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.

Impacts of sequential salinity and heat stress are recovery time-specific in freshwater crustacean, Daphnia pulicaria.

Stressors can interact to affect animal fitness, but we have limited knowledge about how temporal variation in stressors may impact their combined effect. This limits our ability to predict the outcomes of pollutants and future dynamic environmental changes. Elevated salinity in freshwater ecosystems has been observed worldwide. Meanwhile, heatwaves have become more frequent and intensified as an outcome of climate change. These two stressors can jointly affect organisms; however, their interaction has rarely been explored in the context of freshwater ecosystems. We conducted lab experiments using Daphnia pulicaria, a key species in lakes, to investigate how elevated salinity and heatwave conditions collectively affect freshwater organisms. We also monitored the impacts of various recovery times between the two stressors. Daphnia physiological conditions (metabolic rate, Na+-K+-ATPase (NKA) activity, and lipid peroxidation level) and life history traits (survival, fecundity, and growth) in response to salt stress as well as mortality in heat treatment were examined. We found that Daphnia responded to elevated salinity by upregulating NKA activity and increasing metabolic rate, causing a high lipid peroxidation level. Survival, fecundity, and growth were all negatively affected by this stressor. These impacts on physiological conditions and life history traits persisted for a few days after the end of the exposure. Heat treatments caused mortality in Daphnia, which increased with rising temperature. Results also showed that individuals that experienced salt exposure were more susceptible to subsequent heat stress, but this effect decreased with increasing recovery time between stressors. Findings from this work suggest that the legacy effects from a previous stressor can reduce individual resistance to a subsequent stressor, adding great difficulties to the prediction of outcomes of multiple stressors. Our work also demonstrates that cross-tolerance/susceptibility and the associated mechanisms remain unclear, necessitating further investigation.

Selenium bioaccumulation in Daphnia pulex via aqueous and dietary exposure.

Pit lakes are currently being investigated as a way to store and reclaim waste materials in the Alberta Oil Sands (AOS) region, Canada. Lake Miwasin (LM) is a pilot-scale pit lake consisting of treated fine tailings overlayed with oil sands process-affected water (OSPW) blended with fresh surface water. In October 2021, the surface water contained a mean concentration of 1.33 ± 0.04 µg/L dissolved selenium (Se), slightly above the Canadian Council of Ministers of Environment water quality guideline for long-term protection of aquatic life (1 µg Se/L). This study assessed the bioaccumulation of Se by the cladoceran Daphnia pulex under laboratory conditions through both aqueous and dietary exposure routes for comparison to field-collected specimens. In 12-day semi-static tests, lab-cultured D.pulex were exposed to water, and algae grown in media spiked with selenate. Results showed that Se bioaccumulation by lab-cultured D. pulex increased in all exposure treatments from days 5 to 12, with maximum Se concentrations of 3.08-3.47 µg/g dry weight (dw) observed within the exposure range tested. Interestingly, lower Se bioaccumulation concentrations (1.26-1.58 µg/g dw) were observed in the highest dissolved Se and dietary Se treatments, suggesting potential internal regulatory mechanisms. In addition, native D. pulex (LM) collected from Lake Miwasin and cultured in-house were exposed in 8-day semi-static tests to Lake Miwasin surface water and algae cultured in Lake Miwasin surface water. Selenium bioaccumulation in native D. pulex (LM) ranged from 2.00 to 2.04 µg/g dw at day 8 and was not significantly different (p > 0.05) compared to Se concentrations in D. pulex collected from Lake Miwasin (2.15 ± 0.28 µg/g) in summer 2022.

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.

Combined effects of spinetoram and Microcystis aeruginosa on Daphnia pulex offspring: Maternal effects and multigenerational implications.

