Genomic Integration and Germline Transmission of Plasmid Injected into Crustacean Daphnia magna Eggs

The water flea, Daphnia, has been the subject of study in ecology, evolution, and environmental sciences for decades. Over the last few years, expressed sequence tags and a genome sequence have been determined. In addition, functional approaches of overexpression and gene silencing based on microinjection of RNAs into eggs have been established. However, the transient nature of these approaches prevents us from analyzing gene functions in later stages of development. To overcome this limitation, transgenesis would become a key tool. Here we report establishment of a transgenic line using microinjection of plasmid into Daphnia magna eggs. The green fluorescent protein (GFP) gene fused with the D. magna histone H2B gene under the control of a promoter/enhancer region of the elongation factor 1α-1 (EF1α-1) gene, EF1α-1::H2B-GFP, was used as a reporter providing high resolution visualization of active chromatin. Transgenic lines were obtained from 0.67% of the total fertile adults that survived the injections. One of the transgenic animals, which exhibited fluorescence in the nuclei of cells during embryogenesis and oogenesis, had two copies of EF1α-1::H2B-GFP in a head-to-tail array. This is the first report of a transgenesis technique in Daphnia and, together with emerging genome sequences, will be useful for advancing knowledge of the molecular biology of Daphnia.     

Cloning and analysis of a cDNA encoding a two-domain hemoglobin chain from the water flea Daphnia magna

A cDNA encoding a two-domain hemoglobin (Hb) chain of Daphnia magna was cloned and its nucleotide (nt) sequence of 1261 bp was determined. The nt sequence contained 74 bp of the leader sequence, 1047 bp of an open reading frame (ORF), and 119 bp of the 3′-untranslated region (UTR), excluding the polyadenylation tail. A sequence, AATACA, located 24 bp upstream from the polyA sequence was considered to be a polyadenylation signal. cDNA-derived amino acid (aa) sequence revealed that D. magna Hb chain is synthesized as a secretory precursor with a signal peptide of 18 aa. Mature D. magna Hb chain consists of 330-aa residues with a calculated molecular weight of 36 227, which is composed of two large repeated domains, domain 1 and 2. Several key aa that are invariant in all or most of other Hb and required for functional heme-binding are conserved in each of the two domains. The N-terminal extension (pre-A segment) of domain 1 was unusually long and contained an unusual threonine-rich sequence. The homology between the aa sequences of the two domains (24% identity) was much lower than that observed in other two-domain Hb chains from clams or nematode. Hb mRNA level in D. magna reared under low oxygen concentration was more than 12 times higher than that in D. magna reared with sufficient aeration, indicating that the expression of Hb gene is regulated by mRNA level.     

Assimilation of E. coli cells by Daphnia magna on the whole organism level

Consumption of E. coli cells by Daphnia magna was studied. It was found that this organism not only ingested E. coli cells but digested them as demonstrated by the release of ¹⁴CO₂ originating from E. coli grown on ¹⁴C-glucose, and by the transfer of the radioactive label from parental Daphnia to their progenies. In addition the effect of antibiotics on the consumption of E. coli cells by Daphnia magna was studied. In long incubation times, antibiotics inhibited bacterial uptake by Daphnia. The microflora isolated from Daphnia was found to be capable of causing leakage of enzymes out of E. coli cells thus playing at least a partial role in the digestion of E. coli cells by Daphnia.     

Conserved Mechanisms for Germ Cell-Specific Localization of nanos3 Transcripts in Teleost Species with Aquaculture Significance

The importance of the aquaculture production is increasing with the declining global fish stocks, but early sexual maturation in several farmed species reduces muscle growth and quality, and escapees could have a negative impact on wild populations. A possible solution to these problems is the production of sterile fish by ablation of the embryonic primordial germ cells (PGCs), a technique developed in zebrafish. Cell-specific regulation of mRNA stability is crucial for proper specification of the germ cell lineage and commonly involves microRNA (miRNA)-mediated degradation of targeted mRNAs in somatic cells. This study reports on the functional roles of conserved motifs in the 3′ untranslated region (UTR) of the miRNA target gene nanos3 identified in Atlantic cod, Atlantic salmon, and zebrafish. The 3′UTR of cod nanos3 was sufficient for targeting the expression of green fluorescent protein (GFP) to the presumptive PGCs in injected embryos of the three phylogenetically distant species. 3′UTR elements of importance for PGC-specific expression were further examined by fusing truncated 3′UTR variants of cod nanos3 to GFP followed by injections in zebrafish embryos. The expression patterns of the GFP constructs in PGCs and somatic cells suggested that the proximal U-rich region is responsible for the PGC-specific stabilization of the endogenous nanos3 mRNA. Morpholino-mediated downregulation of the RNA-binding protein Dead end (DnD), a PGC-specific inhibitor of miRNA action, abolished the fluorescence of the PGCs in cod and zebrafish embryos, suggesting a conserved DnD-dependent mechanism for germ cell survival and migration.     

