research on daphnia

• Published: 13 January 2010

The water flea Daphnia - a 'new' model system for ecology and evolution?

• Angelika Stollewerk 1  

Journal of Biology volume  9 , Article number:  21 ( 2010 ) Cite this article

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Daphnia pulex is the first crustacean to have its genome sequenced. Availability of the genome sequence will have implications for research in aquatic ecology and evolution in particular, as addressed by a series of papers published recently in BMC Evolutionary Biology and BMC Genomics .

One of the major questions in evolutionary biology is to understand how species have adapted to different environments and how the underlying changes in morphology, physiology and behavior relate to modifications in the corresponding genes. The publication of the first crustacean genome sequence, that of Daphnia pulex [ 1 , 2 ], is part of an effort by the members of the Daphnia Genome Consortium to establish Daphnia as a model system for evolutionary environmental genomics. But can Daphnia rise to the challenge?

The vast number of publications on Daphnia in the literature prove that this animal is no newcomer to scientific research. Daphnia is most probably one of the best-studied subjects in ecology [ 3 ]. Populations can be found in freshwater environments ranging from huge lakes to small temporary pools and seasonally flooded depressions. The ecology of Daphnia has been studied from the point of view of its role as a primary consumer in aquatic food chains, its phenotypic plasticity, and its behavior, toxicology and the evolution of sexual and asexual reproduction. Extensive studies on the population genetics of Daphnia have addressed migration and gene flow, hybridization and inbreeding, among other topics. With the availability of the genome sequence, Daphnia research has now the potential to reach a new level. A number of papers on the D. pulex genome in relation to different aspects of Daphnia biology have been published in BMC Evolutionary Biology and BMC Genomics to accompany the genome release [ 4 – 11 ]. These constitute an initial exploration of the genome, and in this article I review some of the highlights and questions raised.

Daphnia ecology and life style

Daphnia are filter feeders that direct small suspended particles into their mouth by a water current produced by their leaf-like legs (Figure 1 ). Daphnia 's common name of 'water flea' comes from its jump-like movement, which results from the beat of the large antennae used for swimming (Figure 1 ). In a normal growth season Daphnia generates diploid eggs by asexual reproduction (partheno-genesis). These eggs develop directly into larvae in the female brood chamber and are released into the water after about 3 days. In most species the larvae go through four to six larval stages before developing into sexually mature adults. However, the Daphnia life cycle is adapted to extreme environmental conditions such as cold winters or summer droughts. If triggered by external stimuli such as high population density and a scarcity of food, Daphnia can produce haploid resting eggs by meiosis; these require fertilization and a period of extended dormancy in order to develop [ 3 ]. Resting eggs are distributed by wind or animals and development is resumed in response to external stimuli (for example, rising temperature). Cyclic parthenogenesis, in which parthenogenesis and sexual reproduction alternate, is common in most Daphnia species, but lineages have been described that exclusively reproduce asexually (obligate parthenogenesis). Cyclic parthenogenetic Daphnia must contain the molecular tools for the production of both haploid gametes (by meiosis) and diploid eggs (by mitosis), the latter developing parthenogenetically into diploid zygotes. This makes Daphnia an ideal system to study the evolution of the molecular processes of parthenogenesis.

Scanning electron micrograph of a Daphnia larva shortly before hatching . Photograph courtesy of Petra Ungerer.

In this regard, Eads and co-workers (Schurko et al . [ 4 ]) suggest that differences between sexual and asexual reproduction most probably relate to mechanisms that differ between meiosis and mitosis, such as kinetochore orientation, DNA recombination and sister-chromatid cohesion, and have screened the D. pulex genome for genes associated with meiosis. The authors report an inventory of 130 D. pulex genes that are homologous with known genes in other organisms and which represent more than 40 distinct protein-coding genes with diverse roles in meiosis. The majority of these genes are present in multiple copies, and Schurko et al . [ 4 ] speculate that the extra copies may be partly responsible for changes to these meiotic processes that enable parthenogenesis. Parthenogenetic species are present in all major animal phyla and future comparison of the genomes of cyclic and obligate parthenogenetic lineages will shed light on the evolution of the underlying molecular processes.

The offspring produced in parthenogenetic cycles are genetic clones of their mother [ 3 ]. This includes the males, as sex is environmentally determined in Daphnia . The existence of clonal reproduction is a powerful tool for quantitative genetic studies because it facilitates the analysis of genetic variation within and between populations. Genetic variation has been reported in Daphnia for a vast number of traits such as size, aging, behavior (for example, vertical migration, fish-escape behavior), morphology (for example, defensive spines, helmets), and the immune system (for example, resistance against parasites, immune responses), and the great number of duplicated genes in Daphnia seems to correlate with Daphnia 's considerable phenotypic plasticity [ 3 ].

Predators and other enemies

Interestingly, several of the above-mentioned traits can be induced by environmental cues. Changes in both the behavior and the morphology of Daphnia can, for example, be affected by predator-borne chemical cues (kairomones). In the presence of fish kairomones, Daphnia magna gives rise to smaller offspring, whereas chemical cues from the phantom midge Chaoborus flavicans induce the generation of larger progeny. This has been shown to be an adaptive phenotypic plasticity that helps avoid predation as fish and midges prefer different sizes of prey. These observations raise the question of the nature of the molecular response to kairomones. Schwarzenberger et al . [ 5 ] have addressed this question by comparing the expression levels of genes involved in protein biosynthesis and catabolism in D. magna in the presence or absence of kairomones. Interestingly, they found that expression of the cyclophilin gene, which encodes an enzyme involved in protein folding, is upregulated in the presence of fish kairomones but downregulated by Chaoborus kairomones , which correlates with the opposite effects of these kairomones on progeny size. The authors used the D. magna cyclophilin sequence to search the D. pulex genome and identified 16 paralogs, which showed a very high variability. Future research will show whether the differences in cyclophilin expression levels can be linked to the observed phenotypic variations and if additional paralogs are involved in the process.

The first step in kairomone-mediated adaptive changes in behavior and morphology is obviously the reception of the chemical signal by specialized sensory structures of the prey. Our knowledge about chemoreception in aquatic organisms is fragmentary, however. In insects, a conserved chemoreceptor superfamily has been identified which can be subdivided into the gustatory (taste) receptor family and the odorant (smell) receptor family. It is obvious that the sensing of odorants will be different in water than in air as aquatic odorants are hydrophilic-molecules dissolved in water whereas airborne odorants are mainly hydrophobic volatile molecules in gaseous form. Penalva-Arana et al . [ 6 ] have identified 58 orthologs of the insect gustatory receptor family in the D. pulex genome. Interestingly, they found no evidence of genes homologous with insect odorant receptor genes and suggest that the odorant receptor family evolved concomitantly with the transition from sea to land in the lineage leading to the insects.

Predators are not the only natural enemies of Daphnia . The study of parasites (viruses, bacteria and multicellular parasites) has also gained momentum as a result of their influence on Daphnia ecology and evolution [ 3 ]. Parasites can directly or indirectly affect host population dynamics, extinction, and maintenance of genetic diversity, among other features. It has been suggested that hosts continuously evolve to reduce parasite virulence, whereas parasites evolve to keep virulence as close as possible to an optimum level. Variation in resistance to infection has indeed been observed in natural fruit fly populations and has been associated with genetic polymorphisms [ 12 ].

All metazoans seem to have an innate immune system, and in insects, at least four different signaling pathways are involved in the immune response and mediate pathogen recognition, attack on the pathogen, and antiviral RNA interference, among other responses. McTaggart et al . [ 7 ] analyzed the D. pulex genome for genes related to the immune system and identified genes homologous with those in other arthropods. The authors found that the Toll signaling pathway, which is activated by the presence of pathogens, is conserved between insects and Daphnia . The activation of this pathway results in the production-of antibacterial and antifungal proteins. These antimicrobial peptides could not be recovered from the D. pulex genome and thus seem to be less well conserved. In addition, McTaggart et al . [ 7 ] report considerable variation in gene family copy number in Daphnia and insects. These differences might reflect the evolutionary history of host-parasite interactions in the individual lineages. Further comparative studies are needed to uncover evolutionary changes in genes that mediate immune responses as well as taxon-specific expansions of gene families, which will contribute to our understanding of how host genes are evolving in response to parasites.

Although a vast number of ecto-and endoparasites have been described for Daphnia , the non-parasitic symbionts have not been analyzed in detail. Ebert and co-workers (Qi et al . [ 8 ]) have used metagenomics to address this question. Metagenomes - genetic material recovered directly from environmental samples - are sequenced and compared to the databases in order to characterize the biological community of a given habitat. One of the advantages of this approach is the recovery of DNA sequences from organisms that cannot be cultured. Ebert and co-workers [ 8 ] searched the shotgun sequences of three clones of three different Daphnia species ( D. pulex , D. magna and D. pulicaria ) for indications of bacterial and plastid symbionts and found sequences representing a large number of bacterial species in each dataset. The majority of the sequences were from the Proteobacteria but many other taxa were also detected. No clear evidence was found for the presence of symbiotic cyanobacteria or of plastids. Interestingly, the composition of the bacterial communities was similar at genus and higher taxonomic levels in all three Daphnia clones, but different bacterial species were present in individual clones. The D. pulex and D. pulicaria DNA used in this study was isolated from clones cultured in North America, whereas the D. magna cultures originated from a laboratory in Switzerland. Since contamination of the Daphnia cultures by cross-Atlantic exchange is unlikely, the authors suggest that the similarities between the symbiont communities in European and North American Daphnia samples indicate a long-term stability of symbiotic associations.

Environmental challenges

Daphnia species have been studied extensively because of their importance to aquatic ecosystems, and they show a striking ability to contend with environmental challenges. The availability of the D. pulex genome should now be able to provide insights into the adaptation to specific environmental conditions, from the ecological to the genetic level. On screening the D. pulex genome for genes involved in the biochemical response to toxicants, Baldwin et al . [ 9 ] identified 75 genes of the cytochrome P450 family, a protein family important in tolerance and resistance to environmental chemicals. The authors report that the same subgroups of cytochrome P450 genes are present in the Daphnia genome as in insects and nematodes, but they discovered distinct changes in the size and gene composition of each group. Dean and co-workers (Sturm et al . [ 10 ]) screened the Daphnia genome for the presence of members of the ABC transporter superfamily (ATP-binding cassette membrane transport-proteins), which are also involved in bio-chemical defense against toxicants. They found that ABC family representation in Daphnia is as complex as in other metazoans, and that Daphnia most resembles the fruit fly in respect of its ABC transporter genes. Future studies on the expression and function of these genes will uncover their importance in the adaptation of Daphnia to environmental toxicants.

Daphnia and arthropod phylogeny

The D. pulex genome also has the potential to contribute to resolving long-standing debates on arthropod phylogeny. Current views of arthropod phylogenetic relationships are based mainly on two types of datasets - molecular genetic data and morphological characters - and this has led to partly contradictory concepts of arthropod phylogeny. There is now almost universal agreement-that arthropods derive from a common ancestor, and that crustaceans and insects are sister groups [ 13 ]. However, some issues of arthropod relationships remain controversial, for example the question of whether insects, crustaceans and myriapods form a monophyletic-group. Crustaceans show the greatest diversity of body organization and development among arthropods [ 14 ] and therefore the phylogenetic relationships within the crustaceans are far from being resolved. Several morphological and molecular studies have questioned the monophyly of crustaceans, and either Branchiopoda (such as Daphnia ) or Malacostraca (lobster, shrimps) has been hypothesized to be the sister group to insects [ 15 ]. Some recent molecular analyses suggest a sister group relationship of myriapods (millipedes) and chelicerates (spiders) [ 16 ]. Interestingly, this suggestion is supported by recent morphological and molecular studies on the development of the nervous system that reveal a surprising degree of similarity between myriapods and chelicerates [ 17 , 18 ]. The morphological support for an insect-crustacean sister-group relationship is mainly based on the comparative analysis of neural characters in higher crustaceans (malacostracans) and insects. For example, in both insects and malacostracans, stem-cell-like neuroblasts have been detected that divide asymmetrically to generate the cells that contribute to the nervous system [ 14 ]. But are these neural characters representative of all crustacean groups? Are homologous genes required for the development and the function of the nervous system? With the availability of a branchiopod genome and the development of genetic tools for Daphnia these questions can now be addressed.

Furthermore, using genome sequences of a wide range of organisms, the origin and evolution of neural signaling pathways can be traced, which will broaden our understanding of the evolution of nervous systems. The neurotrophin signaling pathway plays a role in neural development, regeneration and neural plasticity in mammals. Analyzing the Daphnia genome, Wilson [ 11 ] shows for the first time that a neurotrophin and both a tyrosine receptor kinase (Trk) and a p75-type neurotrophin-receptor (p75NTR) are present in a protostome, indicating that this pathway existed in the last common ancestor of protostomes and deuterostomes.

To conclude, the initial exploration of the D. pulex genome outlined above proves that with the availability of the genome sequence Daphnia research has entered a new era. New and long-standing questions in ecology and evolution can be addressed and it may finally be possible to link evolutionary and environmental adaptations to the underlying genetic processes.

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Acknowledgements

I am grateful to Dieter Ebert for discussions and comments on the manuscript and I thank Petra Ungerer for the scanning electron micrograph of Daphnia . The work was supported by a grant to AS from the BBSRC.

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Stollewerk, A. The water flea Daphnia - a 'new' model system for ecology and evolution?. J Biol 9 , 21 (2010). https://doi.org/10.1186/jbiol212

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• Published: 23 May 2007

Ecological genomics in Daphnia : stress responses and environmental sex determination

• B D Eads 1 , 2 ,
• J Andrews 1 , 2 &
• J K Colbourne 1  

Heredity volume  100 ,  pages 184–190 ( 2008 ) Cite this article

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Ecological genomics is the study of adaptation of natural populations to their environment, and therefore seeks to link organism and population level processes through an understanding of genome organization and function. The planktonic microcrustacean Daphnia , which has long been an important system for ecology, is now being used as a genomic model as well. Here we review recent progress in selected areas of Daphnia genomics research. Production of parthenogenetic male offspring occurs through environmental cues, which clearly involves endocrine regulation and has also been studied as a toxicological response to juvenoid hormone analog insecticides. Recent progress has uncovered a putative juvenoid cis -response element, which together with microarray analysis will stimulate further research into nuclear hormone receptors and their associated transcriptional regulatory networks. Ecotoxicological studies indicate that mRNA profiling is a sensitive and specific research tool with promising applications in environmental monitoring and for uncovering conserved cellular processes. Rapid progress is expected to continue in these and other areas, as genomic tools for Daphnia become widely available to investigators.

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Introduction.

A meaningful integration of ecology and genomics promises important new insights into the process by which populations respond and adapt to their environment. This research begins by identifying the genetic basis for ecologically relevant traits. Then, explicit and quantifiable associations are made between the heritable molecular variation within populations and individual fitness across varying ecological conditions ( Gibson, 2002 ; Feder and Mitchell-Olds, 2003 ). In practice, two approaches are taken to achieve this level of integration; each presents a different set of advantages and challenges. First, ecological and evolutionary studies are pursued using traditional genetic model species that have well developed genomic tools, such as Drosophila , Caenorhabditis , Arabidopsis and Mus (cf. Landry et al., 2006 ; Shimizu and Purugganan, 2006 ). Although these species offer a staggering amount of molecular biological knowledge, many have ill-defined population boundaries and ecologies. This situation is starting to change owing to increased ecological study of these model organisms. Second, molecular genetic and genomic studies are initiated using organisms that have well-understood ecologies and evolutionary histories ( Crawford, 2001 ; Strauss and Martin, 2004 ; Lai et al., 2006 ), of which the water flea, Daphnia , is one. Here, we outline the progress in the use of Daphnia as a model in evolutionary and ecological functional genomics.

Several attributes make Daphnia an especially tractable system for ecological genomics. In addition to their well-understood ecology and their sentinel roles within freshwater ecosystems, these microcrustaceans are easily collected in large numbers and reared in the lab with a 5- to 10-day generation time. They have a relatively small and sequenced genome (ca. 200 Mb) and perhaps most interesting, they reproduce by cyclical parthenogenesis. Females typically reproduce asexually, but environmental conditions can induce parthenogenetic production of males (genetically identical to their mothers) and haploid gametes that are fertilized and enter a state of extended metabolic dormancy called diapause. This life cycle allows the creation of inbred or outbred lines for mapping studies, and provides tremendous power for dissecting genetically based differences (among clones) from experimental or developmental noise by measuring replicates within a clone.

The ecology and evolution of Daphnia is well explored with respect to habitat differentiation ( Fryer, 1991 ; Wellborn et al., 1996 ; Colbourne et al., 1997 ), but progress has been less in characterizing molecular mechanisms responsible for traits conferring a fitness advantage in defined environments. For instance, diurnal and seasonal variations in predation, parasitism, food availability and temperature interact in complex ways to produce phenotypes such as investment in sexual reproduction ( Cáceres and Tessier, 2004 ). Although production of sexual eggs and males is expected to be coupled at some level as an adaptation to make diapausing embryos, mechanisms of this induction by environmental cues are not well understood. An important first step towards such an investigation is to examine the genetics of environmental sex determination. Similarly, Daphnia are often in temperature- and oxygen-stratified habitats, which has important ecological implications for predator avoidance and access to alternative grazing resources ( Williamson et al., 1996 ). Acclimation to hypoxia via hemoglobin (Hb) synthesis has been a subject of extensive study over the last decades. Here, we discuss new discoveries enabled by genomic approaches that provide a mechanistic linkage between two well-studied phenomena, male production and Hb induction in Daphnia .

A consortium of investigators is progressing rapidly in creating tools for Daphnia genomics. First, the genome sequencing and assembly of a D. pulex genome by the Department of Energy's Joint Genome Institute is complete, and its annotation has begun. The annotation goals of identifying genes and their structures are being guided by a growing number of cDNA libraries and sequences for both D. pulex (Colbourne et al. , submitted) and D. magna ( Watanabe et al., 2005 ). Second, microsatellite DNA markers are deployed for population surveys ( Fox, 2004 ) and quantitative trait locus (QTL) mapping experiments ( Colbourne et al., 2004 ). Many of these markers form the basis of a genetic map ( Cristescu et al., 2006 ) that is being integrated along with large insert BAC libraries to identify chromosomal segments under high and low rates of recombination. Third, both cDNA and oligonucleotide microarrays are being used for gene expression analysis in D. magna and D. pulex . Finally, bioinformatic efforts have resulted in the creation of wFleabase ( Colbourne et al., 2005 ), an online repository of genetic, molecular and genomic data for Daphnia , while laboratory projects to create cell lines and virally induced transgenic lines are also ongoing ( Robinson et al., 2006 ).

These resources are opening the way for the systematic investigation of the genes and pathways underlying ecologically relevant traits. Presentations at a recent meeting of the Daphnia Genomics Consortium summarized progress in the areas of gametogenesis, embryonic patterning, parasitology, toxicology and molecular evolution ( http://conferences.cgb.indiana.edu/daphnia2006/ ). To date, much of the data obtained about molecular mechanisms in the area of ecological genomics stem from physiological rather than evolutionary perspectives, which is reflected in our choice of literature to review. Specifically, we focus on progress in understanding sex determination and environmental stress responses.

Male production pathway as an entry into ecological genomics

The ecological basis of sex determination in Daphnia has long been appreciated ( Banta and Brown, 1939 ) and genetic variation for this trait is substantial ( Yampolsky, 1992 ). Environmental factors such as temperature, photoperiod and crowding stimulate male production by parthenogenesis ( Hobæk and Larsson, 1990 ) and, although varying investments into sexual reproduction have important ecological and evolutionary consequences ( Tessier and Cáceres, 2004 ), molecular studies have only begun. Male production is a promising research avenue for several reasons. First, it provides an opportunity to study the molecular basis of environmental sex determination. Second, it is a basis for broader exploration of invertebrate endocrine function and disruption in general, which has lagged far behind our understanding of vertebrate systems ( Oetken et al., 2004 ). Third, as a classic example of phenotypic plasticity and epigenetic regulation, it offers the possibility of uncovering molecular mechanisms responsible for these ecologically and evolutionarily central mechanisms. Fourth, evidence indicates important couplings between juvenile hormone (JH) pathways and ecdysone pathways in Daphnia ( Mu and LeBlanc, 2004 ), which are likely to be useful for genetic and genomic analyses of these pathways across the Ecdysozoa.

