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.

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.

Resurrection genomics provides molecular and phenotypic evidence of rapid adaptation to salinization in a keystone aquatic species.

Ecologists and evolutionary biologists are increasingly cognizant of rapid adaptation in wild populations. Rapid adaptation to anthropogenic environmental change is critical for maintaining biodiversity and ecosystems services into the future. Anthropogenic salinization of freshwater ecosystems is quickly emerging as a primary threat, which is well documented in the northern temperate ecoregion. Specifically, many northern temperate lakes have undergone extensive salinization because of urbanization and the associated increase in impervious surfaces causing runoff, and the extensive use of road deicing salts (e.g., NaCl). It remains unclear whether increasing salinization will lead to extirpation of species from these systems. Using a "resurrection genomics" approach, we investigated whether the keystone aquatic herbivore, Daphnia pulicaria, has evolved increased salinity tolerance in a severely salinized lake located in Minnesota, USA. Whole-genome resequencing of 54 Daphnia clones from the lake and hatched from resting eggs that represent a 25-y temporal contrast demonstrates that many regions of the genome containing genes related to osmoregulation are under selection in the study population. Tolerance assays of clones revealed that the most recent clones are more tolerant to salinity than older clones; this pattern is concomitant with the temporal pattern of stabilizing salinity in this lake. Together, our results demonstrate that keystone species such as Daphnia can rapidly adapt to increasing freshwater salinization. Further, our results indicate that rapid adaptation to salinity may allow lake Daphnia populations to persist in the face of anthropogenic salinization maintaining the food webs and ecosystem services they support despite global environmental change.

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.

Multispecies coexistence in fragmented landscapes.

Spatial dynamics have long been recognized as an important driver of biodiversity. However, our understanding of species' coexistence under realistic landscape configurations has been limited by lack of adequate analytical tools. To fill this gap, we develop a spatially explicit metacommunity model of multiple competing species and derive analytical criteria for their coexistence in fragmented heterogeneous landscapes. Specifically, we propose measures of niche and fitness differences for metacommunities, which clarify how spatial dynamics and habitat configuration interact with local competition to determine coexistence of species. We parameterize our model with a Bayesian approach using a 36-y time-series dataset of three Daphnia species in a rockpool metacommunity covering >500 patches. Our results illustrate the emergence of interspecific variation in extinction and recolonization processes, including their dependencies on habitat size and environmental temperature. We find that such interspecific variation contributes to the coexistence of Daphnia species by reducing fitness differences and increasing niche differences. Additionally, our parameterized model allows separating the effects of habitat destruction and temperature change on species extinction. By integrating coexistence theory and metacommunity theory, our study provides platforms to increase our understanding of species' coexistence in fragmented heterogeneous landscapes and the response of biodiversity to environmental changes.

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.

RNA-seq analysis reveals changes in mRNA expression during development in Daphnia mitsukuri.

Temporal transcriptional variation is a major contributor to functional evolution and the developmental process. Parthenogenetic water fleas of the genus Daphnia (Cladocera) provide an ideal model to characterize gene expression patterns across distinct developmental stages. Herein, we report RNA-seq data for female Daphnia mitsukuri at three developmental stages: the embryo, juvenile (three timepoints) and adult. Comparisons of gene expression patterns among these three developmental stages and weighted gene co-expression network analysis based on expression data across developmental stages identified sets of genes underpinning each of the developmental stages of D. mitsukuri. Specifically, highly expressed genes (HEGs) at the embryonic developmental stage were associated with cell proliferation, ensuring the necessary foundation for subsequent development; HEGs at the juvenile stages were associated with chemosensory perception, visual perception and neurotransmission, allowing individuals to enhance detection of potential environmental risks; HEGs at the adult stage were associated with antioxidative defensive systems, enabling adults to mount an efficient response to perceived environmental risks. Additionally, we found a significant overlap between expanded gene families of Daphnia species and HEGs at the juvenile stages, and these genes were associated with visual perception and neurotransmission. Our work provides a resource of developmental transcriptomes, and comparative analyses that characterize gene expression dynamics throughout development of Daphnia.

