Characterization and Expression of the Cytochrome P450 Genes in Daphnia magna Exposed to Cerium Oxide Nanoparticles.

As cerium oxide nanoparticles (nCeO2) continue to infiltrate aquatic environments, the resulting health risks to exposed aquatic organisms are becoming evident. Cytochrome P450 (CYP) enzymes are integral to the detoxification processes in these species. Herein, we conducted a genomic analysis of CYPs in Daphnia magna, encompassing phylogenetic relationships, gene structure, and chromosomal localization. We identified twenty-six CYPs in D. magna, categorizing them into four clans and seven families, distributed across six chromosomes and one unanchored scaffold. The encoded CYP proteins varied in length from 99 to 585 amino acids, with molecular weights ranging from 11.6 kDa to 66.4 kDa. A quantitative real-time PCR analysis demonstrated a significant upregulation of CYP4C1.4, CYP4C1.5, CYP4C1.6, CYP4c3.3, and CYP4c3.6 in D. magna exposed to 150 mg/L nCeO2 for 24 h. The transcript levels of CYP4C1.3, CYP18a1, CYP4C1.1, and CYP4c3.9 were notably downregulated in D. magna exposed to 10 mg/L nCeO2 for 48 h. A further transcriptomic analysis identified differential expression patterns of eight CYP genes, including CYP4C1.3, in response to nCeO2 exposure. The differential regulation observed across most of the 26 CYPs highlights their potential role in xenobiotic detoxification in D. magna, thereby enhancing our understanding of CYP-mediated toxicological responses to metal nanoparticles in aquatic invertebrates.

The Chronic Toxicity of Endocrine-Disrupting Chemical to Daphnia magna: A Transcriptome and Network Analysis of TNT Exposure.

Endocrine-disrupting chemicals (EDCs) impair growth and development. While EDCs can occur naturally in aquatic ecosystems, they are continuously introduced through anthropogenic activities such as industrial effluents, pharmaceutical production, wastewater, and mining. To elucidate the chronic toxicological effects of endocrine-disrupting chemicals (EDCs) on aquatic organisms, we collected experimental data from a standardized chronic exposure test using Daphnia magna (D. magna), individuals of which were exposed to a potential EDC, trinitrotoluene (TNT). The chronic toxicity effects of this compound were explored through differential gene expression, gene ontology, network construction, and putative adverse outcome pathway (AOP) proposition. Our findings suggest that TNT has detrimental effects on the upstream signaling of Tcf/Lef, potentially adversely impacting oocyte maturation and early development. This study employs diverse bioinformatics approaches to elucidate the gene-level toxicological effects of chronic TNT exposure on aquatic ecosystems. The results provide valuable insights into the molecular mechanisms of the adverse impacts of TNT through network construction and putative AOP proposition.

Trehalose mediates salinity-stress tolerance in natural populations of a freshwater crustacean.

Salinization poses an increasing problem worldwide, threatening freshwater organisms and raising questions about their ability to adapt. We explored the mechanisms enabling a planktonic crustacean to tolerate elevated salinity. By gradually raising water salinity in clonal cultures from 185 Daphnia magna populations, we showed that salt tolerance strongly correlates with native habitat salinity, indicating local adaptation. A genome-wide association study (GWAS) further revealed a major effect of the Alpha,alpha-trehalose-phosphate synthase (TPS) gene, suggesting that trehalose production facilitates salinity tolerance. Salinity-tolerant animals showed a positive correlation between water salinity and trehalose concentrations, while intolerant animals failed to produce trehalose. Animals with a non-functional TPS gene, generated through CRISPR-Cas9, supported the trehalose role in salinity stress. Our study highlights how a keystone freshwater animal adapts to salinity stress using an evolutionary mechanism known in bacteria, plants, and arthropods.

Exploring the Sublethal Impacts of Cu and Zn on Daphnia magna: a transcriptomic perspective.