Olmstead and LeBlanc (2002 ) were first to demonstrate production of male broods of D. magna in response to the sesquiterpenoid hormone methyl farnesoate (MF), the unepoxidated form of insect JH III. Discovery of MF in crustaceans prompted extensive efforts to characterize the functions of this hormone, which largely appears to mirror the effects of JH in insects ( Laufer and Biggers, 2001 ). Other JH analogs (JHAs) in addition to MF have been shown to induce male production in Daphnia . The insecticides pyriproxyfen, fenoxycarb and methoprene cause male broods ( Tatarazako et al., 2003 ), with orders of magnitude greater potency than MF for pyriproxyfen and fenoxycarb. Interestingly, all of these compounds also induce Hb production in Daphnia ( Rider et al., 2005 ). Therefore, male production and Hb induction appear to be coupled at some level by JHA signaling (see below).

Studies with MF demonstrate that D. magna and D. pulex produce male broods in a dose-dependent manner, with concentrations above 300 n M resulting in all male broods ( Olmstead and LeBlanc, 2002 ; personal observation). Furthermore, the sensitive period for MF induction of male development is 1–2 days before egg laying during the vitellogenic period of oogenesis. We have studied the transcriptional effects on genes from developmentally staged females exposed to MF, by using a microarray platform described elsewhere (Colbourne et al. , submitted). Briefly, females from a single mother were kept in a common garden until their first broods were released. Then, 20 individuals were randomly selected for a 3-day exposure to either 400 n M MF (Echelon Biosciences, Salt Lake City, UT, USA, changed daily) or to methanol carrier (0.004%). Three replicates of each condition were used for RNA isolation, cDNA labeling and hybridization, and analysis according to standard protocols ( http://dgrc.cgb.indiana.edu/microarrays/support/protocols.html ).

Compared to controls, MF-treated animals showed higher steady-state levels of 39 unique transcripts and lower levels of 16 transcripts ( Table 1 ). Twenty-two differentially regulated genes shared significant sequence homologies with proteins archived in the NCBI non-redundant database. In particular, Hb and neuronal acetylcholine receptor transcripts were elevated under MF treatment, in addition to arginine kinase, amylase, cytochrome c oxidase and cytochrome b , several cuticle proteins, actin and a putative ribosomal biogenesis regulatory protein. Control animals had higher levels of glucosamine-6-phosphate deaminase, glucose-6-phosphatase, several proteases, an actin de-polymerizing enzyme (profilin), a putative receptor of unknown function and a homolog of the Rheb G-protein-coupled receptor. Another transcript with lower levels during treatment had putative homology to an ecdysteroid-regulated transcript from Manduca Sexta (GenBank accession AAB08704.1 ). Several other genes differed in response to MF, but whose putative homologs in arthropods (such as the Drosophila melanogaster genes CG6770 and CG31997) have no known function. Subsequent study of these genes may reveal whether they have conserved roles in JH response. Several dozen genes with no sequence similarity to entries in the non-redundant protein database showed differential expression as well.

Taken together, results from this microarray experiment are consistent with current data for MF activity in crustaceans and also offer new avenues for exploration ( Figure 1 ). First, higher steady-state levels of Hb are consistent with transcriptional control of this protein, an area of active investigation explored further below. Second, changes in polysaccharide metabolism-related genes, cuticle genes and cytoskeletal protein genes imply a coordinated shift in carbohydrate usage away from ATP production and towards chitin metabolism. Physiological and biochemical studies would be useful to uncover such systemic changes in metabolism in response to JHA treatment. Furthermore, alteration of chitin metabolism underscores connections between MF and ecdysis due to genetic crosstalk with ecdysone pathways ( Mu and LeBlanc, 2004 ). Third, involvement of the rheb receptor in response to MF is, to our knowledge, a novel observation linking this protein to a crustacean hormonal response. In D. melanogaster , Rheb participates in cell growth and proliferation via the S/G1 mitotic transition, in addition to starvation response ( Patel et al., 2003 ). These biological functions in flies are consistent with a role for Rheb in Daphnia related to molting. Fourth, an intriguing and under-explored connection between secretion of JHA and cholinergic regulation is highlighted by increased acetylcholine receptor mRNA levels under MF treatment. Research into molecular mechanisms of cholinergic hormonal control is only beginning to be investigated in arthropods ( Kuo, 2002 ). Genomic tools available in Daphnia may help to inform research in suitable biochemical and physiological crustacean models of both rheb and cholinergic pathway interactions with JHA. Finally, genes of unknown function that are expressed constitutively in response to MF may be members of the MF pathway, and could be interesting targets of subsequent studies such as time course, dose–response and exposure to other JHA.

Schematic depicting the methyl farnesoate pathway in male production and hemoglobin induction. For further discussion, see Laufer and Biggers (2001) and Mu and LeBlanc (2004) . Arrows (induction or production) and blocked lines (repression) may be direct or indirect effects (possibly both, indicated by dashed lines). Sequence and expression data have been obtained from cDNAs encoding farnesoic acid methyltransferase, two CHH peptides and homologs of D. melanogaster sex determination genes sex lethal ( Sxl ) and transformer-2 ( Tra-2 ) (data not shown).

The MF pathway is an early example of genomic studies investigating the mechanism for environmental sex determination in natural settings. Current work in our laboratories includes additional microarray experiments using both wild-type and naturally occurring isolates that never produce males and are thus unresponsive to MF. Mapping panels from crosses between male and non-male producers will help identify QTL for this trait, which could subsequently be used to study ecological correlates of genetic polymorphisms.

Genomics of environmental stress responses

Research in Daphnia has long focused on the environmental stresses they encounter and mechanisms they use to counter or mitigate them. Prominent among these areas are toxicology, hypoxia, temperature, parasites, predators and ultraviolet radiation. Responses to these challenges have been documented at the level of physiology, genetics, maternal effects, morphology, behavior and life history. Adequately addressing these areas is beyond the scope of this review; our aim instead is to highlight recent progress in uncovering molecular mechanisms underlying these changes.

Acute hypoxia in Daphnia leads to compensation via increased heart rate or ventilation rate ( Pirow et al., 1999 ), but chronic hypoxia leads to induction of Hb synthesis. This synthesis is subunit-specific ( Kimura et al., 1999 ), with changes over time occurring at subunit-specific rates ( Zeis et al., 2003 ). Levels of mRNA for subunits hb2 and hb3 are rapidly and highly induced during hypoxia, while hb1 is unaffected ( Zeis et al., 2003 ). These results, plus the identification of putative binding sites for the mammalian hypoxia-inducible factor 1 (HIF-1; Kimura et al., 1999 ), led to a detailed investigation of Hb gene promoters. Induction of Hb gene expression during hypoxia is dependent on the binding of HIF to particular hypoxia response elements (HRE) in promoter regions of the four known D. magna Hb genes ( Gorr et al., 2004 ). Three HREs upstream of the globin-2 gene were found using heterologous transfection of HIF-expressing human and D. melanogaster cells, showing specific binding of HIF complexes at two sites in vitro and binding of an unknown constitutive transcription factor at the third site ( Gorr et al., 2004 ). A recent report ( Gorr et al., 2006 ) has identified a putative juvenoid response element (JRE) in the promoter of hb2 in D. magna that binds an activated factor in response to JHA. This development is exciting for several reasons. Despite decades of intense interest and research into mechanisms of JHA action in arthropods, a nuclear hormone receptor for JHA has not been identified with certainty. The JRE is a powerful tool to establish the identity of the JHA receptor, which will undoubtedly stimulate molecular genetic research into pathways of JHA activity. These findings also link mechanisms for the ecologically relevant traits of male production and Hb induction for the first time.

The recent discovery of two additional members of the D. magna globin family using Southern blotting ( Nunes et al., 2005 ) indicates that many more discoveries will emerge from functional studies of these molecules. Studies of Daphnia populations that are locally adapted to oxygen-rich or oxygen-starved habitats may help elucidate the ecological implications of lower mass-specific oxygen consumption rates in hypoxia-acclimated animals ( Seidl et al., 2005 ) for community structure and trophic interactions. Furthermore, increased O 2 delivery due to higher Hb content incurs physiological and fitness costs via increased protein synthesis and predation ( Pirow et al., 2001 ), providing an opportunity to link physiological ecology with molecular mechanisms such as HIF binding.

In toxicogenomics, research into endocrine disrupting chemicals such as the JHA compounds discussed above continues to be a high priority ( Oetken et al., 2004 ). In this regard, Daphnia will be useful for elucidating gene expression patterns and associated ecological changes in response to these challenges. The difficulty and potential of combining a genomics approach with environmental monitoring has been a subject of intense interest (for example, Lettieri, 2006 ). Some conceptual issues are as follows: (1) How specific are mechanisms of toxicity or clearance, as revealed by the genes and pathways responding to toxicants? (2) How sensitive and useful is expression profiling at uncovering those genes or pathways? (3) Are traditional toxicological approaches (for example, end points, LC 50 s, dose–response curves) optimal for genomic assays? (4) Are genomic responses to toxicants indicative of changes within a population and ultimately the state of its ecosystem? Data from groups performing toxicological genomics in Daphnia , although preliminary, bear directly on some of these questions. Future studies using more complete microarray platforms with tens of thousands instead of thousands of probes will provide interesting insight into variation among toxicological conditions. Yet, the trends described here are universal enough to provide a meaningful picture.

First, while ‘stress response’ genes or pathways tend to be induced consistently across physiological challenges as diverse as metal toxicity, ordinance (explosive) contamination or nanoparticle exposure, each challenge also provides a unique signature. These observations imply that certain types of molecular response, such as induction of a particular class of chaperones, may be diagnostic of organisms under stress. Also, condition-specific responses are likely, depending on experimental protocols (for example, exposure times and dosages) and the life stages of the animals. Second, low (sub-lethal and environmentally relevant) chronic exposures generally can induce measurable changes in gene expression. Low-dose exposures, along with exposure to simultaneous challenges, are often used as experimental tools for discerning potential mechanisms of toxicity ( Lettieri, 2006 ). Therefore, transcriptional profiling should be useful, in combination with data from other sources, to uncover how toxins work. Beyond that, how genetic background of particular clones might affect outcomes (see Lopes et al., 2006 ), the importance of trans-generational (maternal or grand-maternal) effects, and how sequence, gene expression and/or physiological response vary are unanswered questions.

A recent study of the effect of the fungicide propiconazole on D. magna ( Soetaert et al., 2006 ) illustrates the challenges and potential of environmental monitoring using microarrays. Propiconazole resembles other chemicals studied for toxicity and their effects on development ( Kast-Hutcheson et al., 2001 ), such as diethylstilbestrol ( Baldwin et al., 1995 ) and nonylphenol ( Shurin and Dodson, 1997 ), which kill or deform embryos, often at concentrations similar to those in the environment. Transcriptional profiling of propiconazole treatment of 4- and 8-day-old animals using microarrays constructed from age-specific cDNA libraries established differential expression of over 10% of the arrayed genes ( Soetaert et al., 2006 ). Gene expression varied with both time and dose, which underscores the difficulty of choosing appropriate end points for such studies. However, results are encouraging for a research community poised to apply microarrays to discover the condition dependency of responses to toxicants. For example, transcripts related to development and reproduction were differentially expressed, such as an eightfold decrease in transcript levels of the primary yolk protein vitellogenin. Also, some transcripts either had homologs of unknown function in other organisms or appeared unique to Daphnia . Profiling gene expression across a range of environmentally relevant conditions should elucidate cellular and organism level processes that are vital within natural settings, but that are not typically elicited in laboratory studies of traditional model organisms.

It would be interesting to extend the above observations by profiling offspring from mothers given a propiconazole exposure, which affects embryonic development in Daphnia ( Kast-Hutcheson et al., 2001 ). The quality of the maternal environment influences traits such as size at birth and number of offspring ( Sakwinska, 2004 ), strain-specific immunity to bacterial parasites ( Little et al., 2003 ) and energy allocation to offspring ( LaMontagne and McCauley, 2001 ). Interestingly, mothers reproducing in poor environments produced offspring more than twice as resistant to bacterial infection as mothers in favorable environments ( Mitchell and Read, 2005 ). These findings underscore an important requirement for systematic approaches to quantify sources and consequences of both genetic and environmental variability in ecological studies ( Forbes, 1998 ).

Maternal effects have been experimentally addressed during exposure to predator kairomones, some effects of which occur across generations. The formation of defensive structures like helmets and neckteeth in response to kairomones is a classic example of cyclomorphosis, and depends upon maternal environment to an even greater extent than in the late embryonic environment ( Agrawal et al., 1999 ). A study of heat-shock protein (HSP) induction and the actin and tubulin cytoskeleton in predator-exposed Daphnia ( Pijanowska and Kloc, 2004 ) demonstrated particular maternal effects from kairomone exposure. Upon exposure to the invertebrate predator Chaoborus or to fish kairomones, HSP40, HSP60 and HSP70 all decreased in expression. Using anti-phosphohistone 3 as a control also showed that α -tubulin and actin protein levels decreased in exposed animals, and that tubulin expression decreased in F1 progeny of exposed animals compared to F1 from control animals. Interestingly, Cu and Zn both reduced neckteeth induction in Daphnia neonates exposed to predator kairomones ( Hunter and Pyle, 2004 ), indicating interference by metals in the cyclomorphosis signaling pathway. Employing combinations of different environmental stressors has considerable promise for dissecting signaling pathways and molecular responses to these challenges.

A powerful tool for microevolutionary studies is the hatching of dormant diapause embryos from sediment core samples of ponds and lakes, dubbed ‘resurrection ecology’ ( Kerfoot and Weider, 2004 ). Implications of migration through time, or the effects of overlapping generations on population dynamics, are an active research area (for example, Cáceres and Tessier, 2004 ). In addition, hatching dormant propagules may compare ancestral populations and their descendents, for example before and after introduction of a novel predator or toxicant, to measure genetic differences related to the environmental change ( Weider et al., 1997 ; Hairston et al., 1999 ). For instance, a Daphnia population exposed to variable and well-documented fish predation over 30 years was examined for changes in an adaptive quantitative trait (phototactic behavior, related to diel vertical migration and thus predator avoidance) and in neutral genetic markers ( Cousyn et al., 2001 ). Results demonstrated increased behavioral plasticity in response to predator kairomones, and order of magnitude higher genetic differentiation for the trait than for neutral markers, consistent with a strong role for selection as the driver of rapid, directional changes. Another example demonstrated evolution of resistance to toxic food sources (cyanobacteria) through a reduction in phenotypic plasticity over time ( Hairston et al., 2001 ). In a similar way, combining molecular biology and genomics with paleolimnology may test hypotheses about adaptation in populations exposed to severe metal contamination ( Pollard et al., 2003 ). Monitoring changes in allele frequencies within naive and post-exposed populations is possible even with unhatched or gravely degraded embryos ( Limburg and Weider, 2002 ); the greatest limitation, until now, was determining the sequence identity of candidate genes that have possibly evolved structurally in response to the documented changes in the environment.

Understanding genome structure in light of the stress of ecological competition (for example, for limited carbon or phosphorus) is a primary motivation for research into the functional consequences of rDNA length and copy number, termed biological stoichiometry ( Weider et al., 2005 ). Interest in rDNA per se stems from mechanistic links between copy number and intergenic spacer (IGS) length, and their effects on organismal growth rate and phosphate requirements ( Weider et al., 2005 ). The growth rate hypothesis (GRH) posits that N:P and C:P ratios reflect allocation of P to ribosomal RNA, with higher rates demanding more P for rRNA production and protein synthesis ( Elser et al., 2000 ). Predictions based on the GRH have been supported by numerous studies ( Weider et al., 2005 ), and environmental P concentrations are frequently low enough to limit growth of Daphnia ( Anderson and Hessen, 2005 ). It is not clear whether stressful conditions due to poor food quality (low P content) are sufficient to drive the evolution of rDNA repeat length or copy number. However, evidence from laboratory experiments indicates that this may be the case. For example, by selecting for divergent weight-specific fecundity in D. pulex , Gorokhova et al. (2002 ) were able to demonstrate significant changes in age of first reproduction, size of first clutch, RNA and phosphorus content and IGS length after only five generations.

The growing genomics toolbox for Daphnia is providing opportunities to extend laboratory research from the molecular basis of morphological, physiological and behavioral differences to questions addressing the degree and importance of genetic variation within natural populations. Although Daphnia genomics research is still nascent, understanding the function of key genetic networks modulating traits with clear ecological relevance is advancing. For instance, microarray experiments and sequence analysis are uncovering members of these networks with characterized functions, plus genes that are apparently conserved across arthropods, but have no known function. Other genes are presumably unique in Daphnia . Indeed, at the recent Daphnia Genomics Consortium meeting, we presented data suggesting that at least 57% of over 12 600 cDNA sequences for D. pulex showed no similarity to protein sequences archived within the NCBI non-redundant database.

The structure and function of a genome is a product of the genome's environment, with interactions occurring at multiple scales. Current work – on genes linked within asexual genomes, on genes locked within a co-evolutionary arms race against parasites, and on multiple gene families whose copy numbers may be tied to ecological processes – is paving the way for genome-wide searches for signatures of selection on genomic elements. For example, the attributes of species are important in the macro-evolutionary processes acting on genome composition. These signatures range from adaptive amino-acid substitutions in genes associated with differences between humans and chimpanzees ( Clark et al., 2003 ) to the distribution of elements such as introns and transposable elements as a function of long-term effective population size differences among some major divisions of life ( Lynch and Conery, 2003 ). In a complementary way, research on populations of Daphnia will provide an important micro-evolutionary perspective. This view comes from studying genomic variation at local geographic scales and during recent demographic history, such as the latest demonstration that obligately asexual Daphnia lineages suffer increased deleterious mutations compared to sexual lineages ( Paland and Lynch, 2006 ), which has clear evolutionary implications.

Accession codes

Genbank/embl/ddbj.

NP_600374.1

XP_313747.1

XP_392564.1

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Acknowledgements

We apologize to those whose work could not be cited because of space limitations, and thank the editors for an opportunity to contribute to this special volume on Ecological Genomics. Our work is supported in part by National Science Foundation grants (EF-0328516, DEB-0221837) and by the Center for Genomics and Bioinformatics via the METACyt Initiative and the Indiana Genomics Initiative under the Lilly endowment. This research is also made possible by the Daphnia Genomics Consortium.

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Eads, B., Andrews, J. & Colbourne, J. Ecological genomics in Daphnia : stress responses and environmental sex determination. Heredity 100 , 184–190 (2008). https://doi.org/10.1038/sj.hdy.6800999

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Received : 26 April 2006

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DOI : https://doi.org/10.1038/sj.hdy.6800999

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Zooplankton study challenges traditional views of evolution

by Richard Harth, Arizona State University

In new research, Arizona State University scientists and their colleagues investigated genetic changes occurring in a naturally isolated population of the water flea, Daphnia pulex. This tiny crustacean, barely visible to the naked eye, plays a crucial role in freshwater ecosystems and offers a unique window into natural selection and evolution.

Their findings , reported in the current issue of the journal Proceedings of the National Academy of Sciences , rely on a decade of research. Using advanced genomic techniques, the research team analyzed DNA samples from nearly 1,000 Daphnia.

They discovered that the strength of natural selection on individual genes varies significantly from year to year, maintaining variation and potentially enhancing the ability to adapt to future changing environmental conditions by providing raw material for natural selection to act on.

In seemingly stable environments, there is significant fluctuation in the frequency of gene variants known as alleles at specific chromosomal regions over time, even if the overall strength of selection remains near zero on average over many years. This suggests that such genetic variation allows populations to remain adaptable to environmental changes.

"This study has, for the first time, given us a glimpse into the kinds of temporal changes in gene frequencies that occur even in seemingly constant environments, a sort of ongoing churn of genetic variation distributed across the genome," says Michael Lynch, lead author of the new study.

Lynch is the director of the Biodesign Center for Mechanisms of Evolution and professor in the School of Life Sciences at ASU. Additional researchers on the study include colleagues from ASU, Central China Normal University and the University of Notre Dame.

The power of selection

Daphnia, a form of zooplankton, have fascinated biologists for centuries due to their crucial role in aquatic ecosystems and ability to adapt to environmental stressors. In addition to their value for multigenerational genetic research, Daphnia are widely used model organisms for freshwater toxicity testing because they have a rapid asexual reproductive cycle and are sensitive to various environmental pollutants.

The tiny creatures are a vital food source for fish and help keep algae growth in check. Their ability to adapt quickly to environmental changes could hold clues for how other species—including those important to human food supplies—might respond to pollution, climate change and other human-induced stressors.