Prevalence of kairomone-induced diapause in Daphnia magna from habitats with and without fish

The prevalence of the diapause response of the freshwater crustacean Daphnia magna to chemical cues on fish predation was evaluated in 35 clones originating from 14 European water bodies—inhabited or not inhabited by fish. Clonal lineages of experimental animals were cultured for 4 weeks in the presence or absence of water to which an extract of faeces of crucian carps (Carassius carassius), that were fed with Daphnia, was added. The proportion of females producing diapausing forms during the experimental period was used as a measure of the diapause response. A positive diapause response to fish predation cues was observed in 43% of investigated clones that originated from 71% of the water bodies tested. This indicated that the diapause response to fish scent is a common phenomenon in Daphnia magna from various locations in Europe. Surprisingly, no signs of a stronger diapause reaction to the tested cues were found in Daphnia originating from water bodies inhabited by fish compared with those from fish-free habitats.     

Water fleas require microbiota for survival,growth and reproduction

Microbiota have diverse roles in the functioning of their hosts; experiments using model organisms have enabled investigations into these functions. In the model crustacean Daphnia, little knowledge exists about the effect of microbiota on host well being. We assessed the effect of microbiota on Daphnia magna by experimentally depriving animals of their microbiota and comparing their growth, survival and fecundity to that of their bacteria-bearing counterparts. We tested Daphnia coming from both lab-reared parthenogenetic eggs of a single genotype and from genetically diverse field-collected resting eggs. We showed that bacteria-free hosts are smaller, less fecund and have higher mortality than those with microbiota. We also manipulated the association by exposing bacteria-free Daphnia to a single bacterial strain of Aeromonas sp., and to laboratory environmental bacteria. These experiments further demonstrated that the Daphnia–microbiota system is amenable to manipulation under various experimental conditions. The results of this study have implications for studies of D. magna in ecotoxicology, ecology and environmental genomics.     

Identification of proteins specifically interacting with YB-1 mRNA 3′ UTR and the effect of hnRNP Q on YB-1 mRNA translation

In this study, proteins specifically interacting with the 3′ untranslated region (UTR) of mRNA of the multifunctional Y-box-binding protein 1 (YB-1) were identified. One of these, hnRNP Q, was shown to specifically interact with the regulatory element (RE) in YB-1 mRNA 3′ UTR and to inhibit translation of this mRNA. Its binding to the RE was accompanied by displacement from this element of the poly(A)-binding protein (PABP), a positive regulator of YB-1 mRNA translation, and by enhanced binding of the negative YB-1 mRNA translation regulator — YB-1 itself.     

CRISPR/Cas-Mediated Targeted Mutagenesis in Daphnia magna

The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs) is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18–47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.     

Molecular genetic identification and phylogeny of Daphnia species (Crustacea, Cladocera) from water bodies of the Lake Chany basin

The data on the molecular genetic identification of Daphnia species from the water bodies of the Lake Chany basin are presented. Phylogenetic relationships between these species have been established. The fragments of the mitochondrial DNA genes were used as genetic markers. According to the data obtained, the water bodies examined were inhabited by five Daphnia species, including Daphnia (Daphnia) galeata Sars, D. (D.) longispina O. F. Müller, D. (D.) curvirostris Eylmann, D. (D.) pulex Leydig, and D. (Ctenodaphnia) magna Straus. In addition, longispina a-like individuals that form a separate phylogenetic lineage was identified.     

Identification of short untranslated regions that sufficiently enhance translation in high-quality wheat germ extract

High-quality wheat germ extract (hqWGE) is very useful for the high-yield production of various types of protein. The most important key to high productivity is the design of mRNA templates. Although the design has been refined for straightforward and efficient translation in hqWGE, there is still room for improvement in untranslated regions (UTRs), especially the 3′ UTR length, because a long, cumbersome 3′ UTR is commonly used for translation enhancement. Here we examined some short viral 3′ cap-independent translation enhancers (3′ CITEs) to identify effective ones for efficient translation in hqWGE. We then combined the most effective 3′ CITE and a 5′ enhancer to further increase the translation efficiency. mRNA with the optimal short 3′ and 5′ UTRs, both of whose length was less than 150 nt, exhibited a productivity of 1.4 mg/mL in prolonged large-scale protein synthesis in hqWGE, which was comparable to that of control mRNA with a commonly-used long 3′ UTR (∼1200 nt).