Daphnia uses its circadian clock for short-day recognition in environmental sex determination.

Some organisms have developed a mechanism called environmental sex determination (ESD), which allows environmental cues, rather than sex chromosomes or genes, to determine offspring sex.1,2,3,4 ESD is advantageous to optimize sex ratios according to environmental conditions, enhancing reproductive success.5,6 However, the process by which organisms perceive and translate diverse environmental signals into offspring sex remains unclear. Here, we analyzed the environmental perception mechanism in the crustacean, Daphnia pulex, a seasonal (photoperiodic) ESD arthropod, capable of producing females under long days and males under short days.7,8,9,10 Through breeding experiments, we found that their circadian clock likely contributes to perception of day length. To explore this further, we created a genetically modified daphnid by knocking out the clock gene, period, using genome editing. Knockout disrupted the daphnid's ability to sustain diel vertical migration (DVM) under constant darkness, driven by the circadian clock, and leading them to produce females regardless of day length. Additionally, when exposed to an analog of juvenile hormone (JH), an endocrine factor synthesized in mothers during male production, or subjected to unfavorable conditions of high density and low food availability, these knockout daphnids produced males regardless of day length, like wild-type daphnids. Based on these findings, we propose that recognizing short days via the circadian clock is the initial step in sex determination. This recognition subsequently triggers male production by signaling the endocrine system, specifically via the JH signal. Establishment of a connection between these two processes may be the crucial element in evolution of ESD in Daphnia.

Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.

The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity in Daphnia.

Effect of chlorpyrifos-exposure on the expression levels of CYP genes in Daphnia magna and examination of a possibility that an up-regulated clan 3 CYP, CYP360A8, reacts with pesticides.

Daphnia magna is a test organism used for ecological risk assessments of pesticides, but little is known about the expression levels of cytochrome P450s (CYP)s and their changes after pesticide exposure in the less than 24-h-olds used for ecotoxicity tests. In this study, D. magna juveniles were exposed to 0.2 µg/L of chlorpyrifos under the conditions for acute immobilization test as specified by the OECD test guideline for 24 h, and then the gene expression was compared between the control and chlorpyrifos-exposure groups by RNA-sequencing analysis, with a focus on CYP genes. Among 38 CYP genes expressed in the control group, seven were significantly up-regulated while two were significantly down-regulated in the chlorpyrifos-exposure group. Although the sublethal concentration of chlorpyrifos did not change their expression levels so drastically (0.8 < fold change < 2.6), CY360A8 of D. magna (DmCYP360A8), which had been proposed to be responsible for metabolism of xenobiotics, was abundantly expressed in controls yet up-regulated by chlorpyrifos. Therefore, homology modeling of DmCYP360A8 was performed based on the amino acid sequence, and then molecular docking simulations with the insecticides that were indicated to be metabolized by CYPs in D. magna were conducted. The results indicated that DmCYP360A8 could contribute to the metabolism of diazinon and chlorfenapyr but not chlorpyrifos. These findings suggest that chlorpyrifos is probably detoxified by other CYP(s) including up-regulated and/or constitutively expressed one(s).

Different photoperiodic responses in diapause induction can promote the maintenance of genetic diversity via the storage effect in Daphnia pulex.

Understanding mechanisms that promote the maintenance of biodiversity (genetic and species diversity) has been a central topic in evolution and ecology. Previous studies have revealed that diapause can contribute to coexistence of competing genotypes or species in fluctuating environments via the storage effect. However, they tended to focus on differences in reproductive success (e.g. seed yield) and diapause termination (e.g. germination) timing. Here we tested whether different photoperiodic responses in diapause induction can promote coexistence of two parthenogenetic (asexual) genotypes of Daphnia pulex in Lake Fukami-ike, Japan. Through laboratory experiments, we confirmed that short day length and low food availability induced the production of diapausing eggs. Furthermore, we found that one genotype tended to produce diapausing eggs in broader environmental conditions than the other. Terminating parthenogenetic reproduction earlier decreases total clonal production, but the early diapausing genotype becomes advantageous by assuring reproduction in 'short' years where winter arrival is earlier than usual. Empirically parameterized theoretical analyses suggested that different photoperiodic responses can promote coexistence via the storage effect with fluctuations of the growing season length. Therefore, timing of diapause induction may be as important as diapause termination timing for promoting the maintenance of genetic diversity in fluctuating environments.