Most of the sites examined on the Daphnia genome were shown to experience changing selection pressures over the study period. On average, these pressures tend to balance out to have little overall effect, meaning that no single direction of selection consistently dominates over time. Instead, the genetic advantages or disadvantages of specific traits change from one period to the next.

These findings challenge the traditional belief that measuring genetic diversity (the range of different traits in a population) and genetic divergence (the differences between populations) can easily show how natural selection is consistently operating. Instead, natural selection seems to operate with greater subtlety and complexity than previously thought.

Rethinking genetic variation

The study breaks new ground by pinpointing when and where selection pressures occur within the genome. Other than traits known to be strongly influenced by natural selection , there is little information on how allele frequencies change over time in natural populations.

The multiyear, genome-wide analysis of nearly 1,000 genetic samples from a Daphnia pulex population shows that most genetic sites experience varying selection, with an average effect close to zero, indicating little consistent selection pressure over different times and selection spread across many genomic regions.

These findings challenge the usual understanding of genetic diversity and divergence as indicators of random genetic drift and selection intensity.

Variation and survival

The observed patterns of selection on various gene sites provide a mechanism for maintaining genetic diversity , which is essential for rapid adaptation. The study also revealed that genes located near each other on chromosomes tend to evolve in a coordinated manner. This linkage allows beneficial combinations of gene variants to be inherited together, potentially accelerating the adaptation process.

This effect could help explain how species sometimes adapt faster than scientists would normally expect. On the other hand, the same phenomenon may result in deleterious alleles being swept to higher frequencies by linked beneficial alleles, reducing the overall efficiency of selection in some cases.

The study shows that evolution is more dynamic and complex than previously appreciated. The environment's influence on genes changes frequently, possibly helping species keep the genetic variety needed to adapt to future conditions. This new understanding may prompt scientists to rethink how they study evolution in the wild.

While the study focused on Daphnia pulex, the findings may have implications for understanding how other species might respond to rapid environmental changes, including those driven by human activities, such as pollution and climate change. Assessing the stability of allele frequencies in more stable environments is an important preliminary step. Such studies are critical, as laboratory experiments alone cannot duplicate the complexity of environmental influences acting on wild populations.

Further, understanding how Daphnia evolve may provide insights into the resilience of entire ecosystems. This knowledge could help researchers predict and potentially mitigate the impacts of environmental changes on biodiversity and food webs.

As the world grapples with an accelerating environmental crisis, studies like this one provide crucial insights into nature's capacity for resilience and adaptation. By continuing to study these tiny creatures, the scientists hope to better understand the fundamental mechanisms of evolution and apply these lessons to broader ecological and conservation efforts.

Journal information: Proceedings of the National Academy of Sciences

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February 3, 2011

Copious Genes of Tiny Water Flea Promise a Leap in Understanding Environmental Toxins

With the Daphnia genome in hand, scientists hope to put this key environmental indicator species to even better work

By Katherine Harmon

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Not far from milepost 200 on a stretch of the Pacific Coast Highway near the Oregon Dunes National Recreation Area is a humble water hole known in some biology circles as Slimy Log Pond. It was from this inauspicious pool that a water flea ( Daphnia pulex ) dubbed The Chosen One was plucked in 2000, and became the first crustacean to have its genome sequenced. Analysis of The Chosen One's genome shows that this Lilliputian crustacean contains the most genes of any animal sequenced to date. It also has the potential to accelerate scientists' understanding of synthetic chemicals' effects on the environment —and human health. The world's most common small freshwater feeder—gobbling up algae in lakes and ponds the world over— Daphnia are also a staple in fishes' diets, proving a crucial link in food webs. This miniscule animal—barely visible to the naked eye—has long been an invaluable aquatic indicator species and is used by agencies across the globe to take stock of the health of freshwater systems. As such a well-studied species, Daphnia are poised to become a key model organism to delve deeper in the study of environmental genomics. Improved understanding of the interactions among genes and the environment could also diminish the deleterious effects of chemicals on human health as well. The sequence details , published online February 3 in Science , turned up the most shared genes with humans of any arthropod that has been sequenced to date. This genetic overlap means that the sentinel species could also end up being "a surrogate for humans to show the effects of the chemicals on shared pathways," says John Colbourne of Indiana University Bloomington's Center for Genomics and Bioinformatics and the lead author on the new paper. "The majority of the genome is a reflection of how the animal has evolved to cope with environmental stress." Previous genetic snapshots of Daphnia have hinted at its overall makeup. But whole genome sequences provide "much better information about the function of genes, and allow us to be much more comprehensive in understanding the effects of toxicants," says Chris Vulpe of the Nutritional Science and Toxicology group at University of California, Berkeley, who was not involved in the new study. "It really adds to your ability to understand what's going on" in the environment. Muddy biology Named for the Greek mythological nymph Daphne (who shuns the god Apollo's advances and in Ovid's telling was transformed into a tree), aquatic Daphnia , with its gently branching antennae, generally reproduce without males by passing along a diploid genome (a complete set of chromosomes) to offspring. This consistency creates clone lines, making them excellent candidates for laboratory study. But like the water they often live in, these crustaceans "have a really muddy biology," Colbourne says. That murkiness, however, has turned out to be fertile territory for genetic research, he notes. "The genome is a lot more plastic and a lot more responsive to the environment than we had given it credit for." Researchers working to sequence Daphnia —as part of the Daphnia Genomics Consortium —were expecting to find one about the size of the fruit fly , with its 14,000 genes. So they were stunned to find that the D. pulex genome contains at least 30,907 genes—nearly 8,000 more than the human genome . Some 36 percent of these genes have not previously been identified in any other organism. And researchers found that rather than being evolutionary deadweight, most of these unfamiliar genetic signatures "tend to be the genes that are most responsive to Daphnia 's ecology," Colbourne says. Not all of the crustacean's genes are active at any given time. Rather, a large portion of them are switched on or off with changes in the flea's environment. They are "more or less environment-specific," Colbourne says. Although they are "coding for the same proteins, they're being expressed differently depending on what environmental stresses you expose the animal to." And finding the genes that allow the animal to tolerate outside stressors—whether they are chemicals or UV radiation—could help researchers search for parallel pathways in humans. Modeling complexity One of the reasons the Daphnia genome contains so many genes, the researchers found, is because gene duplication in this species occurs at a much higher rate than in other familiar species—about 30 percent higher rate than in humans and about three times the rate in fruit flies. "There's obviously a selective advantage to having so many genes," Colbourne says. "We were able to discover for the very first time that newly duplicated genes can acquire new functions very, very rapidly." In other species duplicate genes tend to become harmful or irrelevant and thus get weeded out quickly. Daphnia genes stick around longer, suggesting that they are often put to good use—and quickly—responding to environmental factors. In the past, diploid Daphnia have been bred in the lab to cut down on extraneous genetic material that, in the wild, is necessary for their mainly mateless reproductive strategy . But this artificial inbreeding is less than desirable for researchers who are looking to study gene-environment interactions . So members of the research project launched a transcontinental search to find a specimen that was naturally inbred. What they found in The Chosen One was just that—a Daphnia in which "nature has gotten rid of all the bad alleles," simplifying the genome without losing its ecologically attuned adaptations, Colbourne says. This little arthropod and her progeny received such a grand nickname as another Slimy Log Pond candidate line—now known as The Rejected One—was being sidelined. In preliminary analyses The Rejected One was found to have a genome that is quite heterozygous (with more differentiated alleles), Colbourne recalls. Its radical differences, however, did allow for some useful comparison with The Chosen One. "There was actually some great science that was done because of the Rejected One, although it created quite a heart attack in the community," he says. Although D. pulex is the most common species, others, such as D. magna and Ceriodaphnia dubia are usually called on in standardized water quality tests. The D. magna genome sequence is currently in the pipeline, says Vulpe, who uses that species in his research and is part of the larger consortium. For his research, the D. pulex genome "has been an incredible boon to be able to compare and help us understand what's happening" in the D. magna genetics. Beyond death With its substantial genome now decoded, Daphnia might soon play an even more integral role in environmental assessment—beyond simple tests for dissolved oxygen or excessive chlorine. Only a small fraction of tens of thousands of man-made chemicals have been tested for safety, and then they are usually only tested as isolated compounds—rather than in more realistic amalgamations as they often crop up in the environment. "We have so many damn chemicals," Vulpe says. "We're concerned about their effect on humans and on ecosystems ." But with so much analysis that remains to be done, "there's no way that our current methods of screening for the danger of these chemicals can catch up," Colbourne says. If Daphnia prove to be a solid model organism to study the effects of chemicals and environment on genes, they could enable a more efficient high-throughput process for assessing chemicals. The relatively new field of "ecotoxicogenomics"—which Vulpe admits "doesn't really roll off the tongue very well"—is working to catch up to more biologically based genetics. But with the genome sequence of Daphnia , he hopes that it will allow the field to catch up. "We have the sequence of the mouse and human—and we can use genomics in a very powerful way—but unfortunately this has lagged behind in these eco-indicator species." Bringing a genomic approach to studying toxicology promises to create a more "mechanistic understanding" of the field, Colbourne says. Vulpe explains that toxicology has relied on the "kill 'em and count 'em" approach, in which death was the primary endpoint in a chemical's dosage assessment: "We previously asked the question: Did they die?" he says. As researchers are now starting to be able to suss out particular genetic pathways, "it might help us consider endpoints that we hadn't considered," Vulpe adds, such as how chemicals are having more nuanced effects on reproductive or immune systems. Daphnia are of course not a perfect foil for studying chemicals' potential effects on human biology, and their use as screening organisms will have to be validated by further research. "But it's certainly exciting that there is a similarity," Vulpe says. "Who would have thought that a little crab would have been similar to people?"

Study challenges traditional views of evolution

Research uncovers how environmental changes influence genetic variation over time.

Daphnia, a form of zooplankton, have fascinated biologists for centuries due to their crucial role in aquatic ecosystems and ability to adapt to environmental stressors. A new study explores DNA samples from nearly 1,000 Daphnia, revealing new subtleties in the evolutionary processes of natural selection. Graphic by Jason Drees

In new research, Arizona State University scientists and their colleagues investigated genetic changes occurring in a naturally isolated population of the water flea, Daphnia pulex. This tiny crustacean, barely visible to the naked eye, plays a crucial role in freshwater ecosystems and offers a unique window into natural selection and evolution.

Their findings , reported in the current issue of the journal Proceedings of the National Academy of Sciences (PNAS), rely on a decade of research. Using advanced genomic techniques, the research team analyzed DNA samples from nearly 1,000 Daphnia. 

They discovered that the strength of natural selection on individual genes varies significantly from year to year, maintaining variation and potentially enhancing the ability to adapt to future changing environmental conditions by providing raw material for natural selection to act on.

In seemingly stable environments, there is significant fluctuation in the frequency of gene variants known as alleles at specific chromosomal regions over time, even if the overall strength of selection remains near zero on average over many years. This suggests that such genetic variation allows populations to remain adaptable to environmental changes. 

“This study has, for the first time, given us a glimpse into the kinds of temporal changes in gene frequencies that occur even in seemingly constant environments, a sort of ongoing churn of genetic variation distributed across the genome,” says Michael Lynch , lead author of the new study.

Lynch is the director of the  Biodesign Center for Mechanisms of Evolution and professor in the  School of Life Sciences at ASU. Additional researchers on the study include colleagues from ASU, Central China Normal University and the University of Notre Dame.

The power of selection

Daphnia, a form of zooplankton, have fascinated biologists for centuries due to their crucial role in aquatic ecosystems and ability to adapt to environmental stressors. In addition to their value for multigenerational genetic research, Daphnia are widely used model organisms for freshwater toxicity testing because they have a rapid asexual reproductive cycle and are sensitive to various environmental pollutants.

The tiny creatures are a vital food source for fish and help keep algae growth in check. Their ability to adapt quickly to environmental changes could hold clues for how other species — including those important to human food supplies — might respond to pollution, climate change and other human-induced stressors.

Most of the sites examined on the Daphnia genome were shown to experience changing selection pressures over the study period. On average, these pressures tend to balance out to have little overall effect, meaning that no single direction of selection consistently dominates over time. Instead, the genetic advantages or disadvantages of specific traits change from one period to the next.

These findings challenge the traditional belief that measuring genetic diversity (the range of different traits in a population) and genetic divergence (the differences between populations) can easily show how natural selection is consistently operating. Instead, natural selection seems to operate with greater subtlety and complexity than previously thought. 

Rethinking genetic variation

The study breaks new ground by pinpointing when and where selection pressures occur within the genome. Other than traits known to be strongly influenced by natural selection, there is little information on how allele frequencies change over time in natural populations.

The multiyear, genome-wide analysis of nearly 1,000 genetic samples from a Daphnia pulex population shows that most genetic sites experience varying selection, with an average effect close to zero, indicating little consistent selection pressure over different times and selection spread across many genomic regions. 

These findings challenge the usual understanding of genetic diversity and divergence as indicators of random genetic drift and selection intensity.

Variation and survival

The observed patterns of selection on various gene sites provide a mechanism for maintaining genetic diversity, which is essential for rapid adaptation. The study also revealed that genes located near each other on chromosomes tend to evolve in a coordinated manner. This linkage allows beneficial combinations of gene variants to be inherited together, potentially accelerating the adaptation process. 

This effect could help explain how species sometimes adapt faster than scientists would normally expect. On the other hand, the same phenomenon may result in deleterious alleles being swept to higher frequencies by linked beneficial alleles, reducing the overall efficiency of selection in some cases.  

The study shows that evolution is more dynamic and complex than previously appreciated. The environment's influence on genes changes frequently, possibly helping species keep the genetic variety needed to adapt to future conditions. This new understanding may prompt scientists to rethink how they study evolution in the wild. 

While the study focused on Daphnia pulex, the findings may have implications for understanding how other species might respond to rapid environmental changes, including those driven by human activities, such as pollution and climate change. Assessing the stability of allele frequencies in more stable environments is an important preliminary step. Such studies are critical, as laboratory experiments alone cannot duplicate the complexity of environmental influences acting on wild populations. 

Further, understanding how Daphnia evolve may provide insights into the resilience of entire ecosystems. This knowledge could help researchers predict and potentially mitigate the impacts of environmental changes on biodiversity and food webs.

As the world grapples with an accelerating environmental crisis, studies like this one provide crucial insights into nature's capacity for resilience and adaptation. By continuing to study these tiny creatures, the scientists hope to better understand the fundamental mechanisms of evolution and apply these lessons to broader ecological and conservation efforts.

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Daphnia magna model in the toxicity assessment of pharmaceuticals: A review

Affiliations.

• 1 Institute of Biological Bases of Animal Production, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland. Electronic address: [email protected].
• 2 Department of Hydrobiology and Protection of Ecosystems, University of Life Sciences in Lublin, Dobrzańskiego 37, 20-62 Lublin, Poland. Electronic address: [email protected].
• 3 Chair and Department of Toxicology, Faculty of Pharmacy, Medical University of Lublin, Jaczewskiego 8b, 20-090 Lublin, Poland. Electronic address: [email protected].
• 4 Institute of Biological Bases of Animal Production, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland. Electronic address: [email protected].
• 5 Institute of Biological Bases of Animal Production, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland. Electronic address: [email protected].
• PMID: 33127157
• DOI: 10.1016/j.scitotenv.2020.143038

Daphnia magna is one of the most commonly used model organism to assess toxicity of wide range of pharmaceuticals such as antibiotics, anticancer drugs, antidepressants, anti-inflammatory drugs, beta-blockers and lipid-regulating agents. Currently, daphnia toxicity tests based on immobilisation and lethality standardised by OECD, acute immobilisation test and reproduction test, are mainly used in toxicological studies. Detailed analysis of Daphnia biology allows distinguishing the swimming behaviour and physiological endpoints such as swimming speed, distance travelled, hopping frequency, heart rate, ingestion rate, feeding rate, oxygen consumption, thoracic limb activity which could be also useful in assessment of toxic effects. The advantage of behavioural and physiological parameters is the possibility to observe sublethal effects induced by lower concentrations of pharmaceuticals which would not be possible to notice by using OECD tests. Additionally, toxic effects of tested drugs could be assessed using enzymatic and non-enzymatic biomarkers of daphnia toxicity. This review presents scientific data considering characteristics of D. magna, analysis of immobilisation, lethality, reproductive, behavioural, physiological and biochemical parameters used in the toxicity assessment of pharmaceuticals. The aim of this paper is also to emphasize usefulness, advantages and disadvantages of these invertebrate model organisms to assess toxicity of different therapeutic classes of pharmaceuticals. Also, various examples of application of D. magna in studies on pharmaceutical toxicity are presented.

Keywords: Behavioural endpoints; Biochemical parameters; Heart rate; Oxygen consumption; Physiological endpoints.

Copyright © 2020 Elsevier B.V. All rights reserved.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Daphnia ’s challenge: survival and reproduction when calcium and food are limiting

Corresponding editor: Karl Havens

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Alicia Pérez-Fuentetaja, Fawn Goodberry, Daphnia ’s challenge: survival and reproduction when calcium and food are limiting, Journal of Plankton Research , Volume 38, Issue 6, 25 November 2016, Pages 1379–1388, https://doi.org/10.1093/plankt/fbw077

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Calcium levels have declined in boreal lakes in North America and Europe due to soil mineral leaching, logging and climate change. Crustacean zooplankton species with high-calcium demand such as Daphnia , are particularly vulnerable to calcium-related stress. In a factorial design, we tested the effects of three calcium concentrations (2.5, 1.0 and 0.5 mg/L Ca) and two food levels (high = 1.67 mg/L C and low = 0.16 mg/L C) on second-generation calcium-stressed Daphnia pulex  ×  pulicaria . Calcium limitation affected reproduction, molting and population growth, but food quantity was also relevant to how Daphnia dealt with the lack of calcium. When adequate levels of calcium were available (2.5 mg/L Ca), population growth was similar at high and low food, however, individual Daphnia produced fewer neonates at low food. Under high food and low calcium, Daphnia ’s life-history strategy focused on reproduction, with a negative effect on survivorship due to calcium limitation. Alternatively, under low food and low calcium, their strategy was survival and somatic maintenance, minimizing reproduction. Boreal lakes with modest levels of calcium may support Daphnia populations during periods with sufficient food, but if food quantity or quality is too low to mitigate the effects of calcium limitation, Daphnia populations could disappear due to low reproductive output.

Daphnia species dominate the first consumer level in many temperate freshwater ecosystems and link algae with higher trophic levels of invertebrate and fish predators. Because of their abundance and unique trophic position, Daphnia are an efficient conduit for the transfer of energy and nutrients up the food web. One of the vital nutrients that moves up through trophic interactions is calcium, which is needed by consumers for a variety of functions from skeletal support to metabolism, although they can also absorb soluble calcium from water ( Simmons, 1971 ). Natural concentrations of calcium in boreal lakes, particularly in lakes from Scandinavia ( Hessen and Alstad Rukke, 2000b ) and the Laurentian Shield in Canada ( Keller et al ., 2001 ), have been steadily decreasing since the advent of the industrial era, mainly due to the effects of depletion of the base-cation pools from soils due to acid rain impact and recovery, intensive logging ( Likens et al ., 1996 ; Watmough and Dillon, 2003 ; Akselsson et al ., 2007 ) and climate change ( Yao et al ., 2011 ). This is particularly problematic for the Daphnia in these lakes because they have calcium requirements that are orders of magnitude higher than other crustacean zooplankton. Daphnia allocate about 90% of the calcium they acquire to their carapace ( Porcella et al ., 1969 ), and this calcium is mostly obtained from the surrounding water and minimally from diet. In addition to calcium's structural role, Daphnia also require 8–26% of the calcium obtained for physiological processes such as growth, egg production, formation of soft tissues, cell signaling and muscle contractions ( Porcella et al ., 1969 ). Because Daphnia molt their carapace regularly and they can only store less than 10% of their calcium during molting ( Alstad et al ., 1999 ), Daphnia have a critical requirement for this mineral, which must be available in the surrounding medium to support their physiology. In fact, when water calcium levels decrease, total body calcium concentration in daphniids decreases as well ( Muyssen et al ., 2009 ). Therefore, when calcium levels are low in the water, crustaceans must compensate for the lack of calcium needed for molting by a combination of strategies that include delaying reproduction, producing less calcified exoskeletons and/or reducing their potential body size. These responses increase vulnerability to mechanical damage and predation causing population declines ( Hessen et al ., 2000 ; Riessen et al ., 2012 ).

