Epipodite and fat cells as sites of hemoglobin synthesis in the branchiopod crustacean Daphnia magna

In contrast to the malacostracan crustaceans that use hemocyanin as the oxygen carrier, a number of branchiopod crustaceans, such as the water flea Daphnia magna, utilize hemoglobin (Hb) as the respiratory protein. By means of in situ hybridization (ISH) techniques with subsequent signal amplification using catalyzed reporter deposition, sites of Hb synthesis were localized in Daphnia magna. Based on a previously reported Hb-cDNA sequence, a specific ISH probe was designed and hybridized with the Hb-mRNA in histological sections of adult D. magna. The detection of Hb-mRNA was tissue specific and revealed that Hb is synthesized in fat cells, which play a role in fat and glycogen metabolism, and in epithelial cells of the epipodites, which are involved in osmoregulation. Sites of Hb synthesis have been identified in several invertebrate phyla, including Annelida and Nematoda. However, this is the first example in the class Crustacea, and only the second in the phylum Arthropoda. Accepted: 6 September 1999     

Juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna

Daphnids (Daphnia magna) utilize cyclic parthenogenesis as a reproductive strategy. During periods of abundant resources, these organisms reproduce asexually. In response to environmental cues that signal the onset of environmental adversity, daphnids produce males and reproduce sexually. The environmental cues that stimulate the sexual reproductive phase are well known; however, the endocrine signals that transduce these environmental cues remain unknown. The present study was undertaken to test the hypothesis that the crustacean juvenoid hormone, methyl farnesoate, is a male sex determinant in this species. Continuous exposure to aqueous concentrations of methyl farnesoate greater than approximately 30 nM stimulated a concentration-dependent production of male-containing broods of organisms. Short-term exposures to methyl farnesoate during periods of egg and embryo maturation revealed that male sex determination occurred during a specific 12-hour period of ovarian egg development. Exposure of eggs to 400 nM methyl farnesoate during this sensitive developmental period resulted in the production of all-male broods of offspring, while exposure to concentrations as low as 52 nM produced mixed broods of males and females. This active concentration range of methyl farnesoate is consistent with levels measured in the hemolymph of some decapod crustaceans. These results demonstrate that methyl farnesoate is capable of programming daphnid embryos to develop into males and is likely the endocrine factor responsible for initiating the sexual reproductive phase in these organisms.     

Environmental antiecdysteroids alter embryo development in the crustacean Daphnia magna

The role of ecdysteroids in crustacean embryo development and the susceptibility of the developing embryo to the antiecdysteroidal properties of an environmental chemical were evaluated. The agricultural fungicide fenarimol was shown to exhibit antiecdysteroidal activity to the crustacean Daphnia magna by lowering endogenous ecdysone levels and delaying molting in a concentration-dependent fashion that was mitigated by co-exposure to exogenous 20-hydroxyecdysone. Exposure of either gravid maternal organisms or isolated embryos to fenarimol resulted in embryo abnormalities ranging from early partial developmental arrest to incomplete development of antennae and shell spines. Developmental abnormalities were associated with suppressed ecdysone levels in the embryos and the abnormalities could be prevented by co-exposure to 20-hydroxyecdysone. Developmental abnormalities caused by the antiecdysteroid were associated with reduced fecundity of the parental organisms. These results demonstrate that ecdysteroids are critical to normal crustacean embryo development and environmental antiecdysteroids can disrupt normal embryo development and compromise the production of viable offspring. Antiecdysteroidal activity may provide a means by which environmental chemicals impact crustacean species while not affecting vertebrates.     

Humic substances influence sodium metabolism in the freshwater crustacean Daphnia magna

Humic substances are ubiquitous components of natural waters with important roles in alleviating metal toxicity to aquatic organisms. Recent literature reports suggest that humic substances may also exert direct influences on biota. This study investigated the influence of two commercially available humic substances on sodium metabolism in Daphnia magna, a hyperregulating freshwater crustacean. Environmentally realistic levels of Suwannee River natural organic matter (SRN) and Aldrich humic acid (AHA) significantly enhanced sodium transport. This effect was described as an uncompetitive stimulation of sodium influx, as characterised by an increased maximal sodium transport rate (Jmax), accompanied by a decreased uptake affinity (increased Km). SRN exposure also significantly promoted the unidirectional loss of sodium from the daphnids to the water, an effect not observed in the presence of AHA. A 24-h preexposure to AHA before influx measurement had no effect on AHA-induced stimulation of sodium influx. Conversely, 24-h preexposure to SRN resulted in influx values that returned to control (humic-free) levels. Whole-body sodium levels reduced by SRN exposure were also restored to control levels following 24-h SRN preexposure. The significance and potential mechanisms of these actions are discussed, and the toxicological implications of these findings are assessed.     