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.

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.

Evolutionary Insights from a Large-Scale Survey of Population-Genomic Variation.

The field of genomics has ushered in new methods for studying molecular-genetic variation in natural populations. However, most population-genomic studies still rely on small sample sizes (typically, <100 individuals) from single time points, leaving considerable uncertainties with respect to the behavior of relatively young (and rare) alleles and, owing to the large sampling variance of measures of variation, to the specific gene targets of unusually strong selection. Genomic sequences of ∼1,700 haplotypes distributed over a 10-year period from a natural population of the microcrustacean Daphnia pulex reveal evolutionary-genomic features at a refined scale, including previously hidden information on the behavior of rare alleles predicted by recent theory. Background selection, resulting from the recurrent introduction of deleterious alleles, appears to strongly influence the dynamics of neutral alleles, inducing indirect negative selection on rare variants and positive selection on common variants. Temporally fluctuating selection increases the persistence of nonsynonymous alleles with intermediate frequencies, while reducing standing levels of variation at linked silent sites. Combined with the results from an equally large metapopulation survey of the study species, classes of genes that are under strong positive selection can now be confidently identified in this key model organism. Most notable among rapidly evolving Daphnia genes are those associated with ribosomes, mitochondrial functions, sensory systems, and lifespan determination.

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.

Declining calcium concentration drives shifts toward smaller and less nutritious zooplankton in northern lakes.

Zooplankton community composition of northern lakes is changing due to the interactive effects of climate change and recovery from acidification, yet limited data are available to assess these changes combined. Here, we built a database using archives of temperature, water chemistry and zooplankton data from 60 Scandinavian lakes that represent broad spatial and temporal gradients in key parameters: temperature, calcium (Ca), total phosphorus (TP), total organic carbon (TOC), and pH. Using machine learning techniques, we found that Ca was the most important determinant of the relative abundance of all zooplankton groups studied, while pH was second, and TOC third in importance. Further, we found that Ca is declining in almost all lakes, and we detected a critical Ca threshold in lake water of 1.3 mg L-1 , below which the relative abundance of zooplankton shifts toward dominance of Holopedium gibberum and small cladocerans at the expense of Daphnia and copepods. Our findings suggest that low Ca concentrations may shape zooplankton communities, and that current trajectories of Ca decline could promote widespread changes in pelagic food webs as zooplankton are important trophic links from phytoplankton to fish and different zooplankton species play different roles in this context.

Developing a Passive Dosing Method for Acute Aquatic Toxicity Tests of Cationic Surfactant Benzalkoniums (BACs).

Benzalkonium chlorides (BACs) have been of environmental concern due to their widespread use and potential harm. However, challenges arise in defining and controlling the exposure concentration (Cw) in aquatic toxicity tests involving BACs with a long alkyl chain (i.e., #C > 14). To address this, a novel passive dosing method was introduced in the 48 h-acute ecotoxicity test on Daphnia magna and compared to the conventional solvent-spiking method in terms of Cw stability and toxicity results. Among 13 sorbent materials tested for their sorption capacity, poly(ether sulfone) (PES) membrane was an optimal passive dosing reservoir, with equilibrium desorption of BACs to water achieved within 24 h. The Cw of BACs remained constant in both applied dosing methods during the test period. However, the Cw in solvent-spiking tests was lower than the nominal concentration for long-chain BACs, particularly at low exposure concentrations. Notably, the solvent-spiking tests indicated that the toxicity of BACs increased with alkyl chain length from C6 to 14, followed by a decline in toxicity from C14 to 18. In contrast, the passive dosing method displayed similar or slightly increasing toxicity levels of BACs from C14 to C18, indicating higher toxicity of C16 and C18-BACs than that inferred by the solvent spiking test. These findings emphasize the potential of applying this innovative passive dosing approach in aquatic toxicity tests to generate reliable and accurate toxicity data and support a comprehensive risk assessment of cationic surfactants.