In addition to calcium decline, boreal lakes also experience changes in water chemistry that affect algal communities. Boreal lakes are recovering from pH-changes caused by acid rain ( Keller et al ., 1999 ; Jeziorski et al ., 2008 ), and nowadays the trend is of increasing levels of dissolved organic carbon (DOC) as a result of decreased acid deposition and increased temperature, precipitation and plant cover ( Thrane et al ., 2014 ). The shading effect of the water-coloring from increasing DOC levels negatively impacts primary production in these lakes ( Thrane et al ., 2014 ). These changes in water chemistry can induce shifts in phytoplankton community composition to less edible or nutritious species, impacting grazer populations ( Paterson et al ., 2008 ) and directly affecting Daphnia ’s growth rates. Particularly during the juvenile stage, Daphnia is more sensitive to algal food of low quality with structures that interfere with digestion ( Ferrão-Filho et al ., 2000 ). Under suboptimal food conditions, Daphnia tend to allocate their energy to respiration and carapace formation instead of reproduction ( Glazier and Calow, 1992 ) affecting brood production and population dynamics. However, food quantity and quality differ in their effects on Daphnia . Poor quality food may be deficient in a necessary nutrient, such as a protein, while providing sufficient calories. The effect may be that some of the eggs are aborted and substances essential to development are allocated to the remaining eggs in the clutch to maximize hatching ( Urabe and Sterner, 2001 ).

Given Daphnia ’s importance in boreal lakes and the challenges they are facing with changes in water chemistry and food sources, we asked the question of how Daphnia deals with these changes: What mechanisms are at play to adapt to or survive these suboptimal conditions and how can these adjustments at the individual level potentially affect population endurance? To address this question we compared three calcium levels in water with two different food levels in a fully randomized experiment. We reared neonates in the three experimental calcium levels and the broods that these Daphnia produced were used for the experiments. In other words, the neonates used for the experiments were second generation, born to mothers that grew and reproduced at the experimental calcium levels. This approach provided an unusual look into the strategies of individuals that had been born to calcium-stressed mothers and had to survive in a challenging environment that included suboptimal levels of calcium and food. To reduce variability in animal performance due to food quality, the type of food provided to all the experiments was the same, but the quantity varied to limit growth and reproduction.

We used a hybrid Daphnia pulex  ×  pulicaria for our experiment provided by Norman Yan (FLAMES Laboratory, Dorset, Ontario, Canada). This hybrid occurs naturally in McFarlane Lake in the Canadian Shield, which has a calcium concentration of 15.5 mg/L. Lakes in this region range in calcium from 0.9 to 23 mg/L, but many fall within 1–3 mg/L Ca. The Daphnia we used in our experiments was a hybrid of D. pulicaria , which is a widely distributed species in Central and Northern Ontario and can be found over the whole range of calcium concentrations in the lakes from this region (M. Celis-Salgado, FLAMES Laboratory, Dorset, Ontario, Canada, personal communication ).

The Daphnia were cultured in FLAMES medium with a calcium content of 2.531 mg/L ( Celis-Salgado et al ., 2008 ) and the culture was maintained in an incubator at 20°C. The 2.531 mg/L Ca concentration was measured in Blue Chalk and Red Chalk lakes in the Muskoka region of the Canadian Shield in Ontario. Blue Chalk Lake outflows into Red Chalk Lake and these lakes have been routinely sampled for 30 years by the Ontario Ministry of the Environment and Climate Change. These two lakes support a stable and varied population of daphniids. The chemical composition of these lakes was the basis for the development of a formulation to make media that resembled soft water from boreal lakes in the Canadian Shield (FLAMES), which range between 6 and 17 in hardness as mg/L of CaCO 3 ( Celis-Salgado et al ., 2008 ). Given the thriving populations of daphniids in the lakes the water formulation originated from, we determined 2.5 mg/L Ca as an optimal level to support Daphnia in our experiments. This level is not to be construed as an upper limit for Ca tolerance in Daphnia , but rather a realistic Ca level that supports cladocerans well in the Muskoka lakes.

Daphnia with mature eggs were isolated and observed for 8 hours; neonates born within that time period were isolated. Neonates from this cohort were randomly assigned to 20 mL glass vials, containing one neonate per vial with media at different calcium concentrations and with high food levels. These isolated Daphnia were monitored daily until they produced the first clutch of eggs, at which point neonates that were born into a given calcium medium were used to start the experiment. Six treatments were used with 10 individuals per treatment separated into individual vials, for a total of 60 experimental units.

The experiment was a 2 × 3 factorial design to explore how the combined effects of calcium and food affect the growth, reproduction and survival of Daphnia. The FLAMES medium (2.5 mg/L Ca) was considered the optimum calcium treatment for Daphnia in soft water and reduced calcium treatments were obtained by adjusting the concentrations of CaSO 4 in the FLAMES’ recipe. Each calcium treatment was combined with two food levels; high and low. The purpose of these food levels was to create abundant vs. stressful food conditions, although the quality of the food was the same, the quantity in the “low” treatment was designed to limit reproduction ( Lampert, 1978 ). Thus, the treatments were either high or low food (HF, LF) combined with 2.5 mg/L Ca, 1.0 mg/L Ca or 0.50 mg/L Ca. These three levels of calcium were chosen to induce various intensities of Ca-limitation stress in the experimental animals. The acronyms for these treatments henceforth are 2.5 HF, 1.0 HF, 0.5 HF and 2.5 LF, 1.0 LF, 0.5 LF. We used live Ankistrodesmus sp. for the food treatments. The treatments with low food levels had 0.16 mg/L C and the treatments with high food concentration had 1.97 mg/L C (carbon content based on M. Celis-Salgado's measurements on Ankistrodesmus sp. , unpublished data ). The concentration of algal cells for the high food treatment was chosen after conducting a preliminary pilot study to determine the algal density that produced rapid growth in Daphnia and large clutches of eggs. Alternatively, for the low food, we chose the threshold of algal concentration at which adults survived and were able to reproduce but at a reduced rate. In this pilot study the calcium level was optimal (2.5 mg/L Ca). Results from the statistical analysis (two tailed t -test) of the number of neonates produced from each clutch for the 2.5 HF and 2.5 LF treatments showed that HF produced significantly more neonates than LF ( P = 0.0015). The 2.5 HF treatment produced on average 20 neonates after the second clutch in the pilot study, which is deemed normal reproduction ( Taylor and Gabriel, 1992 ), and the 2.5 LF treatment produced on average 7.33 neonates per brood. The carbon levels we used in the HF and LF treatments are in agreement with those measured by Lampert (1978) ; he determined that 0.2 mg/L C was the minimal carbon threshold for egg production (our LF = 0.16 mg/L C), and the upper threshold was 0.7 mg/L C, where Lampert's egg-production curve plateaus (our HF = 1.97 mg/L C).

In the experimental setup, each experimental unit (a glass vial with one neonate in medium containing the calcium and food treatments) was randomly placed in a test-tube rack in a 20°C incubator with a photoperiod of L:D = 15:9 h. Test-tube racks containing all the experimental vials were located in the same incubator shelf and rotated daily. Also, on a daily basis, the experimental Daphnia were placed in a clean vial and given fresh food and media. The experiment ran for 19 days and data were collected every day. The data collected were: survival, molting, presence of eggs, number of neonates produced and neonate length. Lipid and ovary indices were determined for the surviving Daphnia at the end of the experiment by visual inspection. These indices rank from 0 to 3 ( Tessier and Goulden, 1982 ). A score of 0 for the lipid index indicates that individuals are starved while a score of 3 indicates adequate nutrition. For the ovary index, a score of 0 indicates lack of ovary provisioning while a score of 3 indicates well developed ovaries.

Statistical analyses

All data was tested for normality (Kolmogorov–Smirnov test) and for homogeneity of variance (Levene's test). When data violated these assumptions it was transformed. If transformations did not improve normality, non-parametric statistics were used (Kruskal–Wallis test and Mann–Whitney test). A factorial 2 × 3 ANOVA was used to evaluate significant differences between treatments. Post hoc tests were used for pairwise multiple comparisons using the Bonferroni correction at P < 0.0167 for significance (critical value of 0.05 divided by three, for three comparisons), to avoid Type I error to build above α  = 0.05 ( Field, 2009 ).

The intrinsic rate of population increase, r , was calculated using the following equation:

The intrinsic rate of population increase, r , for Daphnia (mean ± SE) at different calcium and food levels. Different letters above bars indicate significant differences ( post hoc Tukey HSD, P < 0.05). Treatments are 2.5 HF= 2.5 mg/L Ca and high food, 1.0 HF = 1.0 mg/L Ca and high food, 0.50 HF = 0.50 mg/L Ca and high food, 2.5 LF = 2.5 mg/L Ca and low food, 1.0 LF = 1.0 mg/L Ca and low food, 0.50 LF = 0.50 mg/L Ca and low food.

Two-way ANOVA on the effects of calcium concentration and food level on the intrinsic rate of population increase (r), number of neonates produced and their length, number of instars and number of egg clutches produced

* indicates significance at α = 0.05, ns indicates no significance.

Number of neonates produced per individual by the experimental Daphnia (mean ± SE). Different letters above bars indicate significant differences ( post hoc Tukey HSD, P < 0.05). For treatment acronyms see Fig. 1 .

Percent survivorship of experimental Daphnia in high-food and low-food treatments at different calcium concentrations.

The process of molting, and thus number of instars, was significantly affected by calcium concentration and food level (two-way ANOVA, Table I ) and an interaction effect was nearly present between calcium and food ( P = 0.05). Daphnia exposed to 2.5 mg/L Ca underwent significantly fewer instars than Daphnia in the lower calcium treatments ( post hoc Tukey HSD, P < 0.05). Similarly, Daphnia in treatments with high food levels also underwent significantly fewer instars than those in treatments with low food levels (pairwise comparison LSD, P = 0.001). However, before their first reproduction, the number of instars the experimental animals underwent was not significantly affected by calcium concentrations (Kruskal–Wallis, P > 0.05) or food levels (Mann–Whitney, P > 0.05). Therefore, before reproduction Daphnia underwent a similar number of molts in all treatments but once they started reproducing, molting differed among treatments with Daphnia in low calcium and low-food treatments molting more frequently than those in high calcium and high food.

Number of days until first reproduction (mean ± S.E). Error bars are not present in the 2.5 mg/L Ca treatments because all individuals reproduced at an age of 7 days.

Lipid and ovary rank for the different treatments. A score of 0 indicates very poor resources; scores from 1 to 3 indicate increasing resources and ovary condition. Only individuals that survived to the end of the experiment are depicted in these graphs. Error bars are not represented in the 0.5 HF treatment because data were taken from only one surviving individual.

The total number of egg clutches produced by Daphnia per treatment was significantly affected by calcium concentration (Table I ) but not by food levels. The Daphnia in 2.5 mg/L Ca produced significantly more clutches (mean = 6 clutches) than in the other two calcium concentrations (1.0 and 0.50 mg/L Ca, mean = 4.5 clutches) ( post hoc Tukey HSD, P < 0.0001).

The number of neonates produced from the first three clutches was significantly affected by calcium and food levels (e.g. first clutch, two-way ANOVA, F 2, 44  = 7.115, P = 0.002 for calcium concentration and F 41, 47  = 5.635, P = 0.022 for food level). However, after the third clutch the number of neonates produced was affected mostly by food level only. After the third clutch, high food treatments produced significantly more neonates than low-food treatments regardless of calcium levels: fourth clutch (two-way ANOVA, F 5,23  = 72.697, P < 0.001), fifth clutch (two-way ANOVA, F 1,22  = 36.847, P < 0.0001) and sixth clutch (Mann–Whitney, P = 0.007).

Natural populations of Daphnia encounter seasonal shortages in the quality or quantity of the food available to them. Both algal quantity and quality may alternate during the season, resulting in food limitation during most of the warmer period. For instance, Daphnia may encounter low quantities of edible algae dominating the seston during the clear-water phase in early summer, and fewer edible algal species becoming dominant and more abundant later in the season ( Müller-Navarra & Lampert, 1996 ). In both situations, Daphnia may experience nutritional stress. Obviously, daphniids have adapted to these seasonal variations and can adjust to a lake's natural food cycles. However, their resilience is truly tested when they encounter a combination of reduced nutrition from food and a shortage of calcium, an essential nutrient for their structural and metabolic integrity. Our results show that the intrinsic rate of population growth ( r ) is driven by both calcium and food, but when calcium levels are adequate, Daphnia can survive with lower quantity (and possibly lower quality) of food. Even when calcium levels were limiting, such as at 1.0 and 0.5 mg/L Ca, the population continued to grow (albeit at a lower rate) as long as quality food was available in sufficient quantities to support reproduction (Fig. 1 ). Similar results have been found by Ashforth and Yan (2008) with D. pulex in high food treatments, where the population grew at all calcium levels except the most limiting (0.1 mg/L Ca). Thus, as calcium levels decline in boreal lakes, calcium-dependent cladoceran populations could manage to survive as long as there are seasonally edible algae of suitable quality to allow for reproduction. Conversely, if the available algae are low in quantity or quality, the stress on Daphnia populations can become severe as the combination of low calcium and poor food may impair population growth.

The Daphnia in our experiments were the second generation reared in the experimental media, thus carrying the metabolic costs of calcium deficiency (for the 1.0 and 0.5 mg/L Ca treatments) for two generations. Our results may offer a more realistic view of what is happening in nature than other studies of this kind, where the experimental Daphnia were from parental stock with adequate calcium supply. The main difference between the ideal conditions of high food and high calcium and a deficiency in one or both of these factors, was the ability of the parental Daphnia to allocate enough resources to their eggs for adequate development. Even under optimal food conditions, reduced calcium availability always resulted in a lower number of neonates (Fig. 2 ), ultimately affecting population growth. At low food levels but sufficient calcium (2.5 LF), experimental Daphnia mothers who had grown and developed under limiting food themselves, could assess food levels and adjust their reproductive output by producing small clutches of eggs. According to the experiments by Gliwicz and Guisande (1992) with clonal D. pulicaria and D. hyalina , the resulting neonates probably would have been better adapted to surviving food shortages. In contrast, in our low calcium treatments (1.0 and 0.5 mg/L Ca), calcium restriction put an additional metabolic burden on the mothers. Parental Daphnia probably expended more energy on calcium uptake under low calcium conditions to support their own ecdysis process ( Hessen et al ., 2000 ) and endured restrictions in the amount of growth they could attain ( Riessen et al ., 2012 ). In addition, they also had to partition any additional calcium they could spare among their eggs to build the neonates’ exoskeletons. The resulting neonates were likely not as well-equipped to survive in the environment as if they had adequate calcium resources. In an experiment looking at prey defenses in D. pulex , Riessen et al . (2012) found that calcium-deficient neonates failed to develop “a full array” of the defenses typically induced in Daphnia by predators like the phantom midge ( Chaoborus sp.) such as increased body size, neck spines and a strong carapace, resulting in a 50–186% increase in their vulnerability to predation. The compounded effect of multi-generational malnutrition, therefore, goes beyond a reduced reproductive rate. Mortality rates increase because Daphnia become more vulnerable to predation and mechanical damage for lack of a well-calcified exoskeleton ( Hessen et al ., 2000 ). In extreme situations when calcium is low and lake conditions preclude the presence of edible algae, neonate production becomes very low and potentially insufficient to ensure population survival. For instance, in our experiment, low levels of calcium and low food had a strong influence on the small number of neonates produced, even though the food we provided was easily grazed (Fig. 2 ). Also, the combined effects of calcium and food levels affected the size of the neonates at birth (Table I ). Although calcium was essential to form the carapace of the neonates, food was necessary for attaining a larger body size by the time they were born and to provide a proper head-start for the neonates. At high-calcium levels, neonate size was similar in high and low-food environments, however, when calcium was limiting, high food levels always produced larger neonates than low-food treatments. Neonate body size upon release from the maternal brood chamber will determine whether they can endure harsh conditions in the environment, such as starvation periods. The maternal investment in the body length of her progeny is therefore crucial for their success ( Tessier and Consolatti, 1989 ). Other studies that used lake water or culture water (calcium levels not a concern), found that low-food conditions resulted in Daphnia producing fewer larger eggs and larger neonates ( Gliwicz and Guisande, 1992 ; Urabe and Sterner, 2001 ). In contrast, when calcium levels are part of the experiment as is the case here, limiting calcium conditions and high food always produced larger neonates than the equivalent low-food treatments, indicating that the interaction of calcium and food determined neonate size (at least for the D. pulex × pulicaria clone we used in these experiments).

Experimental scenarios and summary of main Daphnia responses based on data shown in Figs 1 – 3 ; for justification of inferred strategies see discussion

In order to maximize fitness, Daphnia under stressful conditions had to optimize the allocation of their calcium and food resources. In particular, food was the main factor in the condition of the ovary and the lipid reserves. High food levels resulted in more lipid droplets and better ovary condition (Fig. 5 ). However, the Daphnia at 0.5 LF had a high lipid index and a low ovary index, indicating a strategy of storing resources instead of using them to reproduce. Research by Wacker and Martin-Creuzburg (2007) on D. magna showed that, in order to maximize fitness under poor food quality conditions (but adequate calcium from lake water), lipid resources were allocated between somatic and reproductive tissues, and large amounts of fatty acids were allocated to the eggs for development into neonates. However, in our experiments, where calcium deficiency played a role, the low number of neonates produced by Daphnia at 0.5 mg/L Ca and their small size indicate that the low-food ration combined with the stress of low calcium prevented the allocation of biochemical compounds into the eggs, reducing the energy directed at reproduction ( Guisande and Gliwicz, 1992 ). The result was an investment into longevity (Table II ), perhaps as a strategy to maximize survival in order to wait for a change in the food conditions in the environment. This strategy would have adaptive value in nature, as storing resources will increase an individual's lifespan until better grazing is available.

The strategic allocation of energy to somatic maintenance was also reflected in the higher rates of molting in low calcium and LF treatments compared to high calcium and HF. When food is limiting, the highest energy priority for Daphnia is the formation of the carapace, even though the relative molt mass is higher for them than it would be under optimal food conditions due to the smaller percentage of biomass that comprises storage at low food levels ( Glazier and Calow, 1992 ). The higher molting rate the animals experienced when low food and low calcium were present increased their level of stress, as they were losing much-needed calcium. Dapniids are estimated to lose 40% of their total body calcium when they discard their exuvia and 50% of the remaining calcium is leached into the environment ( Alstad et al ., 1999 ). Therefore, Daphnia ’s investment into carapace formation and molting in low-food environments is a strategy that, when combined with low calcium, limits egg-formation and development and severely restricts reproduction (Figs 1 and 2 ). Daphnia reached maturity at different times in our experiments depending on calcium resources. Those animals that had sufficient calcium levels in their water, reproduced earlier and produced more broods of larger neonates than the Daphnia in calcium-deficient treatments (Fig. 4 ). Interestingly, calcium was important for the production of the first three egg clutches, when young mothers were also growing. However, after the growth of older adults slowed , their own calcium demand declined and they were able to allocate enough calcium for reproduction under limiting conditions when food levels were sufficient. These results indicate that the largest calcium demand was for parental Daphnia growth and development, and once this growth was accomplished, even low levels of calcium could be used for reproduction in the presence of adequate food.