Chemical tolerance related to the ABC transporter gene and DNA methylation in cladocera (Daphnia magna).

We performed multigenerational tests to clarify the chemical tolerance mechanisms of a nontarget aquatic organism, Daphnia magna. We continuously exposed D. magna to a carbamate insecticide (pirimicarb) at lethal or sublethal concentrations (0, 3.8, 7.5, and 15 µg/L) for 15 generations (F0-F14). We then determined the 48 h-EC50 values and mRNA expression levels of acetylcholinesterase, glutathione S-transferase, and ATP (Adenosine triphosphate)-binding cassette transporter (ABCt) in neonates (<24 h old) from F0, F4, F9, and F14. To ascertain the effects of DNA methylation on pirimicarb sensitivity, we measured 5-methylcytosine levels (DNA methylation levels) in neonates of parents in the last generation (F14). In addition, we cultured groups exposed to 0 and 7.5 µg/L (the latter of which acquired chemical tolerance to pirimicarb) with or without 5-azacytidine (de-methylating agent) and determined methylation levels and 48 h-EC50 values in neonates (<24 h old) from the treated parents. The EC50 values (30.3-31.6 µg/L) in F14 of the 7.5 and 15 µg/L groups were approximately two times higher than that in the control (16.0 µg/L). A linear mixed model analysis showed that EC50 and ABCt mRNA levels were significantly increased with generational alterations; further analysis showed that the ABCt mRNA level was positively related to the EC50 . Therefore, ABCt may be associated with altered pirimicarb sensitivity. In addition, the EC50 value and DNA methylation levels in pirimicarb-tolerant clones decreased after exposure to 5-azacytidine, suggesting that DNA methylation contributes to chemical tolerance. These findings improved our knowledge regarding the acquisition of chemical tolerance in aquatic organisms.

Thermal stress-stimulated ZnO toxicity inhibits reproduction of freshwater crustacean Daphnia magna.

Elevated temperatures due to climate change pose a variety of environmental risks to the freshwater ecosystem. At the same time, zinc oxide (ZnO) has become widely used and has entered the freshwater environment. As thermal stress may potentially impact the physicochemical properties of ZnO, its toxicity to freshwater organisms in the face of global warming is poorly understood. The potential effects on reproductive performances, including oogenesis, are of particular concern. In this study, we investigate the reproductive performances and related mRNA abundance of the zooplankton Daphnia magna under conditions of ZnO exposure and heat stress. The results revealed that ZnO and elevated temperature delayed maturity and juvenile production of D. magna. Histological observations indicated that oogenesis was inhibited, and the number and size of oocytes were reduced in the condition of ZnO exposure under heat stress. Eventual offspring in the same treatment exhibited decreased numbers, size, and quality. Congenital juvenile anomalies were increased, such as deformed eye, and impaired antenna and tail spine. Moreover, both ZnO and elevated temperature treatments inhibited expression levels of reproduction-related genes (vtg, EcR and VMO1) and induced the dmrt93b gene involved in the production of male offspring. Furthermore, we found that D. magna tried to cope with ZnO and thermal stress by upregulating hsp90, HIF-1α and HIF-1ß. ZnO and heat stress inhibited the reproductive capacity of D. magna, produced deleterious effects on reproduction-associated physiological pathways, and damaged reproductive outcomes.

Evolutionary Genomics of Sister Species Differing in Effective Population Sizes and Recombination Rates.

Studies of closely related species with known ecological differences provide exceptional opportunities for understanding the genetic mechanisms of evolution. In this study, we compared population-genomics data between Daphnia pulex and Daphnia pulicaria, two reproductively compatible sister species experiencing ecological speciation, the first largely confined to intermittent ponds and the second to permanent lakes in the same geographic region. Daphnia pulicaria has lower genome-wide nucleotide diversity, a smaller effective population size, a higher incidence of private alleles, and a substantially more linkage disequilibrium than D. pulex. Positively selected genes in D. pulicaria are enriched in potentially aging-related categories such as cellular homeostasis, which may explain the extended life span in D. pulicaria. We also found that opsin-related genes, which may mediate photoperiodic responses, are under different selection pressures in these two species. Genes involved in mitochondrial functions, ribosomes, and responses to environmental stimuli are found to be under positive selection in both species. Additionally, we found that the two species have similar average evolutionary rates at the DNA-sequence level, although approximately 160 genes have significantly different rates in the two lineages. Our results provide insights into the physiological traits that differ within this regionally sympatric sister-species pair that occupies unique microhabitats.