Metal contamination of aquatic environments remains a major concern due to their persistence. The water flea Daphnia magna is an important model species for metal toxicity studies and water quality assessment. However, most research has focused on physiological endpoints such as mortality, growth, and reproduction in laboratory settings, as well as neglected toxicogenomic responses. Copper (Cu) and zinc (Zn) are essential trace elements that play crucial roles in many biological processes, including iron metabolism, connective tissue formation, neurotransmitter synthesis, DNA synthesis, and immune function. Excess amounts of these metals result in deviations from homeostasis and may induce toxic responses. In this study, we analyzed Daphnia magna transcriptomic responses to IC5 levels of Cu (120 µg/L) and Zn (300 µg/L) in environmental water obtained from a pristine lake with adjusted water hardness (150 mg/L CaCO3). The study was carried out to gain insights into the Cu and Zn regulated stress response mechanisms in Daphnia magna at transcriptome level. A total of 2,688 and 3,080 genes were found to be differentially expressed (DEG) between the control and Cu and the control and Zn, respectively. There were 1,793 differentially expressed genes in common for both Cu and Zn, whereas the number of unique DEGs for Cu and Zn were 895 and 1,287, respectively. Gene ontology and KEGG pathways enrichment were carried out to identify the molecular functions and biological processes affected by metal exposures. In addition to well-known biomarkers, novel targets for metal toxicity screening at the genomic level were identified.

Reproductive toxicity and molecular responses induced by telmisartan in Daphnia magna at environmentally relevant concentrations.

With aging population increasing globally, the use of pharmaceutically active compounds is rising. The cardiovascular drug telmisartan has been widely detected in various environmental compartments, including biota, surface waters, and sewage treatment plant effluents at concentrations ranging from ng/L to µg/L levels. This study evaluated the effects of telmisartan on the microcrustacean Daphnia magna at a wide range of concentrations (0.35, 0.70, 1.40, 500, and 1000 µg/L) and revealed significant ecotoxicological implications of this drug, even at environmentally relevant concentration. Acute exposure to telmisartan (1.40, 500, and 1000 µg/L) resulted in a notable decrease in heart rate, while chronic exposure accelerated the time to the first brood by 3 days and reduced neonate body size. Molecular investigations revealed marked downregulation of vitellogenin genes (Vtg1 and Vtg2). Non-monotonic dose responses were observed for gene expression, early-stage body length, and the total number of offspring produced, while the heart rate and time to the first brood showed clear concentration-dependent responses. These findings highlight the potential risks, notably to reproductive capacity, associated with exposure to telmisartan in environmentally relevant concentration, suggesting the need for further studies on the potential long-term ecological consequences.

A molecular mechanism for environmental sex determination.

Daphnia produce genetically identical males and females; their sex is determined by environmental conditions. Recently, Kato et al. identified isoform switching events in Daphnia as a gene regulatory mechanism for sex-specific development. This finding uncovers the impact of alternative usage of gene isoforms on this extreme phenotypic plasticity trait.

Differences in alternative splicing events in the adaptive strategies of two Daphnia galeata genotypes induced by fish kairomones.