Development of a bicistronic expression system in the branchiopod crustacean Daphnia magna

The viral 2A peptides have recently been used for bicistronic expression in various organisms. In this system, a single mRNA that codes for two proteins flanking the 2A peptide can be translated simultaneously into each protein by ribosomal skipping at this peptide sequence. Here, we tested the function of the Thosea asigna insect virus 2A (T2A) peptide in the branchiopod crustacean Daphnia magna—an emerging model of evolutionary developmental biology. First, we used transgenic Daphnia that expresses a potential bicistronic RNA containing mCherry and histone H2B‐ green fluorescent protein (GFP) open reading frames upstream and downstream of the T2A sequence, respectively. Microscopic observation revealed difference of localization of the two proteins in the cell, homogenous distribution of mCherry and nuclear localization of H2B‐GFP. Second, we changed localization of mCherry from cytoplasm to plasma membrane by attachment of a consensus myristoylation motif in the bicistronic reporter. RNA that codes for this new bicistronic reporter was injected into eggs. At gastrulation stage, we found spectrally distinct fluorescence with enough intensity and resolution to detect membrane localized mCherry and nuclear GFP. These results indicate that the T2A peptide functions in D. magna and T2A‐mediated bicistronic expression would be a promising tool for evo‐devo studies of this species.     

Optimization of mRNA design for protein expression in the crustacean Daphnia magna

The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3′ UTR failed to induce fluorescence. To assess reporter expression, the length of the 3′ UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3′ end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3′ UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3′ UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3′ UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.     

Development of an RNA interference method in the cladoceran crustacean Daphnia magna

Daphnids are small crustaceans ubiquitous in fresh water; they have been a subject of study in ecology, evolution, and environmental sciences for decades. To understand data accumulated in daphnid biology at the molecular level, expressed sequence tags and a genome sequence have been determined. However, these discoveries lead to the problem of how to understand the functions of newly discovered genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a useful tool to achieve specific gene silencing in nontransformable species. Hence, we established a technique to inject exogenous materials into ovulated eggs and developed a dsRNA-based RNAi method for Daphnia magna. Eggs were collected just after ovulation and injected with dsRNA specific to the Distal-less (Dll) gene, which functions in appendage development in invertebrates and vertebrates. We found that the dsRNA successfully triggered the degradation of Dll mRNAs, which induced the truncation of the second antenna in a dose-dependent manner. This effect was sequence specific in that: (1) an unrelated dsRNA did not induce any morphological abnormalities and (2) two non-overlapping Dll dsRNAs generated the same phenotype. This is the first report of an RNAi technique in D. magna and, together with the emerging genome sequences, will be useful for advancing knowledge of the molecular biology of daphnids.     

Evidence for a cost of immunity when the crustacean Daphnia magna is exposed to the bacterial pathogen Pasteuria ramosa

The deployment of the immune system has the obvious potential to ameliorate infection outcomes, but immune responses can also harm hosts by either damaging host tissues or monopolizing resources, leading to enhanced mortality. To gain insight into such a 'cost of immunity' when the crustacean Daphnia magna is challenged with the bacterium Pasteuria ramosa, we measured survivorship among hosts that resisted infection following exposure to various strains and doses of the parasite. In the first of two experiments, these exposures were: single exposures with relatively non-aggressive strains, double exposures with non-aggressive strains, and exposure to aggressive strains. Mortality increased across this gradient of exposure. In a second experiment, we varied the dose of the most aggressive P. ramosa strain and found that resisting infection when a large dose was applied resulted in greater mortality than when a medium or low dose was applied. Assuming that resistance is accomplished with an immune response, and that more aggressive parasites and/or larger doses of parasites are more immunostimulatory, these data are compatible with a cost of immunity. Indeed, in terms of survival, resisting parasites can be more harmful than infection.     