Triazophos exposure on maternal Daphnia magna at environmental-related concentrations revealed toxic effects to its offspring.

Due to chemical and photochemical stability, triazophos has been frequently detected in rivers and oceans over the years with extensive use for pest control in agriculture, and it has become a worldwide ecological concern to the aquatic environment. Until now, fewer data are available regarding the potential long-term adverse effects of triazophos on aquatic invertebrates, which plays an essential role in aquatic food webs, as a key group for water ecosystems. In this experiment, the F1- and F2 progenies of Daphnia magna were recovered when daphnias (F0) exposure to triazophos at environmental-related concentrations (0.1 and 1.0 µg/L) for 21 d; and the indexes related to phenotypic traits, reproduction and gene expression were measured in tested animals. The results showed that heart rate and total number of neonates in exposed F0-daphnias were significantly lower than those of control group, and the detoxification genes (HR96 and P-gp) were up-regulated while genes related reproduction (Vtg) and molting (Nvd and Shd) were significantly down-regulated. The heart rate and individual size of F1-daphnias (<24 h) were significantly reduced in the treatment group. After 21-d recovery, the heart rate and expression of HR96, P-gp, Vtg, Nvd and Shd were declined in F1-daphnias. There was no obvious difference of morphological traits and heart rate between treatment and control in F2-daphnias (<24 h). In summary, daphnias (F0) exposure to triazophos with environmental dose could raise toxic effects on its offspring (F1), which is mainly manifested by reduced heart rate, the accumulated number and individual size of offspring and decreased expression of genes related to molting and reproduction.

Genotype specific and microbiome effects of hypoxia in the model organism Daphnia magna.

The fitness of the host is highly influenced by the interplay between the host and its associated microbiota. The flexible nature of these microbiota enables them to respond swiftly to shifts in the environment, which plays a key role in the host's capacity to withstand environmental stresses. To understand the role of the microbiome in host tolerance to hypoxia, one of the most significant chemical changes occurring in water ecosystems due to climate change, we performed a reciprocal gut transplant experiment with the freshwater crustacean Daphnia magna. In a microbiome transplant experiment, two genotypes of germ-free recipients were inoculated with gut microbiota from Daphnia donors of their own genotype or from the other genotype, that had been either pre-exposed to normoxic or hypoxic conditions. We found that D. magna individuals had a higher survival probability in hypoxia if their microbiome had been pre-exposed to hypoxia. The bacterial communities of the recipients changed over time with a reduction in alpha diversity, which was stronger when donors were pre-exposed to a hypoxic environment. While donor genotype had no influence on the long-term survival probability in hypoxia, donor genotypes was the most influential factor of the microbial community 3 days after the transplantation. Our results indicate that microbiome influencing factors mediate host fitness in a hypoxic environment in a time depending way.

Climate has contributed to population diversification of Daphnia galeata across Eurasia.

Climate is a fundamental abiotic factor that plays a key role in driving the evolution, distribution and population diversification of species. However, there have been few investigations of genomic signatures of adaptation to local climatic conditions in cladocerans. Here, we have provided the first high-quality chromosome-level genome assembly (~143 Mb, scaffold N50 12.6 Mb) of the waterflea, Daphnia galeata, and investigated genomic variation in 22 populations from Central Europe and Eastern China. Our ecological-niche models suggested that the historic distribution of D. galeata in Eurasia was significantly affected by Quaternary climate fluctuations. We detected pronounced genomic and morphometric divergences between European and Chinese D. galeata populations. Such divergences could be partly explained by genomic signatures of thermal adaptation to distinct climate regimes: a set of candidate single-nucleotide polymorphisms (SNPs) potentially associated with climate were detected. These SNPs were in genes significantly enriched in the Gene ontology terms "determination of adult lifespan" and "translation repressor activity", and especially, mthl5 and SOD1 involved in the IIS pathway, and EIF4EBP2 involved in the target of the rapamycin signalling pathway. Our study indicates that certain alleles might be associated with particular temperature regimes, playing a functional role in shaping the population structure of D. galeata at a large geographical scale. These results highlight the potential role of molecular variation in the response to climate variation, in the context of global climate change.