Confocal Surface-Enhanced Raman Imaging of the Intestinal Barrier Crossing Behavior of Model Nanoplastics in Daphnia Magna.

Nanoplastics (nP) pose hazards to aquatic animals once they are ingested. Significant knowledge gaps exist regarding the nP translocation across the animal intestine, which is the first barrier between the ingested nP and the animal body. We examined the intestinal barrier crossing behavior of nP in an aquatic animal model (Daphnia magna) and determined the translocation mechanism with the help of model "core-shell" polystyrene nanoplastics (nPS) and confocal surface-enhanced Raman spectroscopy (SERS). The Raman reporter (4-mercaptobenzoic acid)-tagged gold "core" of the model nPS enables sensitive and reliable particle imaging by confocal SERS. This method detected SERS signals of model nPS concentration as low as 4.1 × 109 particles/L (equivalent to 0.27 µg/L PS "shell" concentration). The translocation was observed with the help of multilayer stacked Raman maps of SERS signals of the model nPS. With a higher concentration or longer exposure time of the model nPS, uptake and translocation of the plastic particles increased. In addition, we demonstrated that clathrin-dependent endocytosis and macropinocytosis were two major mechanisms underlying the translocation. This study contributes to a mechanistic understanding of nP translocation by using the pioneering model nPS and an analytical toolkit, which undergird further investigations into nP behavior and health effects in aquatic species.

Epidemics in native species influence the outcome of a species invasion.

Invasive species can have large effects on native communities. When native and invasive species share parasites, an epidemic in a native species could facilitate or inhibit the invasion. We sought to understand how the incidence and timing of epidemics in native species caused by a generalist parasite influenced the success and impact of an invasive species. We focused on North American native and invasive species of zooplankton (Daphnia dentifera and Daphnia lumholtzi, respectively), that can both become infected with a fungal parasite (Metschnikowia bicuspidata). In a laboratory microcosm experiment, we exposed the native species to varying parasite inocula (none, low, high) and two invasive species introduction times (before or during an epidemic in the native species). We found that the invasive species density in treatments with the parasite was higher compared to uninfected treatments, though only the early invasion, low-parasite and uninfected treatments exhibited significant pairwise differences. However, invasive resting eggs were only found in the uninfected treatments. The density of the native species was lowest with a combination of the parasite present, and the invasive species introduced during the epidemic. Native infection prevalence in these treatments (late invasion, parasite present) was also higher than prevalence in treatments where the invasive species was introduced before the epidemic. Therefore, the timing of an invasion relative to an epidemic can affect both the native and invasive species. Our results suggest that the occurrence and timing of epidemics in native species can influence the impacts of a species invasion.

Mysterious microsporidians: springtime outbreaks of disease in Daphnia communities in shallow pond ecosystems.

Parasites can play key roles in ecosystems, especially when they infect common hosts that play important ecological roles. Daphnia are critical grazers in many lentic freshwater ecosystems and typically reach peak densities in early spring. Daphnia have also become prominent model host organisms for the field of disease ecology, although most well-studied parasites infect them in summer or fall. Here, we report field patterns of virulent microsporidian parasites that consistently infect Daphnia in springtime, in a set of seven shallow ponds in Georgia, USA, sampled every 3-4 weeks for 18 months. We detected two distinct parasite taxa, closely matching sequences of Pseudoberwaldia daphniae and Conglomerata obtusa, both infecting all three resident species of Daphnia: D. ambigua, D. laevis, and D. parvula. To our knowledge, neither parasite has been previously reported in any of these host species or anywhere in North America. Infection prevalence peaked consistently in February-May, but the severity of these outbreaks differed substantially among ponds. Moreover, host species differed markedly in terms of their maximum infection prevalence (5% [D. parvula] to 72% [D. laevis]), mean reduction of fecundity when infected (70.6% [D. ambigua] to 99.8% [D. laevis]), mean spore yield (62,000 [D. parvula] to 377,000 [D. laevis] per host), and likelihood of being infected by each parasite. The timing and severity of the outbreaks suggests that these parasites could be impactful members of these shallow freshwater ecosystems, and that the strength of their effects is likely to hinge on the composition of ponds' zooplankton communities.