Our experimental results support the notion that boreal lakes that have modest levels of calcium may be able to support Daphnia populations during periods when food is plentiful. If the food quality or quantity is too low to mitigate the effects of calcium limitation, Daphnia populations will decrease or even disappear due to low reproductive output. However, the genetics of different Daphnia species or even the genetic variants of a species can have a significant effect on the animals’ response to low levels of nutrients in natural systems. Environmental factors may influence a genotype and its response may confer a beneficial outcome such as a competitive advantage, a tolerance to metals or to toxic cyanobacteria ( Hietala et al ., 1997 ; Barata et al ., 1998 ; Weider et al ., 2005 ). Therefore, the response to calcium limitation may vary among Daphnia with different genetic make-ups. Yet, despite the various levels of resilience that different species of Daphnia (or clones or hybrids) may have to calcium limitation, the fact remains that the depletion of an essential nutrient needed for structural integrity would eventually take a toll on planktonic community structure. Species that have a low need for calcium will likely dominate in calcium-depleted lakes. For instance, in Daphnia sp., the calcium requirement is 3–8% DW ( Cairns and Yan, 2009 ), but the calcium requirement for copepods is only 0.06% DW ( Alstad et al ., 1999 ), for bosminids is 0.2–0.4% DW ( Cairns and Yan, 2009 ), and for Holopedium , which relies on a gelatin shield, 0.4% DW ( Yan et al ., 1989 ). The higher requirement of calcium for Daphnia puts them at a metabolic disadvantage in boreal low-calcium lakes and gives a competitive advantage to crustacean species with low, or very low requirements for this mineral. In addition to calcium stress, algal quality or quantity may contribute to limiting daphniids or substituting grazer populations that can better deal with both sources of stress. In many boreal lakes, the effect of low calcium is compounded by the presence of contaminants such as toxic metals, for which calcium plays a protective role in cladocerans by decreasing uptake rates ( Altshuler et al ., 2011 ). In addition, low-DOC boreal lakes also are impacted by ultraviolet radiation, and Daphnia are more susceptible to its effects when stressed by low calcium levels ( Hessen and Alstad Rukke, 2000b ). At the ecological level, the high energetic cost of calcium deficiency for Daphnia will affect carbon sequestration in zooplankton (as growth is stalled) and potentially carbon cycling in the pelagia ( Hessen et al ., 2000 ; Hessen and Alstad Rukke, 2000a ). The resulting community structure of calcium-limited crustacean zooplankton will determine the complexity and efficiency of the pelagic food web and ultimately could affect the calcification of the predators in these lakes.

Our experiments using second-generation calcium-stressed D. pulex  ×  pulicaria indicate that the current trend in calcium decline in boreal lakes can alter the zooplankton community by limiting the presence of calcium-dependent cladocerans, such as Daphnia sp. However, the fate of the cladoceran community may be modulated by food availability and by Daphnia ’s life-history response to calcium and food supplies. Our results show that calcium limitation can decrease population growth, but seasonally available algae may mitigate this impact and the population could continue to grow at a slower rate. In the event that calcium levels and edible algae were both too low to attempt reproduction successfully, Daphnia ’s strategy would be to invest in survivorship and longevity and, presumably, to wait for more favorable environmental conditions for reproduction. Overall, we conclude that boreal lakes that experience periods of abundant edible algae could support Daphnia populations periodically, even if water calcium concentrations are below the optimum range. But if the food quality or quantity are also low, Daphnia ’s reproductive output would not be sufficient to maintain a thriving population throughout the ice-free season.

We would like to thank Norman Yan, Martha Celis-Salgado and Dallas Linley of the FLAMES laboratory in Dorset, Ontario (Canada) for inspiring this work and providing the hybrid used in our experiments. F.G. would like to thank the members of her graduate committee at Buffalo State: Howard Riessen, and Randal Snyder.

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Practical Biology

A collection of experiments that demonstrate biological concepts and processes.

Observing earthworm locomotion

Practical Work for Learning

Published experiments

Investigating factors affecting the heart rate of daphnia, class practical.

Thanks to the British Pharmacological Society for providing the teaching notes on this practical.

With modifications made by Prof Richard Handy, University of Plymouth

Lesson organisation

This will depend on access to a healthy culture of Daphnia and on the number of microscopes you have. Students can readily follow this procedure working in pairs. Because of the variability of results between individual Daphnia, it is not appropriate to draw conclusions from one set of results; each pair (or group) of students should carry out more than one investigation to contribute to the class set.

One option is to record a live video of a sample Daphnia , during a time period in which students count the heart beats. Then you replay the video in slow motion and count the heart beats again. This allows students to consider the accuracy of their counting.

If your time or access to chemicals is limited, you could allow the students to work through the procedure in order to evaluate it and then use the example results provided for analysis.

Apparatus and Chemicals

For each group of students:.

Microscope – low power, transmission

Small piece of cotton wool

Pasteur pipette (for water from the Daphnia culture tank)

Chemicals that may affect the heart rate – at low concentrations ( Note 4 )

For the class – set up by technician/ teacher:

Culture of water flea – Daphnia ( Note 1 )

Water from Daphnia culture tank at different temperatures – 0 °C (in an ice bath), 10 °C (by adding ice to a water bath), 20 °C, 30 °C and 40 °C (in water baths) ( Note 2)

Ethanol, 1% and 10%, 10 cm 3 of each ( Note 3 )

Health & Safety and Technical notes

With Daphnia cultured in the laboratory, fed on yeast, Liquifry No.1, Spirulina or egg-yolk medium, there are no significant hazards associated with this procedure. With pond water culture, or other sources of food, more careful hygiene precautions are necessary.

Read our standard health & safety guidance

1 Keeping live cultures of Daphnia : These notes are based on information in the CLEAPSS Laboratory Handbook. You will find more details in section L56. Daphnia are crustaceans, commonly found in ponds and lakes and widely sold as live fish food. These animals are fascinating objects for observation and study in their own right. They feed by filtering minute particles such as bacteria and algae, from the fresh water in which they live.

Daphnia can be kept in any watertight container containing tap water that has been allowed to stand for a few days. Keeping a few Daphnia is not difficult, but cultivating a vigorous, dense colony requires some care. A good supply of oxygen is necessary, either by aeration or by using a large shallow tank to ensure that a large surface area of water is exposed to the air. Warming the water to about 15 °C also ensures rapid growth of the colony.

You can purchase live cultures from suppliers, including pet shops and local aquarists. Some scientific suppliers sell viable dried Daphnia eggs and culture kits. Alternatively, you can collect adult Daphnia by pond dipping; in this case you must observe strict hygiene procedures, since pollutants and the bacteria causing Weil’s disease may contaminate pond water. Stock purchased from aquarists is usually free from this hazard.

The safest, most hygienic and most convenient ways to provide the necessary food for a colony of Daphnia is to feed them on a few drops of a suspension of fresh yeast or of egg-yolk medium (made by blending a hard-boiled egg in 500 cm 3 of water). Alternatively, you can buy food such as Liquifry No 1 or Spirulina powder from aquarists or scientific suppliers.

Small, regular supplies of food are required. Provide only sufficient to cause the water to turn faintly cloudy. After a few days the Daphnia will have filtered out the suspended particles of food, making the water clear once more, which is your cue to add more food. Clear scum from the surface of the water; but leave debris that sinks to the bottom – it may contain Daphnia eggs.

2 Instead of heating water in a water bath, you could surround the Daphnia in the Petri dish with a circular heating coil connected to a 6V battery. This will gradually heat the water in the dish, and the cardiac frequency can be estimated at 5 °C or 10 °C intervals. An additional, larger dish outside the small one could also be filled with water at the appropriate temperature to help reduce heat loss from the experimental chamber.

3 Ethanol (IDA) Hazcard 40A is highly flammable and harmful because of the presence of methanol. Once diluted to 10% and 1%, this is low hazard for the students using the liquid.

4 Physiologically-active compounds: (Refer to Hazcard 3C) Each compound will have different hazards and associated risk control measures. Acetylcholine is an irritant (to eyes, respiratory system and skin) and is used at a concentration of 1 g in 1000 cm 3 of water. L-adrenaline (epinephrine) is toxic by inhalation, in contact with the skin and if swallowed. Used by students at a concentration of 1 g in 1000 cm 3 of water it is low hazard. Caffeine is harmful if swallowed (!). 0.3 g in 1000 cm 3 of water is similar to the concentration of caffeine in an ordinary cup of coffee or a cola drink and so is low hazard for the students (see also Hazcard 103). Aspirin (o-acetylsalicylic acid) is harmful if swallowed, but a soluble tablet dissolved according to the manufacturers’ instructions would give a suitable concentration to use in the investigation at low hazard to the students. In each case, add one drop to 5 cm 3 of water before applying to the Daphnia .

5 Heating due to the microscope lamp: When working with organisms under a microscope, the effects of heating due to the microscope lamp itself can be significant. Turning the lamp on only when observing the Daphnia will help, and LED microscopes produce less heat than those with incandescent lamps.

Ethical issues

Teachers should be careful to introduce these animals in a way that promotes a good ethical attitude towards them and not a simply instrumental one. Although they are simple organisms that may not 'suffer' in the same way as higher animals, they still deserve respect. Animals should be returned promptly to the holding tank after being examined. This supports ethical approaches that are appropriate to field work where pond animals are returned to their habitat after observations have been made.

SAFETY: Take care handling any chemicals that might affect the heart rate of Daphnia . Observe normal, good laboratory hygiene practices when completing the practical.

Preparation

a Take a small piece of cotton wool, tease it out and place it in the middle of a small Petri dish.

b Select a large Daphnia and use a pipette to transfer it onto the cotton wool fibres.

c Immediately add pond water to the Petri dish until the animal is just covered by the water.

d Place the Petri dish on the stage of a microscope and observe the animal under low power. The beating heart is located on the dorsal side just above the gut and in front of the brood pouch (see diagram). Make sure that you are counting the heart beats, and not the flapping of the gills or movements of the gut. The heart must be observed with transmitted light if it is to be properly visible.

e Use a stopwatch to time 20 seconds, and count the number of heart beats in several periods of 20 seconds. The heart beat of Daphnia is very rapid, so count the beats by making dots on a piece of paper in the shape of a letter S. Count the dots and express heart rate as number of beats per minute.

f At the end of the investigation, return the Daphnia to the stock culture.

Investigating the effect of temperature

g Record the temperature of the water in the Petri dish.

h Add pond water at a different temperature to the Petri dish. Allow the Daphnia some time to acclimatise, but keep a check on the temperature of the water in the dish and add more hot or cool pond water if necessary to adjust the temperature.

i Record the heart rate again as in step e .

j Plot a graph of mean frequency of heart beats per minute against temperature.

Investigating the effect of chemicals

k Take a large Daphnia from the stock culture and record its heart beat at room temperature in pond water (as in step e ).

l Add one drop of 1% ethanol to 5 cm 3 of pond water in a beaker. Mix well. Draw the pond water off the Daphnia with a pipette and replace it with 2 or 3 cm 3 of the water containing ethanol ( Note 3 ). Record the rate of heart beat again.

m Repeat step l using 10% ethanol in place of 1%.

n Repeat with other chemicals such as acetylcholine, L-adrenaline (epinephrine), caffeine or aspirin ( Note 4 ).

Teaching notes

Daphnia is poikilothermic, which means that its body temperature and therefore its metabolic rate are affected directly by the temperature of the environment. The change in metabolic rate is reflected in the rate at which the heart beats (cardiac frequency).

The effect of temperature on a metabolic activity may be expressed in terms of the temperature coefficient (Q 10 ). This is the ratio of the rate of activity at one temperature to its rate at a temperature 10 degrees higher.

Within a range of 10 °C above and below ‘normal’ environmental temperatures, the rate of a metabolic process is expected to double for every 10 °C rise in temperature. Daphnia heart rate has a more complex relation to temperature than a single enzyme-controlled reaction, so Q 10 = 2 is not expected. Above 40 °C and 50 °C, the relation between the two rates will not hold because of the deleterious effects of extreme temperature.

There will be considerable variation in the data gathered. Class results for the heart beat at any temperature should be recorded and mean results (and standard deviation) calculated.

Student notes

Example results:

Background information: chemicals and the heart

Acetylcholine: In humans and many other animals, heart rate is slowed by the parasympathetic nervous system (neurotransmitter: acetylcholine) via activation of cell surface receptors in the sinoatrial node (pacemaker) called acetylcholine muscarinic receptors. This occurs after feeding, during sleep, and during breath-holding and swimming underwater. A slowed heart rate and the associated fall in the rate of ejection of blood from the heart is sufficient to maintain body function during rest, and conserves energy in the heart under conditions where its supply (and the supply of oxygen in the blood) are diminished. A drug that slows heart rate is called a negative chronotrope;  this is demonstrated in this experiment, where acetylcholine is used to slow the rate of the Daphnia 's heart.

Noradrenaline and adrenaline: In contrast, heart rate is increased by the sympathetic nervous system (neurotransmitter: noradrenaline) and the hormone adrenaline circulating in the blood via activation of cell surface receptors in the sinoatrial node - pacemaker) (called beta-1 adrenoceptors). This occurs during exercise or fear. The effect is to increase the rate of ejection of blood by the heart. This means that there will be more blood flow to skeletal muscle (in which exercise causes dilatation of blood vessels), so the skeletal muscle cells are supplied with more oxygen and respiratory substrates used to generate energy in respiration where it is needed. A drug that increases heart rate is called a positive chronotrope, and this is demonstrated in this experiment when adrenaline is used to increase heart rate in Daphnia .

One of the ways adrenaline increases heart rate is through the action of what is known as a 'second messenger' or 'transduction component', in this case it is a chemical made in the cell known as cyclic adenosine monophosphate (cAMP). Transduction is the process that follows the action of a drug, hormone or neurotransmitter at a receptor. Thus, when adrenaline activates the beta-1 adrenoceptor in the sinoatrial node, this leads to an increase in cAMP in the sinoatrial node and the result is an increase in heart rate.

Caffeine: Caffeine mimics some of the effects of adrenaline and noradrenaline in the heart. By a different mechanism not involving beta-1 adrenoceptors, caffeine also increases the amount of cAMP in the sinoatrial node. Then cAMP levels increase and this increases the electrical activity of the sinoatrial node, making it depolarize and 'beat' faster. Caffeine has additional effects on the heart. Like adrenaline and noradrenaline, it can affect the main pumping chambers (ventricles), leading to an increase in the rate of contraction and relaxation of each heart beat. This means that, as well as beating faster, the heart's individual beats are associated with an increased volume of blood ejected into the circulation per unit time. This is called increasing cardiac output. Two or three cups of strong coffee or tea contain enough caffeine (and a similar acting compound called theobromine) to cause an increase in human heart rate of 5-20 beats/min.

Ethanol: Ethanol slows heart rate. At the concentrations used in this experiment, ethanol depresses the nervous system by acting as what is known as a non-selective neurodepressant. The amounts of ethanol necessary to achieve this effect in humans would also be sufficient to depress the respiratory centres of the brain, rather like the effect of an overdose of general anaesthetic, resulting in death.

Aspirin : Aspirin has no effect on heart rate. Despite this, aspirin has beneficial effects in the heart. By reducing the ability of platelets to adhere to damaged blood vessel walls, aspirin reduces the chance of coronary artery thrombosis, the event that precipitates a heart attack. People who are take aspirin long-term for medical reasons (because they have cardiovascular disease or diabetes) may have a lower heart rate than controls, simply because they experience less coronary and peripheral thrombosis and thus have a better lubricated cardiovascular system.

Some words of caution The Daphnia ’s heart differs from the human heart in many respects. In terms of heart rate, the Daphnia sinoatrial node is actually a collection of spontaneously active nerves in a body called the cardiac ganglion. This means that it would be risky to extrapolate heart rate findings from Daphnia directly to humans without first validating the model.

Model validation requires examination of a range of positive and negative controls for their effects in the model. To achieve this, the type and extent of the effect in humans at the same drug concentration (the human template) must be known. It is not always possible to obtain such a human template; this is why the outcome of a novel non-human experimental study is of only provisional clinical relevance. Proof of model validity emerges only once human data sets are available.

Health & Safety checked, May 2009

Related experiments

Observing the effects of exercise on the human body Compare the results of this experiment with the results of the investigation into the effects of exercise on human heart rate.

Increased extinction probability and altered physiological characteristics in pirimicarb-tolerant Daphnia magna

• Research Article
• Published: 13 July 2024

Cite this article

• Makoto Ishimota   ORCID: orcid.org/0000-0003-4686-0244 1 ,
• Mebuki Kodama 1 ,
• Naruto Tomiyama 1 &
• Kazutoshi Ohyama 1  

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We evaluated the physiological characteristics of chemical-tolerant cladocerans. Over the course of 26 generations (F25), Daphnia magna was continuously exposed to pirimicarb (carbamate) solutions (0, 3.8, 7.5, and 15 µg/L) in sub-lethal or lethal levels. The 48 h EC 50 values (29.2–29.9 µg/L) for 7.5 and 15 µg/L exposure groups were found to be nearly two times higher than that in the control (17.2 µg/L). Subsequently, we investigated whether the extinction probability changed when the chemical-tolerant daphnids were fed two different types of food, Chlorella vulgaris and Synechococcus leopoliensis . Furthermore, we ascertained how chemical tolerance influences respiration and depuration rates. The 48 h EC 50 value was positively related to the extinction probability when the daphnids were fed S. leopoliensis . Because the measured lipid content of S. leopoliensis was three times lower than that of C. vulgaris , the tolerant daphnids struggled under nutrient-poor conditions. Respiration rates across all pirimicarb treatment groups were higher than those in the control group, suggesting that they may produce large amounts of energy through respiration to maintain the chemical tolerance. Since the pirimicarb depuration rate for 7.5 µg/L exposure groups was higher than that in the control, the altered metabolic/excretion rate may be one factor for acquiring chemical tolerance. These altered physiological characteristics are crucial parameters for evaluating the mechanisms of chemical tolerance and associated fitness costs.

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Data availability

Data, material, and/or code related to the findings of this study are available in the article and supplemental data. Additional documentation is available upon reasonable request.

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We thank our technical staff, Aya Kitahara, for supporting this study and Editage ( https://www.editage.com/ ) for editing and reviewing this manuscript for English.

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Makoto Ishimota, Mebuki Kodama, Naruto Tomiyama & Kazutoshi Ohyama

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All authors discussed the results and contributed to the final manuscript. Makoto Ishimota performed and designed the experiments and analyzed the data. Makoto Ishimota wrote the first draft of this manuscript. Naruto Tomiyama, Mebuki Kodama, and Kazutoshi Ohyama discussed the results and commented on the manuscript. All the authors have read and approved the final version of the manuscript.

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Ishimota, M., Kodama, M., Tomiyama, N. et al. Increased extinction probability and altered physiological characteristics in pirimicarb-tolerant Daphnia magna . Environ Sci Pollut Res (2024). https://doi.org/10.1007/s11356-024-34386-4

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• Life Science

Study Challenges Traditional Views of Evolution

Research uncovers how environmental changes influence genetic variation over time.

In new research, Arizona State University scientists and their colleagues investigated genetic changes occurring in a naturally isolated population of the water flea, Daphnia pulex . This tiny crustacean, barely visible to the naked eye, plays a crucial role in freshwater ecosystems and offers a unique window into natural selection and evolution.

Their findings, reported in the current issue of the journal Proceedings of the National Academy of Sciences (PNAS) , rely on a decade of research. Using advanced genomic techniques, the research team analyzed DNA samples from nearly 1,000 Daphnia . 

They discovered that the strength of natural selection on individual genes varies significantly from year to year, maintaining variation and potentially enhancing the ability to adapt to future changing environmental conditions by providing raw material for natural selection to act on.

In seemingly stable environments, there is significant fluctuation in the frequency of gene variants known as alleles at specific chromosomal regions over time, even if the overall strength of selection remains near zero on average over many years. This suggests that such genetic variation allows populations to remain adaptable to environmental changes. 

“This study has, for the first time, given us a glimpse into the kinds of temporal changes in gene frequencies that occur even in seemingly constant environments, a sort of ongoing churn of genetic variation distributed across the genome,” says Michael Lynch, lead author of the new study.

Lynch is the director of the Biodesign Center for Mechanisms of Evolution and professor in the School of Life Sciences at ASU. Additional researchers on the study include colleagues from ASU, Central China Normal University, and the University of Notre Dame.

The power of selection

Daphnia , a form of zooplankton, have fascinated biologists for centuries due to their crucial role in aquatic ecosystems and ability to adapt to environmental stressors. In addition to their value for multigenerational genetic research, Daphnia are widely used model organisms for freshwater toxicity testing because they have a rapid asexual reproductive cycle and are sensitive to various environmental pollutants.

The tiny creatures are a vital food source for fish and help keep algae growth in check. Their ability to adapt quickly to environmental changes could hold clues for how other species—including those important to human food supplies—might respond to pollution, climate change, and other human-induced stressors.

Most of the sites examined on the Daphnia genome were shown to experience changing selection pressures over the study period. On average, these pressures tend to balance out to have little overall effect, meaning that no single direction of selection consistently dominates over time. Instead, the genetic advantages or disadvantages of specific traits change from one period to the next.

These findings challenge the traditional belief that measuring genetic diversity (the range of different traits in a population) and genetic divergence (the differences between populations) can easily show how natural selection is consistently operating. Instead, natural selection seems to operate with greater subtlety and complexity than previously thought. 

Rethinking genetic variation

The study breaks new ground by pinpointing when and where selection pressures occur within the genome. Other than traits known to be strongly influenced by natural selection, there is little information on how allele frequencies change over time in natural populations.