An In Vivo Whole-Transcriptomic Approach to Assess Developmental and Reproductive Impairments Caused by Flumequine in Daphnia magna.

Among veterinary antibiotics, flumequine (FLU) is still widely used in aquaculture due to its efficacy and cost-effectiveness. Although it was synthesized more than 50 years ago, a complete toxicological framework of possible side effects on non-target species is still far from being achieved. The aim of this research was to investigate the FLU molecular mechanisms in Daphnia magna, a planktonic crustacean recognized as a model species for ecotoxicological studies. Two different FLU concentrations (2.0 mg L-1 and 0.2 mg L-1) were assayed in general accordance with OECD Guideline 211, with some proper adaptations. Exposure to FLU (2.0 mg L-1) caused alteration of phenotypic traits, with a significant reduction in survival rate, body growth, and reproduction. The lower concentration (0.2 mg L-1) did not affect phenotypic traits but modulated gene expression, an effect which was even more evident under the higher exposure level. Indeed, in daphnids exposed to 2.0 mg L-1 FLU, several genes related with growth, development, structural components, and antioxidant response were significantly modulated. To the best of our knowledge, this is the first work showing the impact of FLU on the transcriptome of D. magna.

Transcriptomics and the origin of obligate parthenogenesis.

Despite the presence of obligately parthenogenetic (OP) lineages derived from sexual ancestors in diverse phylogenetic groups, the genetic mechanisms giving rise to the OP lineages remain poorly understood. The freshwater microcrustacean Daphnia pulex typically reproduces via cyclical parthenogenesis. However, some populations of OP D. pulex have emerged due to ancestral hybridization and introgression events between two cyclically parthenogenetic (CP) species D. pulex and D. pulicaria. These OP hybrids produce both subitaneous and resting eggs parthenogenetically, deviating from CP isolates where resting eggs are produced via conventional meiosis and mating. This study examines the genome-wide expression and alternative splicing patterns of early subitaneous versus early resting egg production in OP D. pulex isolates to gain insight into the genes and mechanisms underlying this transition to obligate parthenogenesis. Our differential expression and functional enrichment analyses revealed a downregulation of meiosis and cell cycle genes during early resting egg production, as well as divergent expression patterns of metabolism, biosynthesis, and signaling pathways between the two reproductive modes. These results provide important gene candidates for future experimental verification, including the CDC20 gene that activates the anaphase-promoting complex in meiosis.

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.

Uncovering the Genomic Basis of Infection Through Co-genomic Sequencing of Hosts and Parasites.

Understanding the genomic basis of infectious disease is a fundamental objective in co-evolutionary theory with relevance to healthcare, agriculture, and epidemiology. Models of host-parasite co-evolution often assume that infection requires specific combinations of host and parasite genotypes. Co-evolving host and parasite loci are, therefore, expected to show associations that reflect an underlying infection/resistance allele matrix, yet little evidence for such genome-to-genome interactions has been observed among natural populations. We conducted a study to search for this genomic signature across 258 linked host (Daphnia magna) and parasite (Pasteuria ramosa) genomes. Our results show a clear signal of genomic association between multiple epistatically interacting loci in the host genome, and a family of genes encoding for collagen-like protein in the parasite genome. These findings are supported by laboratory-based infection trials, which show strong correspondence between phenotype and genotype at the identified loci. Our study provides clear genomic evidence of antagonistic co-evolution among wild populations.

No recombination suppression in asexually produced males of Daphnia pulex.

