutionary, as well as developmental genomics.
The authors would like to thank Dr. Michael Pfrender (Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA) for kindly providing us D. melanica. This work was supported by National Institutes of Health grant 1R01AG037969-01 awarded to RCP and JLD. The funding agency had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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