The multiyear, genome-wide analysis of nearly 1,000 genetic samples from a Daphnia pulex population shows that most genetic sites experience varying selection, with an average effect close to zero, indicating little consistent selection pressure over different times and selection spread across many genomic regions. 

These findings challenge the usual understanding of genetic diversity and divergence as indicators of random genetic drift and selection intensity.

Variation and survival

The observed patterns of selection on various gene sites provide a mechanism for maintaining genetic diversity, which is essential for rapid adaptation. The study also revealed that genes located near each other on chromosomes tend to evolve in a coordinated manner. This linkage allows beneficial combinations of gene variants to be inherited together, potentially accelerating the adaptation process. 

This effect could help explain how species sometimes adapt faster than scientists would normally expect. On the other hand, the same phenomenon may result in deleterious alleles being swept to higher frequencies by linked beneficial alleles, reducing the overall efficiency of selection in some cases.  

The study shows that evolution is more dynamic and complex than previously appreciated. The environment's influence on genes changes frequently, possibly helping species keep the genetic variety needed to adapt to future conditions. This new understanding may prompt scientists to rethink how they study evolution in the wild. 

While the study focused on Daphnia pulex , the findings may have implications for understanding how other species might respond to rapid environmental changes, including those driven by human activities, such as pollution and climate change. Assessing the stability of allele frequencies in more stable environments is an important preliminary step. Such studies are critical, as laboratory experiments alone cannot duplicate the complexity of environmental influences acting on wild populations. 

Further, understanding how Daphnia evolve may provide insights into the resilience of entire ecosystems. This knowledge could help researchers predict and potentially mitigate the impacts of environmental changes on biodiversity and food webs.

As the world grapples with an accelerating environmental crisis, studies like this one provide crucial insights into nature's capacity for resilience and adaptation. By continuing to study these tiny creatures, the scientists hope to better understand the fundamental mechanisms of evolution and apply these lessons to broader ecological and conservation efforts.

  - This press release was originally published on the  Arizona State University  website and has been edited for style and clarity

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Miniaturizing nanotoxicity assays in daphnids.

Simple Summary

1. introduction, 2. materials and methods, 2.1. culturing of daphnids and toxicity exposures, 2.2. sample homogenization and biochemical assays, 2.3. feeding and imaging of daphnids, 2.4. statistical analysis, 3.1. exposure to silver nano ink in different vessels and volumes impacts mortality, 3.2. physiology responses following exposure to the silver nano ink, 4. discussion, 5. conclusions, supplementary materials, author contributions, institutional review board statement, informed consent statement, data availability statement, acknowledgments, conflicts of interest.

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Kakavas, D.; Panagiotidis, K.; Rochfort, K.D.; Grintzalis, K. Miniaturizing Nanotoxicity Assays in Daphnids. Animals 2024 , 14 , 2046. https://doi.org/10.3390/ani14142046

Kakavas D, Panagiotidis K, Rochfort KD, Grintzalis K. Miniaturizing Nanotoxicity Assays in Daphnids. Animals . 2024; 14(14):2046. https://doi.org/10.3390/ani14142046

Kakavas, Dimitrios, Konstantinos Panagiotidis, Keith D. Rochfort, and Konstantinos Grintzalis. 2024. "Miniaturizing Nanotoxicity Assays in Daphnids" Animals 14, no. 14: 2046. https://doi.org/10.3390/ani14142046

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Daphnia as a model organism to probe biological responses to nanomaterials—from individual to population effects via adverse outcome pathways

Katie reilly.

1 School of Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham, United Kingdom

Laura-Jayne A. Ellis

Hossein hayat davoudi, suffeiya supian, marcella t. maia.

2 Brazilian Nanotechnology National Laboratory (LNNano), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, Brazil

Gabriela H. Silva

Zhiling guo, diego stéfani t. martinez, iseult lynch.

Guotao Peng , Tongji University, China

The importance of the cladoceran Daphnia as a model organism for ecotoxicity testing has been well-established since the 1980s. Daphnia have been increasingly used in standardised testing of chemicals as they are well characterised and show sensitivity to pollutants, making them an essential indicator species for environmental stress. The mapping of the genomes of D. pulex in 2012 and D. magna in 2017 further consolidated their utility for ecotoxicity testing, including demonstrating the responsiveness of the Daphnia genome to environmental stressors. The short lifecycle and parthenogenetic reproduction make Daphnia useful for assessment of developmental toxicity and adaption to stress. The emergence of nanomaterials (NMs) and their safety assessment has introduced some challenges to the use of standard toxicity tests which were developed for soluble chemicals. NMs have enormous reactive surface areas resulting in dynamic interactions with dissolved organic carbon, proteins and other biomolecules in their surroundings leading to a myriad of physical, chemical, biological, and macromolecular transformations of the NMs and thus changes in their bioavailability to, and impacts on, daphnids. However, NM safety assessments are also driving innovations in our approaches to toxicity testing, for both chemicals and other emerging contaminants such as microplastics (MPs). These advances include establishing more realistic environmental exposures via medium composition tuning including pre-conditioning by the organisms to provide relevant biomolecules as background, development of microfluidics approaches to mimic environmental flow conditions typical in streams, utilisation of field daphnids cultured in the lab to assess adaption and impacts of pre-exposure to pollution gradients, and of course development of mechanistic insights to connect the first encounter with NMs or MPs to an adverse outcome, via the key events in an adverse outcome pathway. Insights into these developments are presented below to inspire further advances and utilisation of these important organisms as part of an overall environmental risk assessment of NMs and MPs impacts, including in mixture exposure scenarios.

1 Introduction

The zooplankton cladoceran Daphnia has captivated biologists for centuries because of its importance in aquatic ecosystems, and its flexibility to cope with, and respond to, environmental stressors. Daphnia are a well-established and widely used model organism for freshwater toxicity testing as they are well characterised, have a rapid parthenogenetic reproductive cycle and show sensitivity to a range of environmental xenobiotics. Daphnia are also a non-sentient species, meaning that their use in toxicity testing is considered acceptable as a strategy for the reduction, replacement and refinement (NC3Rs) of traditional animal testing, making them an optimal model in ecotoxicology ( Colbourne et al., 2022 ; NC3Rs, 2022 ). A broad set of behavioural and morphological changes can be observed in Daphnia when exposed to environmental stimuli, which forms the foundation of the defined and standardised protocols for chemical toxicity testing, such as the OECD 202 (Acute toxicity) and 211 (Reproduction) tests and the EPA testing of chemicals ( OECD, 2004 ; OECD, 2012 ; Maxwell et al., 2014 ). Endpoints evaluated encompass responses such as immobilisation and lethality, which are measured in the acute immobilisation tests in the OECD 202 assay. Changes in life history traits during the chronic test (OECD 211) are also measured, including reproductive changes (such as an increase or decrease in the number of neonates per adult daphnid, or a delay between broods), and growth trends. Further to the standard test end points, phenotypic changes can be observed such as additional spines on the helmet, variability in lipid deposits and behavioural changes such as swimming activity ( Colbourne et al., 2011 ; Chevalier et al., 2015 ; Karatzas et al., 2020 ; Tkaczyk et al., 2021 ). Due to the historic use of Daphnia for chemical testing, they are also an optimal model organism for testing challenging and emerging toxicants such as nanomaterials (NMs) and microplastics (MPs) ( Nasser and Lynch, 2019 ; Zimmermann et al., 2020 ).

NMs, as described by the European Commission, have at least one dimension less than 100 nm ( European Commission. Joint Research Centre, 2020 ). NMs exist in the environment from a range of sources, including naturally occurring (e.g., volcanic ash), incidental particles formed as a result of human activities (e.g., combustion particles, secondary MPs) or can be engineered/manufactured by industry at the nanoscale to exploit specific properties ( Jeevanandam et al., 2018 ). During synthesis, NMs are normally coated with ligands to control their size and limit their agglomeration ( Buesser and Pratsinis, 2012 ). They are distinguished from other non-nanoscale materials by their unique physico-chemical properties ( Haase and Lynch, 2018 ). Being small in size, NMs have a larger surface areas per unit mass than bigger particles, which makes them highly reactive and more dynamic in environmental systems, giving them the ability to interact with different molecules and biological systems ( Rosenkranz et al., 2009 ; Markiewicz et al., 2018 ; Nasser et al., 2020 ) which can transform their original identity ( Lowry et al., 2012 ; Spurgeon et al., 2020 ).

MPs are a significant environmental concern due to their ubiquitous presence, increased biological interactions (compared to macroscale plastic) and difficulties in sampling. Although the size classification of microplastic is often discussed within the literature, the most frequently used definition of MPs is the National Oceanic and Atmospheric Administration (NOAA) definition of less than 5 mm ( NOAA, 2023 ), but discussions within the research community are underway to re-evaluate this in line with the advanced analytical methods now being developed and implemented ( Hartmann et al., 2019 ). MP are also often reported by morphology, categorised as beads or spheres, fibres and fibre bundles, pellets, film, foam or fragments and are introduced into the environment as either primary or secondary plastic ( Rochman et al., 2019 ).

In theory, the considerations applied to NMs for ecotoxicity testing can also be applied to MPs, as the physical interactions and surface conditioning of MPs will also occur in their local environments. Although a relatively emerging field, Daphnia have already been used for a range of MP toxicity studies to date, to elucidate the potential impacts that MPs induce in freshwater environments ( Nasser and Lynch, 2016 ; Schür et al., 2020 ; Zimmermann et al., 2020 ; Kelpsiene et al., 2022 ).

Nanomaterials and microplastics are challenging toxicants to assess due to their physical nature and the surface area of the particles, which makes them an interesting lens from which to review the development in the field of ecotoxicology. Daphnia are a fantastic model for toxicity assessment due to their filter feeding mechanism which means that particle uptake is highly likely, and their transparent bodies then enables a range of optical methods to be applied and developed to quantify the uptake of the physical toxicants, which in turn leads to novel approaches compared to those available for assessment of toxicity of the soluble chemicals that have historically been assessed ( Nasser and Lynch, 2019 ). The particle surface also poses an interesting aspect of the ecotoxicological assessment; the surface of the particles is dynamic and will be affected by the local environment including by biomolecules released by the Daphnia , leading to a more changeable and complex relationship between the toxicant and model organism than that for soluble chemicals ( Wheeler et al., 2021 ; Reilly et al., 2022 ). These dynamic surface-driven properties, which are shared between microplastics and nanomaterials, have enabled interesting developments across the field of ecotoxicology, leading to the development of techniques for assessing in situ transformations and eco-corona evolution. Technological advances such as lab on a chip ( Section 7 ) and conceptual frameworks for identification of key (molecular) events that contribute to adverse outcome pathways ( Section 6 ) provide exciting avenues for further research and development ( Mortensen et al., 2022 ).

2 Nanomaterial transformations in the environment and the role of Daphnia

When NMs are released into the environment, they interact with many environmental components and go through various dynamic transformation processes which can change their physico-chemical properties ( Abbas et al., 2020 ; Malakar et al., 2021 ; Wheeler et al., 2021 ), and significantly impact their toxicity, reactivity, fate and transport in both environmental and biological systems ( Ellis and Lynch, 2020 ; Wheeler et al., 2021 ; Reilly et al., 2022 ). Transformation processes such as adsorption of molecules/ions and macromolecules, agglomeration, oxidation/reduction (redox) reactions, sulfidation and dissolution all occur in biological and environmental systems and can greatly affect the behaviour of NMs ( Lowry et al., 2012 ; Spurgeon et al., 2020 ). The physicochemical transformation of NMs under different environmental conditions are driven by several variables such as ionic strength, kinetics, pH, stability, synthesis method, valency, capping agent, and cation type ( Mitrano et al., 2015 ; Louie et al., 2016 ; Goswami et al., 2017 ). To understand how NMs behave in ecosystems and to determine their toxicity and fate, we must first understand the life cycle and mechanism of NMs transformation processes upon their release into environmental compartments and their interaction with the surrounding environmental components ( Figure 1 ).

Transformation processes of NMs in the environment. Reproduced from ( Batley et al., 2013 ) with permission from ACS Publications.

2.1 Chemical transformations

Dissolution is a key chemical transformation process driven by the release of water-soluble molecules or ions from NMs. The equilibrium solubility (amount of dissolved matter) and the kinetics of particle dissolution (rate of solubility) of NMs will affect their toxicity, behaviour and environmental fate. In general, NMs that readily dissolve were more toxic than poorly soluble NMs, as once bioaccumulated in organisms they undergo rapid dissolution, leading to oxidative stress from the release of reactive oxygen species (ROS) via a so-called trojan horse mechanism. Sulfidation is a major transformation process for many metal NMs, particularly when enhanced concentrations of sulfide are present, such as the ones found in sub-oxic or anoxic sediments or in some parts of wastewater treatment plants ( Lead et al., 2018 ). Sulfidation can reduce solubility, and change particle size and surface charge of NMs and in most studies, the sulfidation of NMs reduces their toxicity ( Devi et al., 2015 ; Starnes et al., 2015 ). Photochemically induced reactions are another main driver of NM transformation, including photolysis, photo-oxidation and photo-catalysis, with sunlight playing an important role in the dissolution of NMs ( Goswami et al., 2017 ; Kansara et al., 2022 ; Xu et al., 2023 ).

2.2 Physical transformations

The physical transformation of NMs leads to alterations in their stability when interacting with environmental components, due to changes in the local ionic strength and pH or due to interactions with sediments and NOM, and are affected by other physical parameters such as sunlight exposure and temperature ( Kansara et al., 2022 ). Agglomeration and sedimentation/deposition are the main physical transformation processes that NMs undergo once released into the environment ( Abbas et al., 2020 ). Agglomerates are particle clusters held together by electrostatic reactions or chemical bonds, they cluster together due to the attractive forces between particles, and this can occur during use, production, storage or upon release of NMs into the environment ( Hartmann et al., 2014 ). The surface area to volume ratio, and thus NMs reactivity, is reduced by the agglomeration process, which will affect their toxicity, transport in porous media, reactivity, sedimentation and uptake by organisms ( Lowry et al., 2012 ). Lead et al. (2018) demonstrated in their review that although many studies showed that agglomeration usually reduces the bioavailability of NMs, however, in some cases it can enhance bioaccumulation by increasing the ingestion rate or by making the particles size more accessible ( Martinez et al., 2022 ). Abbas et al. (2020) also highlighted that at realistic environmental concentrations, homoagglomeration (single NMs only) was proven to be quantitatively unimportant, suggesting that heteroagglomeration (mixture of NMs/particle types) could be more important due to the higher environmental concentrations of natural colloids such as clay. Agglomeration leads to a reduction in NMs number concentration in water or soil suspensions favouring the deposition of agglomerated large particles ( Abbas et al., 2020 ). Larger, denser, particles tend to settle faster than smaller particles and therefore gravitational settling will reduce NM migration ( Hartmann et al., 2014 ; Yin et al., 2015 ; Giorgi et al., 2021 ).

2.3 Biological and macromolecular NM transformations

The major biologically mediated transformation processes that NMs undergo upon their release into natural environments are eco/bio-corona interactions and biodegradation ( Abbas et al., 2020 ). Biomolecules are readily adsorbed onto NM surfaces in ecosystems, and NMs that are taken up by biological organisms can be transformed upon their interaction with biomolecules ( Lowry et al., 2012 ; Xu et al., 2023 ). Additionally, metal ions can be transformed into NMs due to the presence of functional groups and reductive enzymes, for example, metal ions can be transformed into their corresponding NMs by a reducing agent, such as ascorbic acid (C 6 H 8 O 6 ), which occurs naturally ( Abbas et al., 2020 ).

The process of biodegradation is driven by the ability of microorganisms to decompose an organic substance. The interaction of NMs with microbes, extracellular polymeric substances (EPS) and extracellular enzymes determines their relative significance and rate of the biodegradation processes. Biodegradation of NMs core components and surface coatings is a possible transformation pathway of NMs in surrounding environments, particularly for carbon-based NMs including fullerene and carbon nanotubes (CNTs) ( Abbas et al., 2020 ). The biodegradation of organic surface coatings is relevant to all types of manufactured NMs and to MPs, and the gut microbiota of aquatic organisms are likely to play a key role ( Nasser et al., 2020 ). Biomodification is an additional process that can affect the fate and toxicity of NMs, which includes processes that are indirectly mediated by organisms after NMs are taken up by the organisms ( Kelpsiene et al., 2022 ).

In natural environments organisms such as Daphnia produce and secrete a variety of tissue extracts that contain biomolecules (e.g., proteins and polysaccharides). These secreted biomolecules can form coronas on NMs and MPs as the biomolecules adsorb onto the surface of the particles. These coronas are dynamic in nature, and result from the adsorption of different types of available proteins, metabolites and polysaccharides, which also exchange between bound and free forms ( Westmeier et al., 2016 ; Grintzalis et al., 2019 ; Kelpsiene et al., 2022 ). Environmental and biological constitutes that have a molecular weights spanning from 10 to 2,000,000 Da adsorb onto NMs surfaces forming the eco-corona, and affecting the stability, identity, uptake and toxicity of NMs towards Daphnia ( Nasser et al., 2020 ). The adsorbed proteins can also facilitate NMs entry into cells through the receptor-mediated endocytosis process ( Lowry et al., 2012 ). Furthermore, identifying proteins present on the surface of NMs can provide important insights into their biological interactions, including uptake and which mechanistic pathways are induced by NM exposures as part of an ecotoxicity assessment ( Ellis and Lynch, 2020 ; Wheeler et al., 2021 ). Naturally occurring biomolecules (e.g., natural organic matter (NOM), humic substances) also play a major and similar role when interacting with NMs. Therefore, the fate and behaviour of NMs is highly dependent on understanding the characteristics of these macromolecular processes. Interactions with NOM and macromolecules can increase NMs persistence in aquatic systems ( Wang et al., 2016 ).

2.4 Environmental testing conditions—bridging the gap between field and laboratory

Current guidelines for NMs ecotoxicology tests do not prioritise the use environmentally transformed NMs in environmentally representative waters, for example, natural waters that are complex matrices containing natural organic matter and other biopolymers, the concentrations and compositions of which go through various environmental changes which can impact the physiochemical properties and toxicity of the NMs they interact with. Estimates for NM toxicity based on simplified salt only media thus under or over-estimate the impacts of NMs by not testing the appropriate NMs forms ( Selck et al., 2016 ; Ouyang et al., 2022 ). To allow for comparisons between toxicity studies, the Organisation for Cooperation and Development (OECD) promotes the use of a fully defined testing medium for exposures in ecotoxicity testing. As with many biological elements, there is a narrow concentration range between deficiency and toxicity of key elements which needs to be carefully considered, as the testing medium needs to be suitable for the test species (i.e., both algae and Daphnia during chronic testing). Medium composition can also impact the toxicant in question, for example, when testing metal toxicity, it is important to ensure there are no chelating agents, such as EDTA, present that would react with the metals and therefore change the metal bioavailability during the exposure. Media such as the OECD ‘M4’ and ‘M7’ can be modified by removing the EDTA, or an alternative medium can be used that contains no chelating agents ( OECD, 2004 ).

The OECD 202 and 211 tests (along with most current test guidelines) were designed for testing of chemical toxicants, and as such, modification of medium for emerging pollutants such as NMs and MPs, with their large reactive surfaces, and more environmentally realistic testing scenarios is needed ( Giusti et al., 2019 ; Ellis et al., 2020a ; Nasser et al., 2020 ). This includes the potential addition of NOM, which is the decaying plant and animal matter present in natural waters and soils, and has been described as containing varying fractions of humic acids, fulvic acids, polymeric substances and a hydrophilic fraction, and which has been widely reported to have strong absorption to colloidal materials ( Afshinnia et al., 2018 ; Tayyebi Sabet Khomami et al., 2020 ), or the use of medium conditioned by pre-filtration through Daphnia guts (or other relevant organisms used for toxicity testing, including oysters, worms, etc.) which is often termed “conditioned medium,” for example, ( Nasser and Lynch, 2016 ). Although the addition of NOM is not recommended by the OECD due to its heterogenous nature, NOM can act as a stabiliser for NMs within the testing medium, preventing agglomeration of the NMs and therefore maintaining the bioavailable fraction. On the other hand, NOM can sorb chemicals in test solutions which can ultimately affect the fate and bioavailability of the toxicants within the test solutions by decreasing the concentration in the dissolved phase and changing the exposure pathway. The addition or exclusion of NOM is therefore test dependent and should be considered during the toxicity test design stage.