Obligate parthenogenesis (OP) is often thought to evolve by disruption of reductional meiosis and suppression of crossover recombination. In the crustacean Daphnia pulex, OP lineages, which have evolved from cyclical parthenogenetic (CP) ancestors, occasionally produce males that are capable of reductional meiosis. Here, by constructing high-density linkage maps, we find that these males show only slightly and nonsignificantly reduced recombination rates compared to CP males and females. Both meiosis disruption and recombination suppression are therefore sex-limited (or partly so), which speaks against the evolution of OP by disruption of a gene that is essential for meiosis or recombination in both sexes. The findings may be explained by female-limited action of genes that suppress recombination, but previously identified candidate genes are known to be expressed in both sexes. Alternatively, and equally consistent with the data, OP might have evolved through a reuse of the parthenogenesis pathways already present in CP and through their extension to all events of oogenesis. The causal mutations for the CP to OP transition may therefore include mutations in genes involved in oogenesis regulation and may not necessarily be restricted to genes of the "meiosis toolkit." More generally, our study emphasizes that there are many ways to achieve asexuality, and elucidating the possible mechanisms is key to ultimately identify the genes and traits involved.

RNA-seq analysis to identify genes related to resting egg production of panarctic Daphnia pulex.

BACKGROUND: The genus Daphnia switches its reproductive mode from subitaneous egg production to resting egg production in response to environmental stimuli. Although this life history trait is essential for surviving unsuitable environments, the molecular mechanism of resting egg production is little understood. In this study, we examined genes related to induction of resting egg production using two genotypes of panarctic Daphnia pulex, the JPN1 and JPN2 lineages, which differ genetically in the frequency of resting egg production. We reared these genotypes under high and low food levels. At the high food level, individuals of both genotypes continually produced subitaneous eggs, whereas at the low food level, only the JPN2 genotype produced resting eggs. Then, we performed RNA-seq analysis on specimens of three instars, including before and after egg production. RESULTS: These results showed that expressed genes differed significantly between individuals grown under high and low food levels and among individuals of different instars and genotypes. Among these differentially expressed genes (DEGs), we found 16 that changed expression level before resting egg production. Some of these genes showed high-level expression only before resting egg production and one gene was an ortholog of bubblegum (bgm), which is reportedly up-regulated before diapause in bumblebees. According to gene ontology (GO) enrichment analysis, one GO term annotated as long-chain fatty acid biosynthetic process was enriched among these 16 genes. In addition, GO terms related to glycometabolism were enriched among down-regulated genes of individuals holding resting eggs, compared to those before resting egg production. CONCLUSIONS: We found candidate genes highly expressed only before resting egg production. Although functions of candidate genes found in this study have not been reported previously in Daphnia, catabolism of long-chain fatty acids and metabolism of glycerates are related to diapause in other organisms. Thus, it is highly probable that candidate genes identified in this study are related to the molecular mechanism regulating resting egg production in Daphnia.

Characterization of Sulfoxaflor and Its Metabolites on Survival, Growth, Reproduction, Biochemical Markers, and Transcription of Genes of Daphnia magna.

Sulfoxaflor is a promising neonicotinoid. However, the negative implications of sulfoxaflor on nontarget aquatic organisms have been rarely studied. In this study, the risks of sulfoxaflor and its main metabolites X11719474 and X11519540 on Daphnia magna were characterized, including acute toxicity, reproduction, swimming behavior, biochemical markers, and gene transcription. Acute toxicity measurements indicated that X11719474 and X11519540 have high toxicity than the parent compound sulfoxaflor. Chronic exposure reduced reproduction and delayed the birth of the firstborn D. magna. Swimming behavior monitoring showed that exposure to three compounds stimulated swimming behavior. The induction of catalase, superoxide dismutase, and acetylcholinesterase activities was observed with oxidative stress, whereas malondialdehyde content was remarkably increased with exposure to sulfoxaflor, X11719474, and X11519540. Moreover, transcriptomics profiles showed that sulfoxaflor, X11719474, and X11519540 induced KEGG pathways related to cellular processes, organismal systems, and metabolisms. The findings present valuable insights into the prospective hazards of these pesticides and emphasize the critical importance of conducting a systematic evaluation of combining antecedents and their metabolites.

Experimental test of the influence of light availability on the evolution of eye size and behaviour in Daphnia.