Sequence divergence and expression of a transformer gene in the branchiopod crustacean,Daphnia magna

Environmental decline triggers a switch in reproductive strategy of Daphnia magna from asexual to sexual reproduction; however, the molecular basis of such environmental sex determination remains largely unknown. In insects most closely related to branchiopod crustaceans, orthologs of the Drosophila transformer gene such as dipteran transformer (tra), honeybee feminizer (fem) and complementary sex determiner (csd) function as sex determining factors. Therefore, we cloned a D. magna transformer gene (dmagtra) and analyzed its expression. The predicted amino acid sequence has 380 amino acids including an arginine–serine-rich region, which is characteristic of insect orthologs of Tra. Residues 180 to 205 are highly conserved with known Tra orthologs. The domain organization of DmagTra is distinct from known Tra orthologs; moreover, dmagtra does not display any detectable sexual dimorphic differences in expression or splicing patterns. We infer from these results that dmagtra may not be responsible for sex determination in D. magna.     

Detection of triclosan using 2-domain hemoglobin promoter of Daphnia magna

Triclosan has been widely used as an antimicrobial agent. However, triclosan was found to cause toxicity, including muscle contraction disturbances, carcinogenesis, and endocrine disorders. In addition, it was found to affect central nervous system function adversely and even have ototoxic effects. Conventional methods for detecting such triclosan can be performed easily. However, the conventional detection methods are inadequate in precisely reflecting the impact of toxic substances on stressed organisms. Therefore, a test model for the toxic environment at the molecular level through the organism is needed. From that point of view, Daphnia magna is being used as a ubiquitous model. D. magna has the advantages of easy cultivation, a short lifespan and high reproductive capacity, and high sensitivity to chemicals. Therefore, the protein expression pattern of D. magna that appear in response to chemicals can be utilized as biomarkers for detecting specific chemicals. In this study, we characterized the proteomic response of D. magna following triclosan exposure via two-dimensional (2D) gel electrophoresis. As a result, we confirmed that triclosan exposure completely suppressed D. magna 2-domain hemoglobin protein and evaluated this protein as a biomarker for triclosan detection. We constructed the HeLa cells in which the GFP gene was controlled by D. magna 2-domain hemoglobin promoter, which under normal conditions, expressed GFP, but upon triclosan exposure, suppressed GFP expression. Consequently, we consider that the HeLa cells containing the pBABE-HBF3-GFP plasmid developed in this study can be used as novel biomarkers for triclosan detection.     

Resistance to a bacterial parasite in the crustacean Daphnia magna shows Mendelian segregation with dominance

The influence of host and parasite genetic background on infection outcome is a topic of great interest because of its pertinence to theoretical issues in evolutionary biology. In the present study, we use a classical genetics approach to examine the mode of inheritance of infection outcome in the crustacean Daphnia magna when exposed to the bacterial parasite Pasteuria ramosa. In contrast to previous studies in this system, we use a clone of P. ramosa, not field isolates, which allows for a more definitive interpretation of results. We test parental, F1, F2, backcross and selfed parental clones (total 284 genotypes) for susceptibility against a clone of P. ramosa using two different methods, infection trials and the recently developed attachment test. We find that D. magna clones reliably exhibit either complete resistance or complete susceptibility to P. ramosa clone C1 and that resistance is dominant, and inherited in a pattern consistent with Mendelian segregation of a single-locus with two alleles. The finding of a single host locus controlling susceptibility to P. ramosa suggests that the previously observed genotype-genotype interactions in this system have a simple genetic basis. This has important implications for the outcome of host-parasite co-evolution. Our results add to the growing body of evidence that resistance to parasites in invertebrates is mostly coded by one or few loci with dominance.     

Organization and repression by juvenile hormone of a vitellogenin gene cluster in the crustacean, Daphnia magna

Two Daphnia magna vitellogenin (VTG) genes in neighboring but opposite orientations were identified. One was the gene for DmagVTG1, a previously characterized VTG polypeptide with a superoxide dismutase (SOD)-like domain at its NH(2)-terminus [Kato et al., Gene 334 (2004) 157-165]. Both genes had a 17-exon and 16-intron structure in the same configuration. DmagVTG2, a polypeptide encoded by the other gene, also had a SOD-like domain at its NH(2)-terminus. The amino acid sequences of the two VTG domains were highly homologous (95.5% identity), while those of the SOD-like domains were less homologous (62.4% identity). The VTG domains are phylogenetically related to insect VTGs while the SOD-like domains are related to viral and bacterial SODs. The intergenic region of 2.6kb between the two genes contains sequences resembling known juvenile hormone (JH)-responsive and ecdysone-responsive elements. JH agonists, pyriproxyfen and fenoxycarb, strongly repressed the expression of VTG genes in neonate daphnids.     