The increasing occurrence of harmful algal blooms (HABs) in freshwater ecosystems detrimentally affect global water environments. Zooplankton's role in controlling HABs is hindered by contaminant exposure, necessitating research into combined stressors' ecological impacts. The response of Daphnia, a freshwater keystone species, to environmental stressors can be influenced by its maternal effects. Here, we investigated the combined effects of the world-widely used insecticide spinetoram and non-toxic HABs species Microcystis aeruginosa on the life-history traits of D. pulex offspring produced from different maternal food conditions. Four maternal groups were established, with each group receiving a specific blend of C. vulgaris (Ch) and M. aeruginosa (Ma) in varying proportions: A (100% Ch), B (90% Ch + 10% Ma), C (80% Ch + 20% Ma), and D (70% Ch + 30% Ma). The offspring from the third brood were gathered, and a 21-day experiment was carried out, involving various feeding groups (AA, AD, BA, BB, CA, CC, DA, and DD). Results demonstrated that grazing on M. aeruginosa by D. pulex induced maternal effects on their offspring, with the continuous exposure group showing an enhanced tolerance to M. aeruginosa. This study also unveiled that spinetoram could interfere with the molting of D. pulex, leading to developmental retardation. The Recovery Group exhibited an intriguing phenomenon: under the influence of both concentrations of the pesticide spinetoram (0.18, 0.35 µg L-1), D. pulex produced more offspring. This might be due to a combined strategy of allocating more energy towards reproduction in response to low-quality food and a potential hormetic effect from low concentrations of spinetoram. Assessing the interplay of combined stressors across multiple generations, encompassing harmful algal blooms (HABs) and environmental pollutants, is essential for predicting population responses to evolving environmental conditions. This understanding is vital for the protection and management of aquatic environments and ecosystems.

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.

Declining aqueous calcium and fish predation risk interactively modify the phenotypic plasticity in Daphnia pulex.

Aqueous calcium (Ca) decline is threatening freshwater ecosystems worldwide. There are great concerns about the possible ecological consequences of Ca limitation combined with biological pressures like predation. Here we investigated the interactions between Ca restriction and fish predation risk on the phenotypic plasticity in the keystone herbivore Daphnia, together with physiological responses underlying the plastic trait changes. Fish predation risk induced D. pulex to mature earlier and produce more but smaller offspring at adequate Ca. Declining Ca inhibited the expression of defensive traits, with the inhibitive degree showing a linear or threshold-limited dynamic. The presence of predation risk mitigated the negative effect of declining Ca on reducing body size but exacerbated the delay in maturity, indicating a life history trade-off for larger body size rather than the current reproduction in multi-stressed Daphnia. Actin 3-mediated cytoskeleton and AMPK ß-mediated energy metabolism were highly correlated with these plastic trait changes. Altered phenotypic plasticity in planktonic animals is expected to trigger many ecological impacts from individual fitness to community structure, thus providing new insights into the mechanisms underlying decreased Ca affecting lake ecosystems.

Differential Toxicity of Arsenic in Daphnia pulex Under Phosphorus and Food Limitation.

The on-going anthropogenic degradation of freshwater habitats has drastically altered the environmental supply of both nutrients and common pollutants. Most organisms living in these altered habitats experience interactive effects of various stressors that can initiate adjustments at multiple levels impacting their fitness. Hence, studies measuring response to a single environmental parameter fail to capture the complexities of the status quo. We tested both the individual and the interactive effect of arsenic (As) exposure, food quantity, and dietary phosphorus (P)-supply on six life-history traits (Juvenile Growth Rate; Adult Growth Rate; Age and Size at Maturity, Lifespan, and Fecundity) as surrogates for organismal fitness in the keystone aquatic grazer Daphnia pulex. We also tested the effect of food quantity and P-supply on somatic As accumulation in Daphnia. Our results indicated an influence of P-supply on neonatal growth and an influence of As and food quantity on growth and maintenance later in life. Maturation was strongly influenced by all three variables, with no reproduction observed in the presence of two or more environmental stressors. We found a strong interaction between As and dietary P, with increased P-supply intensifing the toxicity effect of As. No such effects were seen between As and food quantity, indicating a differential role of quantity versus quality on As toxicity. We found a nominal effect of diet on somatic As accumulation. The results from the present study emphasize the importance of considering such interactions between co-occurring environmental stressors and the dietary status of organisms, to better predict and manage impacts and risks associated with common environmental toxicants in highly vulnerable ecosystems. Environ Toxicol Chem 2024;43:1807-1819. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Environmental Stress and the Morphology of Daphnia pulex.