BACKGROUND: Daphnia galeata is a suitable model organism for investigating predator-induced defense. Genes and pathways exhibiting differential expression between fish kairomone-treated and untreated groups in D. galeata have been identified. However, understanding of the significance of alternative splicing, a crucial process of the regulation of gene expression in eukaryotes, to this mechanism remains limited. This study measured life-history traits and conducted short-read RNA sequencing and long-read isoform sequencing of two Korean D. galeata genotypes (KB1 and KE2) to uncover the genetic mechanism underlying their phenotypic plasticity under predation stress. RESULTS: KB1 exhibited strategies to enhance fertility and decrease body length when exposed to fish kairomones, while KE2 deployed an adaptive strategy to increase body length. Full-length transcriptomes from KB1 and KE2 yielded 65,736 and 57,437 transcripts, respectively, of which 32 differentially expressed transcripts (DETs) were shared under predation stress across both genotypes. Prominent DETs common to both genotypes were related to energy metabolism and the immune system. Additionally, differential alternative splicing (DAS) events were detected in both genotypes in response to fish kairomones. DAS genes shared between both genotypes may indicate their significant role in the post-transcriptional stress response to fish predation. Calpain-3, involved in digestion and nutrient absorption, was identified as a DAS gene in both genotypes when exposed to fish kairomones. In addition, the gene encoding thymosin beta, which is related to growth, was found to be a statistically significant DAS only in KB1, while that encoding ultraspiracle protein, also associated with growth, was only identified in KE2. Moreover, transcripts encoding proteins such as EGF-like domain-containing protein, vitellogenin fused with superoxide dismutase, and others were identified overlapping between DAS events and DETs and potentially elucidating their association with the observed phenotypic variation in each genotype. CONCLUSIONS: Our findings highlight the crucial role of alternative splicing in modulating transcriptome landscape under predation stress in D. galeata, emphasizing the requirement for integrating gene expression and splicing analyses in evolutionary adaptation studies.

Cytotoxicity and Genotoxicity Effects of a Magnetic Zeolite Composite in Daphnia magna (Straus, 1820).

Zeolite type 5A combined with the magnetic properties of maghemite nanoparticles facilitate the rapid absorption of heavy metals, which makes them an interesting proposal for the remediation of water contaminated with lead and arsenic. However, the physicochemical analysis related to concentration and size for the use of this magnetic zeolite composite (MZ0) in water bodies and the possible toxicological effects on aquatic fauna has not yet been carried out. The main objective of the research work is to determine lethal concentrations that cause damage to Daphnia magna based on LC50 tests, morphology, reproductive rate, and quantification of the expression of three genes closely involved in the morphological development of vital structures (Glass, NinaE, Pph13). To achieve this objective, populations of neonates and young individuals were used, and results showed that the LC50 for neonates was 11,314 mg L-1, while for young individuals, it was 0.0310 mg L-1. Damage to morphological development was evidenced by a decrease in eye size in neonates, an increase in eye size in young individuals, variations in the size of the caudal spine for both age groups, and slight increases in the heart size, body, and antenna for both age groups. The reproductive rate of neonates was not affected by the lower concentrations of MZ0, while in young individuals, the reproductive rate decreased by more than 50% from the minimum exposure concentration of MZ0. And for both ages, Glass gene expression levels decreased as the MZ0 concentration increased. Also, the MZ0 evidenced its affinity for the exoskeleton of D. magna, which was observed using both light microscopy and electron microscopy. It is concluded that MZ0 did not generate significant damage in the mortality, morphology, reproductive rate, or gene expression in D. magna at lower concentrations, demonstrating the importance of evaluating the possible impacts on different life stages of the cladoceran.

The genome-wide signature of short-term temporal selection.

Despite evolutionary biology's obsession with natural selection, few studies have evaluated multigenerational series of patterns of selection on a genome-wide scale in natural populations. Here, we report on a 10-y population-genomic survey of the microcrustacean Daphnia pulex. The genome sequences of [Formula: see text]800 isolates provide insights into patterns of selection that cannot be obtained from long-term molecular-evolution studies, including the following: the pervasiveness of near quasi-neutrality across the genome (mean net selection coefficients near zero, but with significant temporal variance about the mean, and little evidence of positive covariance of selection across time intervals); the preponderance of weak positive selection operating on minor alleles; and a genome-wide distribution of numerous small linkage islands of observable selection influencing levels of nucleotide diversity. These results suggest that interannual fluctuating selection is a major determinant of standing levels of variation in natural populations, challenge the conventional paradigm for interpreting patterns of nucleotide diversity and divergence, and motivate the need for the further development of theoretical expressions for the interpretation of population-genomic data.