Pathways linking nutrient enrichment, habitat structure, and parasitism to host-resource interactions.

Human activities simultaneously alter nutrient levels, habitat structure, and levels of parasitism. These activities likely have individual and joint impacts on food webs. Furthermore, there is particular concern that nutrient additions and changes to habitat structure might exacerbate the size of epidemics and impacts on host density. We used a well-studied zooplankton-fungus host-parasite system and experimental whole water column enclosures to factorially manipulate nutrient levels, habitat structure (specifically: mixing), and presence of parasites. Nutrient addition increased infection prevalence, density of infected hosts, and total host density. We hypothesized that nutrients, mixing, and parasitism were linked in multiple ways, including via their combined effects on phytoplankton (resource) abundance, and we used structural equation modeling to disentangle these pathways. In the absence of the parasite, both nutrients and mixing increased abundance of phytoplankton, whereas host density was negatively related to phytoplankton abundance, suggesting a mixture of bottom-up and top-down control of phytoplankton. In the presence of the parasite, nutrients still increased phytoplankton abundance but mixing no longer did, and there was no longer a significant relationship between host density and phytoplankton. This decoupling of host-resource dynamics may have resulted from reduced grazing due to illness-mediated changes in feeding behavior. Overall, our results show that the impact of one human activity (e.g., altered habitat structure) might depend on other human impacts (e.g., parasite introduction). Fortunately, carefully designed experiments and analyses can help tease apart these multifaceted relationships, allowing us to understand how human activities alter food webs, including interactions between hosts and their parasites and resources.

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.

Increased virulence due to multiple infection in Daphnia leads to limited growth in 1 of 2 co-infecting microsporidian parasites.

Recent outbreaks of various infectious diseases have highlighted the ever-present need to understand the drivers of the outbreak and spread of disease. Although much of the research investigating diseases focuses on single infections, natural systems are dominated by multiple infections. These infections may occur simultaneously, but are often acquired sequentially, which may alter the outcome of infection. Using waterfleas (Daphnia magna) as a model organism, we examined the outcome of sequential and simultaneous multiple infections with 2 microsporidian parasites (Ordospora colligata and Hamiltosporidium tvaerminnensis) in a fully factorial design with 9 treatments and 30 replicates. We found no differences between simultaneous and sequential infections. However, H. tvaerminnensis fitness was impeded by multiple infection due to increased host mortality, which gave H. tvaerminnensis less time to grow. Host fecundity was also reduced across all treatments, but animals infected with O. colligata at a younger age produced the fewest offspring. As H. tvaerminnensis is both horizontally and vertically transmitted, this reduction in offspring may have further reduced H. tvaerminnensis fitness in co-infected treatments. Our findings suggest that in natural populations where both species co-occur, H. tvaerminnensis may evolve to higher levels of virulence following frequent co-infection by O. colligata.

Chronic aquatic toxicity assessment of diverse chemicals on Daphnia magna using QSAR and chemical read-across.

We have modeled here chronic Daphnia toxicity taking pNOEC (negative logarithm of no observed effect concentration in mM) and pEC50 (negative logarithm of half-maximal effective concentration in mM) as endpoints using QSAR and chemical read-across approaches. The QSAR models were developed by strictly obeying the OECD guidelines and were found to be reliable, predictive, accurate, and robust. From the selected features in the developed models, we have found that an increase in lipophilicity and saturation, the presence of electrophilic or electronegative or heavy atoms, the presence of sulphur, amine, and their related functionality, an increase in mean atomic polarizability, and higher number of (thio-) carbamates (aromatic) groups are responsible for chronic toxicity. Therefore, this information might be useful for the development of environmentally friendly and safer chemicals and data-gap filling as well as reducing the use of identified toxic chemicals which have chronic toxic effects on aquatic ecosystems. Approved classes of drugs from DrugBank databases and diverse groups of chemicals from the Chemical and Product Categories (CPDat) database were also assessed through the developed models.