Sulphate conjugation is the main route of pentachlorophenol metabolism in Daphnia magna

• 1.1. Daphnia magna were exposed for 24 hr to ¹⁴C-labelled pentachlorophenol (PCP) at an initial concentration of 20μg/l in the incubation water. Occurrence of free PCP and its metabolites were measured both from the animals and the water.
• 2.2. Hydrophilic metabolites excreted into water were analysed, after acid or enzymatic hydrolyses, with a liquid-liquid extraction and TLC.
• 3.3. PCP was metabolized and excreted, perhaps solely, via the sulphate conjugation. The average excretion rate, 2.65nmol/g/hr, accounted for 35% of the absorption rate measured at the start of exposure.
• 4.4. Neonate daphnids had an equal ability to metabolize PCP as the older animals. Bioconcentration in young animals was, however, only 23% of that in adult ones.
• 5.5. Effect of naturally humic water on metabolization and excretion of PCP was negligible.

     

Eye movements in Daphnia magna

Summary Three types of behavior of the compound eye of Daphnia magna are characterized: flick, a transient rotation elicited by a brief flash of light; fixation, a maintained eye orientation in response to a stationary light stimulus of long-duration; tracking, the smooth pursuit of a moving stimulus. The magnitudes of the flick and fixation responses vary with stimulus position and are generally proportional to stimulus intensity, although at high intensities there is an attenuation of both behaviors. When the stimulus is placed at a position 80° dorsal to the eye axis, there is no response; this area is called the null region. For stationary stimuli in other positions, the direction of the response is such as to bring the stimulus closer to the null region. During tracking, the relative positions of the eye and stimulus change; the eye velocity is approximately half that of the moving stimulus. The regions of the eye in which these behaviors may be induced are different, being largest for flick and smallest for tracking. It is proposed that flick and fixation responses are a means for rotating the eye so that the stimulus is within the area surrounding the null region which is used for tracking.     

The effect of a pathogen epidemic on the genetic structure and reproductive strategy of the crustacean Daphnia magna

Host–parasite coevolution is potentially of great importance in producing and maintaining biological diversity. However, there is a lack of evidence for parasites directly driving genetic change. We examined the impact of an epidemic of the bacterium Pasteuria ramosa on a natural population of the crustacean Daphnia magna through the use of molecular markers (allozymes) and laboratory experiments to determine the susceptibility of hosts collected during and after the epidemic. Some allozyme genotypes were more heavily infected than others in field samples, and the population genetic structure differed during and after the epidemic, consistent with a response to parasite‐mediated selection. Laboratory studies showed no evidence for the evolution of higher resistance, but did reveal an intriguing life‐history pattern: host genotypes that were more susceptible also showed a greater tendency to engage in sex. In light of this, we suggest a model of host–parasite dynamics that incorporates the cycles of sex and parthenogenesis that Daphnia undergo in the field.     

Calcium limitation in Daphnia magna

The role of ambient calcium concentrations on survival, moulting,growth and egg production was assessed in the cladoceran Daphniamagna. A threshold for survival was found in the range 0.1–0.5mg Ca l^–1, even when ionic strength of the medium waskept constant. Accumulated length and length specific dry weightwas retarded at low Ca (0.5–1.0 mg Ca l^–1) at foodconcentrations above incipient limiting level. For lower foodlevels, the effect of Ca on growth was less clear. The effectof low Ca on growth rate was most manifest during the firstdays after hatching, reflecting the higher Ca demands of theearly juveniles. Age-specific egg production was strongly reducedat Ca concentrations <10 mg Ca l^–1. This was partlyan indirect effect of reduced growth rates, but could also bean effect of higher energetic costs associated with Ca uptakein a Ca-poor medium. The high Ca demands in D.magna may notbe representative of other Daphnia species. Nevertheless, highspecific Ca content seems to be a common property of Daphniaspp. and Ca deficiency could be a major determinant of speciessuccess and community structure among crustacean zooplankton;it also puts constraints on carbon sequestration in the pelagicfood web of softwater lakes.     

Haemoglobin in male Daphnia magna

• 1.1. To define the respiratory function of haemoglobin in male Daphnia magna, the swimming activity, the depression of oxygen uptake by treatment with carbon monoxide and in vivo oxygenation of Hb at various oxygen pressures were investigated.
• 2.2. The P₅₀, values for the purified Hbs from male and female red animals were 2.0 and 2.7 torr in 0.1 M phosphate buffer (pH 7.2) at 20°C, respectively.
• 3.3. The isoelectric focusing patterns of the purified Hbs from male and female red animals showed only small differences in Hb components of high PI values.