The toxic effects induced by benzethonium chloride on Daphnia carinata over two generations: Resistance and higher sublethal toxicity.

With the widespread utilization of the sanitizing product benzethonium chloride (BEC) throughout the coronavirus pandemic, concerns have emerged regarding its potential hazards. Nevertheless, the long-term and multigenerational toxic effects of BEC on aquatic organisms remains unexplored. This study investigates acute and chronic toxicity, oxidative stress, mitochondrial membrane potential, ATP concentrations, and gene expression using Daphnia carinata as the model organism. Meanwhile, hierarchical clustering analysis was utilized to investigate phenotypic effects among different treatment groups. The integrated biomarker response index version 2 (IBRv2) was employed to estimate the deviation in toxic effects over two generations. These results indicated that D. carinata in the second generation exhibited higher survival rate and lower levels of oxidative stress than the first generation. However, the higher sublethal effects were found in the second generation as follows, the weakened growth performance, mitochondrial membrane potential depolarization, reduced ATP concentrations, and down-regulated gene expression. The mitochondrial toxicity induced by BEC may account for the distinct toxic effects exhibited in two generations. The findings here can assist with the evaluation of potential risk for BEC on aquatic organisms, and provide new insight into the cross-generational toxicity mechanisms of pollutants in aquatic ecosystems.

Genome-Wide Allele Frequency Changes Reveal That Dynamic Metapopulations Evolve Differently.

Two important characteristics of metapopulations are extinction-(re)colonization dynamics and gene flow between subpopulations. These processes can cause strong shifts in genome-wide allele frequencies that are generally not observed in "classical" (large, stable, and panmictic) populations. Subpopulations founded by one or a few individuals, the so-called propagule model, are initially expected to show intermediate allele frequencies at polymorphic sites until natural selection and genetic drift drive allele frequencies toward a mutation-selection-drift equilibrium characterized by a negative exponential-like distribution of the site frequency spectrum. We followed changes in site frequency spectrum distribution in a natural metapopulation of the cyclically parthenogenetic pond-dwelling microcrustacean Daphnia magna using biannual pool-seq samples collected over a 5-yr period from 118 ponds occupied by subpopulations of known age. As expected under the propagule model, site frequency spectra in newly founded subpopulations trended toward intermediate allele frequencies and shifted toward right-skewed distributions as the populations aged. Immigration and subsequent hybrid vigor altered this dynamic. We show that the analysis of site frequency spectrum dynamics is a powerful approach to understand evolution in metapopulations. It allowed us to disentangle evolutionary processes occurring in a natural metapopulation, where many subpopulations evolve in parallel. Thereby, stochastic processes like founder and immigration events lead to a pattern of subpopulation divergence, while genetic drift leads to converging site frequency spectrum distributions in the persisting subpopulations. The observed processes are well explained by the propagule model and highlight that metapopulations evolve differently from classical populations.

Long-term balancing selection for pathogen resistance maintains trans-species polymorphisms in a planktonic crustacean.

Balancing selection is an evolutionary process that maintains genetic polymorphisms at selected loci and strongly reduces the likelihood of allele fixation. When allelic polymorphisms that predate speciation events are maintained independently in the resulting lineages, a pattern of trans-species polymorphisms may occur. Trans-species polymorphisms have been identified for loci related to mating systems and the MHC, but they are generally rare. Trans-species polymorphisms in disease loci are believed to be a consequence of long-term host-parasite coevolution by balancing selection, the so-called Red Queen dynamics. Here we scan the genomes of three crustaceans with a divergence of over 15 million years and identify 11 genes containing identical-by-descent trans-species polymorphisms with the same polymorphisms in all three species. Four of these genes display molecular footprints of balancing selection and have a function related to immunity. Three of them are located in or close to loci involved in resistance to a virulent bacterial pathogen, Pasteuria, with which the Daphnia host is known to coevolve. This provides rare evidence of trans-species polymorphisms for loci known to be functionally relevant in interactions with a widespread and highly specific parasite. These findings support the theory that specific antagonistic coevolution is able to maintain genetic diversity over millions of years.