AbstractMorphological variation is sometimes used as an indicator of environmental stress in animals. Here, we assessed how multiple morphological traits covaried in Daphnia pulex exposed to five common forms of environmental stress (high temperature, presence of predator cues, high salinity, low food abundance, and low Ca). We measured animal body length, body width, head width, eyespot diameter, and tail spine length along with mass in animals of five different ages (3, 6, 9, 12, and 15 d). There were strong allometric relationships among all morphological traits in reference animals and strong univariate effects of environmental stress on body mass and body length. We found that environmental stressors altered bivariate relationships between select pairwise combinations of morphological traits, with effects being dependent on animal age. Multivariate analyses further revealed high connectivity among body size-related traits but that eyespot diameter and tail spine length were less tightly associated with body size. Animals exposed to natural lake water with and without supplemental food also varied in morphology, with body size differences being suggestive of starvation and other unknown nutritional deficiencies. Yet our results demonstrate that the scaling of body morphological traits of Daphnia pulex is largely invariant with possible context-dependent plasticity in eye size and tail spine lengths. The strong coordination of traits indicates tight molecular coordination of body size during development despite strong and varied environmental stress.

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.

Photoperiodic adaptation of aanat and clock gene expression in seasonal populations of Daphnia pulex.

Changes in day-length entrain the endogenous clock of organisms leading to complex responses to photoperiod. In long-lived organisms experiencing several seasons this response of the clock to photoperiod is phenotypically plastic. However, short-lived organisms often experience a single season without pronounced changes in day-length. For those, a plastic response of the clock to different seasons would not necessarily be adaptive. In aquatic ecosystems, zooplankton species like Daphnia live only for some weeks, i.e. one week up to ca. two months. However, they often show a succession of clones that are seasonally adapted to environmental changes. Here, we found that 16 Daphnia clones per each of three seasons ( = 48 clones) from the same pond and year differed in clock gene expression with a homogenous gene expression pattern in ephippia-hatched spring clones and a bimodal expression pattern in summer and autumn populations indicating an ongoing adaptation process. We clearly demonstrate that spring clones were adapted to a short, and summer clones to a long photoperiod. Furthermore, we found that gene expression of the melatonin-synthesis enzyme AANAT was always lowest in summer clones. In the Anthropocene, Daphnia's clock might be disturbed by light-pollution and global warming. Since Daphnia is a key-organism in trophic carbon transfer, a disruption of its clock rhythm would be devastating for the stability of freshwater ecosystems. Our results are an important step in understanding the adaptation of Daphnia's clock to environmental changes.

Urban runoff mortality syndrome in zooplankton caused by tire wear particles.

Stormwater runoff from roadways is a global threat to water quality, aquatic organisms, and ecosystems. Tire tread wear particles (TWP) from roadway runoff may lead to urban runoff mortality syndrome (URMS) in some aquatic organisms. We tested the hypothesis that urban runoff from roadways can kill zooplankton. Both roadway runoff and TWP leachate were acutely lethal to a model species, the water flea Daphnia pulex. Life table experiments further revealed the lowered survival rates, intrinsic rate of increase, average life span, and net productive rate of D. pulex when exposed to roadway runoff and TWP leachate. The tire rubber antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) mainly contributed to the TWP toxicity. The toxicity of TWP and 6PPD extracted varied with time in nature. Cladocerans and rotifers were more sensitive to TWP and 6PPD than copepods. These results demonstrate the presence of URMS in zooplankton, which may cascade through food webs and affect aquatic ecosystems.