Given the role of standardised tests in ranking the toxicity of chemicals, including NMs and MPs, as well as the use of data from Daphnia toxicity assays for environmental modelling and establishment of threshold levels for pollutants, a deeper understanding of the inherent variability in the test systems is needed. Similarly, the standardised test media has been developed to optimise the test population health, which does not take into consideration any deficiencies in species health or fitness that occur due to natural environmental variation and adaption to the environment. Utilisation of laboratory cultured daphnids, whose conditions are optimised for health and fitness and where there is no competition for food and no predation, could mean that we underestimate the potential toxicity of chemicals to real environmental populations, especially when looking at sublethal toxicity markers such as growth and reproduction due to the lack of variability in other parameters such as temperature and food availability. However, examples emerging in the literature are showing that the “as engineered pristine” NM have fewer toxic consequences in environmentally realistic medium compared to the standard culture media used in standard toxicity testing ( Schultz et al., 2018 ; Ellis and Lynch, 2020 ; Schultz et al., 2020 ). Thus, standardised Daphnia tests following the OECD protocols overestimate NMs toxicity, which can be resolved through using environmentally transformed NMs in representative natural water compositions ( Nasser and Lynch, 2019 ; Ellis and Lynch, 2020 ; Nasser et al., 2020 ). Conversely, wild daphnids from the field are believed by aquatic toxicologists to have a higher resistance towards pollution compared to Daphnia that have been cultured in the laboratory over long periods ( Abdullahi et al., 2022 ; Eastwood et al., 2022 ). This is due to the exposure of wild Daphnia to a much wider range of natural stressors than their lab-cultured equivalents where conditions are closely controlled, leading the wild daphnids to have resistance towards pollutants, decrease of water quality and competition for a limited of food supply, resulting in a greater overall “fitness” and ability to survive in changing environments ( Barata et al., 2000 ). Taking this into consideration, a precautionary approach should be applied when applying lab observations to field studies and vice versa .

According to Brans et al. (2018) , human-induced and natural stressors induce changes in energy metabolism and stress physiology in populations of a wide array of species. Urbanization is a pervasive process with 476,000 ha of arable land are lost annually by the expansion of urban areas ( World Resources Institute, 1996 ; Grimm et al., 2008 ). Urbanization alters both biotic and abiotic ecosystem properties within and surrounding the urban centre ( Miles et al., 2019 ; Ruas et al., 2022 ). Differential selection of stress-coping mechanisms results from stressful environments like those found in cities. For instance, city ponds are exposed to the urban heat island effect and receive polluted run-off, with the result that several stressors may act together and affect the life traits of organisms inhabiting these ecosystems, which might acquire genetic differentiation for physiological traits enabling them to cope better with higher overall stress levels ( Pavlaki et al., 2014 ). Evidence from 62 Daphnia genotypes from replicated urban and rural populations in garden ponds revealed that urban Daphnia have significantly higher concentrations of total body fat, proteins and sugars than their rural counterparts ( Brans et al., 2018 ) highlighting that environmental conditions contribute to Daphnia fitness. This can be further explored through study of acclimation, also called adaptation, resistance, or tolerance, which has been defined as the ability of organisms to cope with stress, either natural such as temperature changes, salinity variation, oxygen level fluctuations, and plant toxins or chemicals arising from anthropogenic inputs of many different classes of contaminants into the environment ( Biagianti-Risbourg et al., 2013 ; Akbar et al., 2022 ). The capacity of physiological adaptation or acclimation toward a stressor is related to the stress syndrome. Physiological acclimation to toxicant conditions also depletes energy reserve levels ( Biagianti-Risbourg et al., 2013 ). For example, in Daphnia organisms pre-exposed to zinc (and having acquired a tolerance toward this metal) did not mobilize their energy reserves further following a laboratory exposure to zinc (0.1 and 1.0 μM) compared with non-exposed animals ( Canli, 2005 ).

Therefore, the culturing history of the Daphnia , or other test species, in addition to the environmental conditioning of the NMs or MPs in the exposure medium can have a significant impact on the toxicity response and the overall impact on the ecosystem, and this should be considered at the test design stage to determine the adequate levels of comparability and environmental relevance of the exposure.

3 The development of Daphnia as a model organism: lifecycle, reproduction and multigenerational approaches

One of the most important issues to address in toxicological testing is how exposure, whether it be acute or chronic, impacts the organism and the subsequent effects to their offspring. When environmental conditions deteriorate, for example, due to an influx of environmental pollution or predator stress, daphnids can develop different phenotypes and can switch from clonal to sexual reproduction ( Colbourne et al., 2011 ; Eastwood et al., 2022 ).

Under favourable environmental conditions (e.g., within the optimal range shown in Table 1 ), Daphnia reproduce parthenogenetically (clonally). Parthenogenesis is a type of asexual reproduction in which the offspring develops from unfertilized eggs. Female Daphnia produce genetically identical daughter clones, which are released from the brood pouch as neonates ( Ebert, 2005 ). The reproduction process continues while the environmental conditions continue to support their growth. Daphnia can change to sexual reproduction under stressful conditions, such as overcrowding, low food, toxicant exposures or variations in abiotic factors such as temperature and pH ( Alekseev and Lampert, 2001 ; Abdullahi et al., 2022 ). This results in the development of ephippia, or resting eggs, that can remain dormant in the sediment for long periods of time (years) and may hatch when conditions improve ( Eastwood et al., 2022 ).

Recommended conditions for optimal culture growth of Daphnia as outlined in the OECD test guideline for acute toxicity (OECD 202).

As a consequence of NMs induced stress, the genetic processes are altered, which can be easily monitored by identification of epigenetic (heritable from one generation to the next) changes in subsequent generations. These changes are due to modifications of the histone proteins of chromatin and DNA methylation, which results in altered gene expression ( Feil and Fraga, 2012 ). This makes Daphnia an ideal model organism for studying the effects of NM toxicity, as epigenetic developmental programs can be used to monitor the effects in the offspring as hereditary traits ( Asselman et al., 2017 ). Having the ability to monitor the offspring after NM parental exposure provides invaluable information regarding the molecular events that occur for survival, growth, reproduction, and adaptation to change ( Abdullahi et al., 2022 ).

An advantage of using multiple generations that follow a germline after parental exposure, is that the genes expressed at an early stage of exposure might not be the same genes as those directly associated with phenotypic effects over a chronic exposure time scale. Therefore, capturing the phenotypical events in the offspring will also identify the longer-term causable effects ( van Straalen and Feder, 2012 ). Multigenerational studies also help to demonstrate how maternal effects of exposure to what is considered a sub lethal concentration, results in a trade-off between growth, reproduction, and survivorship over all generations, which ultimately defines the natural selection of the strongest surviving daphnids.

Several multigenerational studies using Daphnia , in the presence of either chemical or NM exposure have each reported an increased toxic effect in the immediate post-parental exposure generations compared to the exposed parental generation ( Arndt et al., 2014 ; Jeong et al., 2016 ; Kim H. Y. et al., 2017 ; Ellis et al., 2020b ). A study investigating the species difference between D. magna , pulex and galeata over five generations exposed to silver nanoparticles demonstrated that NM exposure had the most negative effects on the first generations, with notable changes between increased toxicity and tolerance in the subsequent generations ( Völker et al., 2013 ). The altered toxicity in the latter generations provides evidence that the ecological risk and safety assessments underestimate NM toxicity using only single generation acute and chronic tests. Ellis et al. (2020b) also identified that the transgenerational responses of multiple germlines showed a direct link with maternal exposure time to ‘sub-lethal’ effect concentrations of NMs (identified from standard OECD acute toxicity tests which chronically presented as lethal) including increased survival and production of males for sexual reproduction in the subsequent germlines ( Ellis et al., 2020b ). Multigenerational studies using only pristine engineered NMs have manifested adverse toxicological outcomes in multiple generations post maternal exposure to Daphnia that could not have been predicted from the single standard 1-generation reproductive studies ( Ellis et al., 2020b ; Ellis and Lynch, 2020 ; Karatzas et al., 2020 ). Collectively, the research demonstrates the importance of updating standard toxicity testing to reflect scientific advances and increase trust in regulation by monitoring the effects in the transgenerational germlines ( Jeong et al., 2016 ; Ellis et al., 2020b ; Nederstigt et al., 2022 ).

4 Feeding behaviour of daphnids and bioaccumulation potential of NM

Daphnia are filter-feeders that have the ability to ingest particulates of up to 50 μm in size through mechanical sieving mechanisms ( Geller and Müller, 1981 ). Daphnia mainly feed on phytoplankton, such as green algae (considered as a high-quality food, such as Scenedesmus sp.), bacteria and organic detritus (considered as a low-quality food). However, their non-selectivity in the uptake process increases the bioaccumulation of environmentally unfriendly materials along the higher trophic levels ( Schwarzenberger and Fink, 2018 ). Therefore, Daphnia are likely recipients of contaminants, including NMs and MPs and, as primary consumers, they are vital for energy transfer in the food chain ( Martinez et al., 2022 ; Yin et al., 2023 ).

4.1 Nano-intestinal interactions and gut chemistry

The gut luminal chemistry is of particular interest to comprehend the fate and adverse effects of NMs on the organism after NM ingestion. The pH (6.8–7.2), ionic strength (e.g., Na + , Ca 2+ , and Mg 2+ ), the presence of NOM, the cuticle chemistry, and redox chemistry are factors that interfere in the absorption of particulates in the gut, and they act as barriers in NMs exposure during uptake from the gut into the tissue ( Van Der Zande et al., 2020 ). From the external environment to the gut, NMs are likely to acquire an eco-corona which is generally expected to reduce their toxicity ( Ekvall et al., 2021 ), although if the acquired corona results in some particle agglomeration the particles may become a more attractive food source and thus be taken up to a greater extent resulting in increased toxicity ( Nasser and Lynch, 2016 ). Depending on their location in the intestine, NMs can acquire an unique eco-corona profile ( Chetwynd et al., 2020 ; Cui et al., 2020 ), and undergo different transformations, for example, a pH-dependent dissolution ( Cao et al., 2022 ). Due to the particularities of NMs, they are normally dispersed in the luminal liquid, rather than dissolved, and a wide diversity of macromolecules (solid-phase food, exudates, digestive enzymes, and proteins) present in or from the external environment can also be considered as additional colloidal components that can contribute to its dispersibility or not ( Nasser et al., 2020 ; Van Der Zande et al., 2020 ). The composition of the digestive tract and the interaction forces between NMs and gut lumen matrix determine NMs bioavailability, potentiating or mitigating NM toxicity to daphnids ( Cui et al., 2020 ). Consequently, the physicochemical characteristics of NMs and natural biological constituents must be studied in terms of their colloidal chemistry to determine their colloidal behaviour and impacts on daphnids’ physiology ( Christenson, 1984 ).

4.2 Enzymes as biochemical markers of nanotoxicity

The median effective concentration leading to immobility (EC 50 ) or lethality (LC 50 ) are the OECD standardised endpoints considered in acute toxicity assessment ( OECD, 2004 ), while body burden, reproductive, and growth rate are the end points for the chronic assessment ( OECD, 2012 ). However, these apical endpoints are less sensitive than the biochemical ones ( De Coen and Janssen, 1997 ). To enable a better understanding of the adverse outcomes on food metabolism and stress response, we need to identify suitable biomarkers for ecotoxicity ( Schwarzenberger and Fink, 2018 ). Biochemical markers can work as early indicators (sub-lethal toxicological effects) of perturbance on organisms metabolism, resulting in alterations in enzyme activity or expression, and can be identified using enzyme assays ( Galhano et al., 2020 ; Galhano et al., 2022 ), or a range of omics techniques ( Taylor et al., 2018 ; Zhang et al., 2018 ; Bhagat et al., 2022 ), respectively. Here, we summarise the main findings reported for two major enzymes classes (digestive and antioxidant) from Daphnia nanoecotoxicology studies ( Table 2 ).

Effects of exposure of D. magna to NMs on the activity and/or expression of antioxidant and digestive enzymes.

↑ increase of enzyme activity/gene expression related, while ↓ implies decrease.

4.2.1 Digestive enzymes (food metabolism)

In addition to the mechanisms involved in the digestive process mentioned earlier, digestive enzymes play an important role in the metabolism of ingested food, breaking down food particles and increasing the efficiency of digestion. Overall, several enzymes are secreted to metabolize proteins such as trypsin and chymotrypsin, sugars such as cellulase, α-amylase and β-galactosidase; and lipids such as esterase ( Lv et al., 2017 ). When exposed to NMs and/or pollutants, their activity is changed to maintain the homeostasis of organism’s metabolism. However, depending on the dosage level which they are exposed, these compounds can affect digestive physiology and food metabolism in Daphnia ( Qi et al., 2022 ). NMs were shown to target mainly intestine epithelium and peritrophic membrane ( Mattsson et al., 2016 ). Recent evidence depicted that NMs can penetrate cell membrane without disrupting it, and be endocytosed ( Santo et al., 2014 ). NMs physicochemical characteristics (e.g., shape, size, and surface chemistry) are determinant to the chemical transformations they undergo in the digestive tract of daphnids and later elicited biological responses ( Liu et al., 2019 ).

An inhibition on the activity of amylase and esterase in the treatment with a low and higher concentration of TiO 2 NMs in acute toxicity assessment was observed, and this effect was even more evident during a chronic assay. Exposure to the NMs has shown to affect the nutrition, growth, and reproductive processes in daphnids ( Fouqueray et al., 2012 ) and this was demonstrated by a dose-dependent decrease in the enzymes’ activity after exposure to fullerene (C 60 ) ( Lv et al., 2017 ). In another work, the activity of enzymes was monitored over days, which confirmed a reduction of their activity in D. magna ( Tao et al., 2016 ). In D. pulex , zinc oxide NMs (ZnO NMs), bulk (ZnO), and ionic species (Zn 2+ ) dysregulated the expression of genes related to chymotrypsin, carboxypeptidases, and serine protease enzymes ( Lin et al., 2019 ). Disruption of intestinal structure of daphnids after interaction with stressors commonly impacts on energy acquisition and causes high metabolic costs, via reduction of the available energy reserves (carbohydrates, lipids, and proteins), to keep the basal metabolism. The adsorption of NMs to active sites or surface of trypsin enzyme was suggested as a possible mechanism to inactivate digestive macromolecules by a comparable experimental and theoretical study ( Zhang et al., 2014 ).

4.2.2 Antioxidant enzymes (oxidative stress)

Daphnids initially respond to the intake of foreign materials (NMs and contaminants) by producing ROS, which results in oxidative stress. Prolonged exposure to these stressors can lead to lipid peroxidation, protein inactivation, and DNA damage. Antioxidant enzymes modulate their activity to reduce the damage caused. Superoxide dismutase (SOD) is the first defence line in detoxification that produces the substrate for catalase (CAT) to metabolize. Then, CAT, glutathione-s-transferase (GST), and glutathione peroxidase (GPx) remove harmful metabolites generated from this process transforming them into less toxic compounds, such as water and oxygen ( Galhano et al., 2022 ). Proteins associated with oxidative stress response can be found in the NM eco-corona, giving insights into the mechanistic pathways associated with NM toxicity ( Nasser and Lynch, 2016 ; Fadare et al., 2019 ; Ellis and Lynch, 2020 ).

Similarly, Daphnia exposed to C 60 (fullerenes) increased SOD activity after 48 h, and decreased after 72 h, indicating oxidative stress damage and possibly the beginning of lipid peroxidation since simultaneously a dramatic increase of malondialdehyde (MDA) was obtained ( Lv et al., 2017 ). Increasing dose of TiO 2 NMs exposed to daphnids augmented the activity of CAT, GST, and GPx but no effect was observed for SOD ( Kim et al., 2010 ). CAT response was similar to those observed for GST and GPx, but in long-term exposure to the NMs, their activity recovered due to acclimation ( Fouqueray et al., 2012 ). In contrast, ZnO NMs increasingly inhibited the activity of enzymes as exposure duration increased. Similarly, the genes corresponding to SOD and GST were upregulated initially and later downregulated, while MDA content increased over time, indicating that the detoxification was overwhelmed and possibly led to GST inactivation ( Mwaanga et al., 2014 ; Chen et al., 2017 ). Exposure to quantum dots (QDs) functionalized with indolicidin (an antimicrobial peptide) also affected enzyme efficiency, but SOD adapted to the stress condition, while CAT was greatly induced after 15 days and GST activity slightly increased during period of exposure ( Falanga et al., 2018 ).

The role of surface chemistry on enzyme activity was investigated using gold NMs (AuNMs). Negatively charged AuNMs (MPA-AuNMs) caused a significant increase in gst expression, related to GST, while no effect was noted from positively charged (PAH-AuNMs) after 24 h ( Qiu et al., 2015 ). However, in another work, exposure to PAH-AuNMs, induced gst expression compared to MPA-AuNMs ( Dominguez et al., 2015 ). Both studies considered that the toxicity may be associated with the AuNMs colloidal behaviour, because PAH-AuNMs were more stably dispersed in the test medium than the MPA-AuNMs, they were more bioavailable to be absorbed and cause damage to the daphnid gut. PAH-AuNMs was slightly toxic even at a low exposure dose (5 μg L −1 ) and increased significantly at 10 and 50 μg L −1 ( Bozich et al., 2014 ). The contrasting responses in these studies may have resulted from the distinct exposure conditions (i.e., the concentration used).

Combining NMs with inorganic or organic contaminants is an attractive approach to investigate the oxidative damage triggered in a real-world-like scenario ( Galhano et al., 2022 ). For example, exposure to TiO 2 NMs in a range of concentration (from 10 to 100 μg L −1 ) of copper (Cu 2+ ) resulted in induction of CAT, reaching a maximum of 10 and 20 μg L −1 in the presence and absence of the NMs respectively. Inhibition of CAT occurred as the dose of Cu 2+ increased ( Fan et al., 2012 ). Similar behaviour was described for SOD, but no statistical difference was found between the exposure in the presence and absence of TiO 2 NMs, meaning that the presence of NMs had no additional effect on the activity of this enzyme. However, an inhibitory effect was observed in Na+/K+ ATPase transporter in mixture conditions compared to the condition without TiO 2 NMs. With respect to the mortality rate, the co-exposure of TiO 2 NMs with Cu 2+ increased the toxicity compared to the treatment which organisms were exposed to Cu 2+ only ( Fan et al., 2012 ). In another study, TiO 2 NMs with varied percentage of free {001}facets combined with Cu 2+ reduced SOD activity, but just a slight decrease was observed in Na+/K+ ATPase activity and no change in this transporter activity in the single exposure condition to the NM ( Liu et al., 2015 ). In Mwaanga et al. (2014) work, NOM was added to the test medium and reduced the inhibitory effect of ZnO and CuO NMs on GST activity, possibly, by diminishing the accessibility of NMs to adsorb GST.

Galhano et al. (2020) integrated an individual and subcellular level approach to effectively assess the toxicity of TiO 2 NMs (12.5–100 μg L −1 Ti) or AgNPs (25–125 μg L −1 Ag), under environmentally relevant conditions, i.e., transformed NMs. The authors observed significant alterations in the activity of GST and CAT mainly after exposure to AgNPs dispersed in wastewater compared to test water. Later, Galhano et al. (2022) evidenced that only in the case of AgNPs dispersed in the wastewate and effluent was SOD activity decreased. The differences in the enzymatic activity of antioxidant enzymes under the conditions of exposure were indicated as resulting from the difference in the physicochemical characteristics of the TiO 2 NMs under the different exposure conditions (7.8 and 4,761.4 μg L −1 Ti in water, 6.3 μg L −1 Ti in wastewater-borne, 17.3 and 5,467.5 μg L −1 Ti in effluent) and AgNPs (81.7 and 105.4 μg L −1 Ag in water, 30.3 μg L −1 Ag in wastewater-borne, 56.4 and 80.5 μg L −1 Ag in effluent), the complexity of the matrices and the aging of the effluents used ( Galhano et al., 2022 ). Stable agglomeration of TiO 2 NMs may have reduced their bioavailability. Interestingly, the author also observed a toxicity coming from the background effluent that proved to be relevant and should be considered in further studies ( Galhano et al., 2022 ).