Copy number and sequence variation in rDNA of Daphnia pulex from natural populations: insights from whole-genome sequencing.

Ribosomal DNA (rDNA) has a vital role in ribosome biogenesis as it contains the genes that encode ribosomal RNA (rRNA) separated by intergenic spacers (IGSs). The rRNA genes occur in hundreds to tens of thousands of copies per haploid genome in eukaryotes and are generally highly conserved with low variation within species. Due to the repetitive nature and large size of rDNA arrays, detecting intraindividual variation can be difficult. In this study, we use whole-genome sequences of 169 Daphnia pulex individuals from 10 natural populations to measure the copy number and sequence variation in rDNA. This revealed that variation in rDNA copy number between individuals spans an order of magnitude. We further observed a substantial level of sequence variation within individual genomes. As expected, single-nucleotide polymorphisms occurred in regions of lower functional constraint such as the IGS and expansion segments of the rRNA genes. The presence of strong linkage disequilibrium among variants facilitated identification of haplotypes within each population. Although there was evidence of recombination among haplotypes from different populations, it is insufficient to eliminate linkage disequilibrium within populations. Estimating copy number and haplotype diversity within individuals revealed that the level of intraindividual sequence variation is not strongly correlated with copy number. The observed patterns of variation highlight a complex evolutionary history of rDNA in D. pulex. Future research should explore the functional implications of rDNA copy number and sequence variation on organismal phenotypes.

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.

A common multi-host parasite shows genetic structuring at the host species and population levels.

Although individual parasite species commonly infect many populations across physical space as well as multiple host species, the extent to which parasites traverse physical and phylogenetic distances is unclear. Population genetic analyses of parasite populations can reveal how parasites move across space or between host species, including helping assess whether a parasite is more likely to infect a different host species in the same location or the same host species in a different location. Identifying these transmission barriers could be exploited for effective disease control. Here, we analysed population genetic structuring of the parasite Pasteuria ramosa in daphniid host species from different lakes. Outbreaks occurred most often in the common host species Daphnia dentifera and Daphnia retrocurva. The genetic distance between parasite samples tended to be smaller when samples were collected from the same lake, the same host species and closer in time. Within lakes, the parasite showed structure by host species and sampling date; within a host species, the parasite showed structure by lake and sampling date. However, despite this structuring, we found the same parasite genotype infecting closely related host species, and we sometimes found the same genotype in nearby lakes. Thus, P. ramosa experiences challenges infecting different host species and moving between populations, but doing so is possible. In addition, the structuring by sampling date indicates potential adaptation to or coevolution with host populations and supports prior findings that parasite population structure is dynamic during outbreaks.

The cuticle proteome of a planktonic crustacean.

The cuticles of arthropods provide an interface between the organism and its environment. Thus, the cuticle's structure influences how the organism responds to and interacts with its surroundings. Here, we used label-free quantification proteomics to provide a proteome of the moulted cuticle of the aquatic crustacean Daphnia magna, which has long been a prominent subject of studies on ecology, evolution, and developmental biology. We detected a total of 278 high-confidence proteins. Using protein sequence domain and functional enrichment analyses, we identified chitin-binding structural proteins and chitin-modifying enzymes as the most abundant protein groups in the cuticle proteome. Structural cuticular protein families showed a similar distribution to those found in other arthropods and indicated proteins responsible for the soft and flexible structure of the Daphnia cuticle. Finally, cuticle protein genes were also clustered as tandem gene arrays in the D. magna genome. The cuticle proteome presented here will be a valuable resource to the Daphnia research community, informing genome annotations and investigations on diverse topics such as the genetic basis of interactions with predators and parasites.

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.