There exists extensive variation in eye size. Much work has provided a connection between light availability and differences in eye size across taxa. Experimental tests of the role of the light environment on the evolution of eye size are lacking. Here, we performed a selection experiment that examined the influence of light availability on shifts in eye size and the connection between eye size and phototactic (anti-predator) behaviour in Daphnia. We set-up replicate experimental populations of Daphnia, repeatedly evaluated phenotypic shifts in eye size during the ~50-day experiment, and performed a common garden experiment at the end of the experiment to test for evolutionary shifts in eye size and behaviour. Our phenotypic analyses showed that eye size rapidly diverged between the light treatments; relative eye size was consistently larger in the low versus high light treatments. Selection on eye size was also modified by variation in density as increases in Daphnia density favoured a larger eye. However, we did not observe differences in eye size between the light treatments following two generations of common garden rearing at the end of the experiment. We instead observed strong shifts in anti-predator behaviour. Daphnia from the low light treatment exhibited decreased phototactic responses to light. Our results show that decreased light relaxes selection on anti-predator behaviour. Such trends provide new insights into selection on eye size and behaviour.

Development of a yeast reporter gene assay to detect ligands of freshwater cladoceran Daphnia magna ultraspiracle, a homolog of vertebrate retinoid X receptors.

Endocrine-disrupting chemicals (EDCs) often affect homeostatic regulation in living organisms by directly acting on nuclear receptors (NRs). Retinoid X receptors (RXRs), the most highly conserved members of the NR superfamily during evolution, function as partners to form heterodimers with other NRs, such as retinoic acid, thyroid hormone, and vitamin D3 receptors. RXRs also homodimerize and induce the expression of target genes upon binding with their natural ligand, 9-cis-retinoic acid (9cRA), and typical EDCs organotin compounds, such as tributyltin and triphenyltin. In the present study, we established a new yeast reporter gene assay (RGA) to detect the ligands of freshwater cladoceran Daphnia magna ultraspiracle (Dapma-USP), a homolog of vertebrate RXRs. D. magna has been used as a representative crustacean species for aquatic EDC assessments in the Organization for Economic Corporation and Development test guidelines. Dapma-USP was expressed along with the Drosophila melanogaster steroid receptor coactivator Taiman in yeast cells carrying the lacZ reporter plasmid. The RGA for detecting agonist activity of organotins and o-butylphenol was improved by use of mutant yeast strains lacking genes encoding cell wall mannoproteins and/or plasma membrane drug efflux pumps as hosts. We also showed that a number of other human RXR ligands, phenol and bisphenol A derivatives, and terpenoid compounds such as 9c-RA exhibited antagonist activity on Dapma-USP. Our newly established yeast-based RGA system is valuable as the first screening tool to detect ligand substances for Dapma-USP and for evaluating the evolutionary divergence of the ligand responses of RXR homologs between humans and D. magna.

Divergence of the Host-Associated Microbiota with the Genetic Distance of Host Individuals Within a Parthenogenetic Daphnia Species.

The taxonomic composition of the microbiota in the gut and epidermis of animals is known to vary among genetically and physiologically different host individuals within the same species. However, it is not clear whether the taxonomic composition diverges with increasing genetic distance of the host individuals. To unveil this uncertainty, we compared the host-associated microbiota among the genotypes within and between genetically distant lineages of parthenogenetic Daphnia cf. pulex across different physiological states, namely, well-fed, starved, and dead. Metagenomic analysis with 16S rRNA showed that, regardless of the host genotypes, diversity of the host-associated microbiota was high when the host individuals were fed food and gradually decreased when they were starved until they died. However, the difference in the host-associated microbiota, that is, ß-diversity, was significant among the genotypes within and between the host lineages when they were fed. Although some bacteria in the microbiota, such as Limnohabitans, Rhodococcus, and Aeromicrobium, were found abundantly and commonly in all host genotypes; others, such as those of Holosoporacea, were found only in the genotypes of a specific lineage. Accordingly, the ß-diversity tended to increase with increasing genetic distance of the host individuals. These results support an idea that the host-associated microbiota diverged with genetic divergence in the host species and that at least some bacteria are highly dependent on the genetically specific metabolites produced by the host individuals.