4.3 Challenges and perspectives for enzyme activity studies in Daphnia

Most studies on nanobio-interfacial interactions in the gut have been carried out on animal models, and cultures of intestinal cells from invertebrates are not yet available, representing a great challenge for the advancement of research on the underlying mechanisms of absorption and bioavailability of NMs in invertebrates. The small size of daphnids and sample contamination (e.g., mucus and carapace) are the main limiting factors that influence data collection ( Mattsson et al., 2016 ; Van Der Zande et al., 2020 ). Besides investigating nano-intestinal epithelial cells interaction, understanding NMs interactions with Daphnia gut microbiota are necessary, since ingested NMs and other stressors change the composition and functioning of microorganisms that inhabit the gut ( Akbar et al., 2020 ; Cui et al., 2020 ). The alteration of life history traits have also been shown to mediate the toxicity response ( Li et al., 2019 ), however, the understanding of life history and gut microbiome influence NM and MP toxicity are still in their infancy ( Li et al., 2019 ; Akbar et al., 2020 ; Varg et al., 2022 ).

Despite biochemical markers being promising tools for early aquatic toxicity assessment ( Michalaki et al., 2022 ), the biochemical and physiological responses generally do not correlate and sometimes there is no consistency in the results. Therefore, it is essential to push the scientific community to harmonise experimental procedures to produce reliable data, following the Findable, Accessible, Interoperable, and Reusable (FAIR) principles ( Wilkinson et al., 2016 ), which will later allow linking physiological responses with molecular patterns and (sub) organismal responses and facilitate computational modelling and predictive (nano) toxicology. This will allow computational modelling of current data, identification of biomarkers that predict adverse outcomes and decision making for regulatory purposes ( Taylor et al., 2018 ).

4.4 Bioaccumulation of NMs in aquatic food webs and quantification of particle uptake

Uptake and bioavailability studies are significant for studying the behaviour of NMs in natural environments and for linking the biological effects to the environmental chemistry of NMs ( Lead et al., 2018 ). In addition to microorganisms, crustaceans such as Daphnia are involved in the degradation of organic matter and nutrient recycling, working as shredders in natural ecosystems and acting as pivotal components of the food web ( Ebert, 2005 ). Further to already being an established model species for regulatory testing, Daphnia are an advantageous model for NM and MP toxicity testing due to their clear body which allows visualisation of the uptake and potential retention of NMs and MP within the Daphnia gut. Fluorescently labelled industrial beads have been widely used to undertake initial toxicity assessments of MPs, which uses the fluorescence as a proxy for the MPs to theoretically determine uptake, translocation, and potential storage in the organism’s tissue ( Figure 2 ) ( Rosenkranz et al., 2009 ). However, the potential for dye to leach from the beads can confound the results, as dyes can be retained in the lipid deposits and other tissues within the Daphnia leading to incorrect tracing of the MPs and to misreporting of translocation of MPs in cases where this has not occurred ( Schür et al., 2019 ). For example, Nile red is often used as a stain to identify MP in environmental samples, but this dye is also widely used for lipid staining within Daphnia which can lead to overestimation of internalised MPs concentrations and retention. Furthermore, the change in internal biological conditions (such as pH) can affect the fluorescent signalling from the dye which can significantly impact the results or could further impact the dye leaching from the particles ( Triebskorn et al., 2019 ; Davis et al., 2020 ).

An example of the use of fluorescence to determine uptake of 1–5 µm polyethylene MP particles by D. magna visualised in two daphnids exposed to different concentrations of the MPs (50 and 500 mg/L, respectively) for 24 h. (A,C) brightfield, and (B,D) fluorescence imaging of the first and second daphnids, respectively. Taken with an Olympus optical microscope with a Green Fluorescent Protein (GFP) filter cube and dichroic mirror and a DP74 colour camera and viewed using CellSens software (×70 magnification).

Quantifying the internalised concentration of particles during exposures can vary depending on the material type but is very useful data to collect as the internalised concentration can be used to more accurately determine a dose response relationship. Internalised concentration, or body load, is needed for Toxicokinetic-Toxicodynamic (TK-TD) modelling such as Dynamic Energy Budgets (DEB) which can link several aspects of life history trait observations to potential changes in the population distribution. Some of the more widely used methods for visualisation of particle accumulation and damage are transmission electron microscopy (TEM) to determine the uptake, potential translocation and retention of particles within organisms tissues as per the example shown in Figure 3 ( Ellis and Lynch, 2020 ) and ICP-MS analysis for quantification of metal, or metal doped, particles in tissue ( Schmiedgruber et al., 2019 ; Ellis and Lynch, 2020 ).

An example of the use of TEM images to determine the accumulation of NM in Daphnia guts and potential translocation. Uptake and localisation of (A) freshly dispersed PVP-coated Ag NMs in HH combo medium showing microvilli interactions, (B) freshly dispersed PVP-coated Ag NMs in HH combo found in the gut lumen space; and (C) freshly dispersed uncoated TiO 2 NMs in HH combo, showing evidence of NM translocation in the bush border. Reproduced from ( Ellis and Lynch, 2020 ) with permission from the Royal Society of Chemistry.

5 Daphnia genome and key NMs toxicity pathways

Direct investigations of how NMs are associated with adverse outcomes and disease in humans are very limited ( Lima et al., 2022 ), mainly due to the issues around how chemicals including NMs are regulated under existing chemical frameworks and the ethical limitations of testing directly on humans ( Hansen, 2017 ; Oksel Karakus et al., 2021 ). Due to the shared mammalian biology, information safeguarding human health relies on the toxicological information from invertebrate (mice and rat models) in vivo studies to protect human health ( Festing and Wilkinson, 2007 ; Franco, 2013 ), as well as fish and algae, which have been traditionally used to set regulatory limits to safeguard environmental health ( Brunner et al., 2009 ; Kaltenhäuser et al., 2017 ).

The principles of the 3Rs (Replacement, Reduction and Refinement) were developed over 50 years ago providing a framework for performing more humane animal research ( https://nc3rs.org.uk ) ( Burden et al., 2017 ; Sneddon et al., 2017 ). Advancement of animal genome research over the last 20 years has led to significant understanding of the relationship between simple organisms and their changing environment ( van Straalen and Feder, 2012 ; Stracke and Hejnol, 2023 ). Moreover, the advancement in evolutionary developmental biology and ecological functional genomics, has identified a possibility to reduce the use of animal experimentation using simple model organisms including invertebrates, due to the large amount of the genome that is conserved across species. Genomics is used to identify genetic variation under natural selection ( Mitchell-Olds and Feder, 2003 ; Leinonen et al., 2013 ; Grummer et al., 2019 ) in model test organisms, which are accessible to both laboratory and field studies along defined environmental gradients ( Spanier et al., 2017 ). Furthermore, comparative studies into the evolution and conservation of genes and genomes, provides significant information on genetic diversity and similarities among major groups of organisms from simple organisms to larger invertebrates ( Ros-Rocher et al., 2021 ).

5.1 Daphnia genome

Characterisation of Daphnia genomes enables the progress of molecular ecotoxicology for evaluating pollutants and NMs impacts, by analysing molecular pathways related to their defence mechanism response ( Lee et al., 2019 ). The complete genome sequence of D. pulex ( Colbourne et al., 2011 ) and D. magna have been elucidated but the search for key gene families related to stressors exposure is ongoing through screening of likely molecular biomarkers ( Orsini et al., 2016 ). Identification of genomic expression profiles enables understanding of how genes are regulated under different ecological conditions and how these expressions are linked to phenotypic change ( Rozenberg et al., 2015 ; Hales et al., 2017 ; Ros-Rocher et al., 2021 ). Phenotypical variation is understood to be largely due to gene and environment interactions that were shaped by evolution, or by environmental stress that predictably disrupt the normal functioning of genes ( Hodgins-Davis and Townsend, 2009 ; Moyerbrailean et al., 2016 ; Alexander-Dann et al., 2018 ).

The expression profile (when compared to a non-treated control organism) of all genes that are expressed in response to a particular initiating event, is called a “molecular phenotype.” The transcription of the “molecular phenotype” is based on the evolutionary history of populations ( Ravindran et al., 2019 ). Hence, the transcriptional responses of Daphnia to environmentally exposed NMs are a rich source of both phenotypic and genotypic information about the mechanisms of adaptation. This approach aligns human- and eco-toxicology towards a more general understanding of how exposure to NMs disrupts biological processes that otherwise ensure animal (including human) health. Evidence is growing on the feasibility of classifying the effect of NMs on humans, based on gene expression monitoring using distantly related environmentally relevant model organisms such as Daphnia ( Amorim et al., 2023 ). Therefore, we can use comparative genomics to confirm that model organisms retain a greater number of ancestral gene families that are highly conserved and are shared with humans which are also closely linked to human diseases ( Marwal and Gaur, 2020 ; Colbourne et al., 2022 ). Consequently, a chemical hazard assessment framework built upon key events may be informative for a greater diversity of species, by exploring the use of the homology between ecological model test species and humans to understand original molecular interactions and responses to emerging pollutants such as MPs and NMs.

5.2 Conserved biochemical pathways as a basis for understanding NM toxicity

The challenging ecological environments in which the multiple species of Daphnia inhabit, make Daphnia , an optimal genomic model for monitoring stress and adaptive changes ( Vandegehuchte et al., 2010 ; Vandenbrouck et al., 2010 ; Colbourne et al., 2011 ) to their reproductive nature (as discussed in section 4 ). Understanding the genomic traits in Daphnia has already given great insight into developmental plasticity, causing altered morphology and behaviour in response to environmental stress ( Akbar et al., 2022 ; Sha and Hansson, 2022 ) and adaptation to environmental toxicity ( van Straalen and Feder, 2012 ). Therefore, due to their high degree of phenotypic plasticity, physiology and ecological importance ( Lee et al., 2019 ), understanding the mechanism of action (MOA) as a result of daphnids exposure to NMs, is critical for the prediction of the selectivity and sensitivity in other species. Furthermore, this understanding will lead to the development of adverse outcome pathways (AOPs), by understanding what NMs concentrations cause harm ( Russo et al., 2018 ), which can then be imposed onto standardized toxicological tests and risk assessments.

Access to the Daphnia genome sequence has enabled researchers to study specific gene changes in response to a multitude of environmental influences, and to discover the MOAs of several chemicals ( Garcia-Reyero et al., 2009 ; Garcia-Reyero and Perkins, 2011 ; Giraudo et al., 2017 ) rendering gene transcription profiling one of the most powerful tools in developmental biology. The advancement of high-throughput RNA-sequencing (RNA-seq) provides whole transcriptome profiling, which allows the unbiased detection of novel transcripts in a sample at a given time ( Giraudo et al., 2017 ).

Over the past decade, developmental work using phylogenetic mapping relationships and amino acid homology have identified that genomic regions under natural selection show evolutionary relationships among conserved genes from different species ( van Straalen and Feder, 2012 ). Although there are major differences between vertebrates and invertebrates, there is a growing body of evidence that distantly related species share many ancestral genes by common descent that serve the same biochemical pathways ( Rogozin et al., 2014 ). The homology between genes (similarity in genes due to shared ancestry between species) are analysed using Gene Ontology ( http://geneontology.org/ ), gene orthologs database ( https://www.orthodb.org/ ), or pathway enrichment tools (such as DAVID, GSEA or Reactome). This analysis catalogues genes shared among species by descent across the animal kingdom to infer functional conservation, which helps to highlight highly conserved key genes involved in stress response pathways. Therefore, the homology-based approach identifies genes “in common” from model species and aligns them with similar orthologues to gene family members across large evolutionary distances ( Spanier et al., 2017 ; Adrian-Kalchhauser et al., 2020 ). Comparative genomics studies have confirmed that crustaceans retain a greater number of ancestral gene families that are shared with humans than insects (such as Drosophila ), including genes responsible for growth, reproduction, and maintenance ( Rogozin et al., 2014 ; Colbourne et al., 2022 ). Indeed, Daphnia retain a disproportionately large number of ancestral gene families that are linked to human diseases, despite more than 780 million years since present day crustaceans and mammals last shared a common ancestor ( Colbourne et al., 2022 ).

Several studies have presented preliminary links, using qualitative gene expression profiling and Daphnia , between NM exposure-related harm and human disease ( Asselman et al., 2013 ; Aksakal and Arslan, 2019 ; Ellis et al., 2020b ). Such research uses gene expression to help bridge the gap between distantly related species by understanding how exposure to pollutants disrupts key conserved biological processes ( Mav et al., 2018 ). The most common homologous genes observed in these studies have been those associated with metal detoxification, oxidative stress, energy production, DNA repair ( Poynton et al., 2007 ) and general maintenance ( Asselman et al., 2013 ; Qiu et al., 2015 ), which all served as biomarkers that are shared between species. The results from these studies collectively recognise that genes associated with growth, reproduction, homeostasis, xenobiotic detoxification, and metabolism, all provide mechanistic insights into NM-organism interactions and represent pathways encoding for cellular functions that are known to be induced by NM exposure studies.

Although further research is needed, comparing common genes and the biochemical pathways that link the differential transcriptomes, shared by common descent among species, offers meaningful insights into the connections between model test species and environmental health exposure, which can help to establish areas for development in NM risk assessment.

6 Adverse outcome pathways—gaps in terms of ecotoxicity and NMs

Relating molecular responses to phenotypic effects is crucial in environmental risk assessment. A promising approach is the AOP framework, which describes key steps of toxic mechanisms resulting in adverse effects in animals and populations ( Ankley et al., 2010 ; Mortensen et al., 2022 ). AOP starts with a molecular initiating event (MIE) that can be triggered by various environmental contaminants. These MIEs are linked to a series of key events (KEs) that can result in an adverse outcome via different signalling pathways with different levels of biological organisation, e.g., molecular, cellular, tissue, organ, organism, and population ( Villeneuve et al., 2014 ; Rugard et al., 2020 ). These interlinked pathways can be assembled into AOPs and serve as a foundation for the development of a mechanistic understanding of toxicity and disease. Therefore, AOPs play an essential role in the ecological risk assessment of environmental contaminants ( Ankley et al., 2010 ; Khan et al., 2020 ).

To date, the majority of AOP-building effort has been focussed in human health and mammalian studies, but the application of AOPs for model organisms such as Daphnia is increasingly being investigated ( Song et al., 2020 ). While in principle AOPs are chemical-agnostic, and focussed more on mechanisms, such as endocrine disruption or ion-channel blocking, for example, work is underway to establish NMs-specific AOPs, focussing on key aspects such as NMs-biomolecule interactions, NM-membrane interactions, and NMs-induced disruption of enzyme activity, for example, Jagiello et al. found that only 8 of 331 available AOPs in the AOP-Wiki are specific for NMs (namely, AOP numbers 144, 173, 207, 208, 209, 210, 241, and 319), meaning that only 2.4% of all AOPs in the AOP-Wiki have (as of 2022) been assessed or considered for their direct relevance to NMs, and thus there is a need to evaluate whether remaining AOPs might be applicable fully or partially to NMs ( Jagiello et al., 2022 ).

NMs induced formation of ROS is one of the most significant reasons for adverse effects from NMs. Likewise, oxidative stress is well known to contribute to pollutant-induced cell damage and toxicity ( Balážová et al., 2020 ). ROS can target multiple cellular components, including mitochondria, membrane lipids, DNA, structural proteins, and enzymes, resulting in different adverse outcome. NM exposure to Daphnia has caused NM accumulation in the gut, which led to accumulated ROS ( Kim et al., 2010 ; Nasser et al., 2020 ), decreased growth ( Karimi et al., 2018 ; Ellis et al., 2020b ), and decreased fertility and reproduction ( Mendonça et al., 2011 ; Olkova, 2022 ). When Cu (II) was absorbed on TiO 2 , the oxidative stress increased, and intestinal damage was found ( Liu et al., 2015 ). It has been reported that CNT exposure can cause ingestion of CNTs, leading to impacted gut and poor food assimilation, which in turn, may lead to poor nutrition and cause adverse effects to growth, moulting, and eventually reproduction ( Arndt et al., 2014 ). Thus, it is likely that gut accumulation of NMs can lead, via oxidative stress, reduced growth and thus reduced fertility, to reduced reproductive success and population decline. Within the RiskGONE, NanoSolveIT and CompSafeNano projects we are documenting the key events in this proposed AOP for discussion with the AOP community.

NMs accumulation in the gut of Daphnia can cause blockage of gut, leading to reduced feeding, followed by decreased supply of oxygen and triggering of antioxidant pathways (e.g., decreased SOD, upregulation of NOX5, increased ROS), which result in decreased moulting and thus the decreased growth, fertility, and reproduction ( Sasaki et al., 2019 ). ROS production induced oocyte apoptosis-associated reproduction decline has been reported previously, whereby increased ROS damages cellular components, leading to energy (ATP) shortage, DNA breaks, telomere shortening, spindle instability, chromosomal abnormalities, dysregulation of autophagy and proteasome system, which may contribute to the reduced developmental competence compared to normal oocytes ( Sasaki et al., 2019 ). In addition, excessive ROS formation can induce increased DNA damage, apoptosis, follicular atresia, and decreased oogenesis, reducing fecundity ( Chatterjee and Bhattacharjee, 2016 ). Therefore, excessive ROS production can lead to oocyte apoptosis-associated reproduction decline. Calorific reduction related to NMs accumulation in the gut (as discussed in Section 4 ) may also trigger a cascade of signalling pathway alteration, including inhibition of FAR gene expression, activation of DMRT and DMRT genes and sex determination genes (doublesex1), and changes in chitin and ceramide metabolism, which subsequently lead to decreased carapace shedding, Daphnia maturation, sex communication, and induced male production.

We propose that all these KEs are triggered by the MIE of physical blockage of the gut. Absorption of NMs onto the surface of aquatic organism is a key step in determining their bioavailability. Absorption of AuNMs on the carapace and appendages of D. magna and the resulting mechanical disruption of the feeding appendages was observed after AuNM exposure ( Botha et al., 2016 ). TiO 2 NMs were taken up mainly by endocytosis, resulting in their accumulation in abdominal areas and the gut of D. magna ( Tan et al., 2017 ). Elimination of NMs is reportedly difficult; the excretion rate constant of AgNMs in daphnids was much lower than that of Ag ions ( Zhao and Wang, 2010 ). The main elimination routes for AgNMs in Daphnia were excretion (63%) and faecal production ( Zhao and Wang, 2010 ). The monodispersed NMs can easily get deep inside the organisms and are harder to be excreted compared with aglomerated NMs.

The application of omics technologies can be of great value for elucidating how contaminants cause adverse effects in an organism. For example, proteomics provides a systematic qualitative and quantitative mapping of the whole proteome in cells and tissues and enables identification of differentially expressed proteins (DEPs) as biomarkers for AOPs. In addition, transcriptomics (single organism), metabolomics, and lipidomics can also be used for future studies for identifying the AOPs ( Ankley et al., 2010 ; Vinken, 2019 ).

7 Innovative approaches to assessing NMs toxicity using Daphnia microfluidics

Microfluidics and lab-on-a-chip are promising technologies to address many of the limitations in toxicity assays. With these innovative approaches the manipulation of small volumes of liquids/fluids under a network of miniaturized channels allows them to 1) mimic the microenvironment conditions, 2) enables easy manipulation of cells and organisms to measure biological specimens and biomolecular targets, and 3) facilitates automatic extraction of relevant data in a fast and easy way. Additionally, these devices can be combined with sensors, cameras, computers, and smartphones becoming a powerful toolbox in toxicological sciences. Initially, lab-on-a-chip devices were applied in toxicity studies to miniaturize and refine in vitro assays. These devices reduce and automate manual handling procedures, miniaturize testing, decrease the required amount of reagent and chemicals for testing and improve performance due to the real-time monitoring capability. Microfluidics technology creates the potential for multiplexed analysis, single-cell, and gradient cytotoxicity assessments. Furthermore, this technology combines sensors and digital cameras for cytotoxicity studies with real-time data collection, allowing the monitoring of many cellular parameters, such as mortality, cell-substrate adhesion, electrophysiology, cell division and kinetics responses of cytotoxicity in a label-free manner ( Garcia-Hernando et al., 2020 ; Wlodkowic and Jansen, 2022 ).

Despite the evolution of microfluidics devices and lab-on-a-chip technologies in the biomedical field, its potential in ecotoxicology has emerged just recently. Ecotoxicology testing using in vivo assays is, in general, labour intensive, whereby the manipulation of organisms is mainly manual, which may increase data variability and decrease reproducibility. In this way, microfluidics automation of organisms sorting, collection and positioning and chemical addition and dilution, combined with powerful data collection and analysis tools offer a significant upgrade to ecotoxicological tests ( Campana and Wlodkowic, 2018a ; Abreu et al., 2022 ). However, there are also some limitations for its development, such as organism size, which is an important parameter to the development of micro/millifluidic devices, currently limited to organisms < mm in size ( Campana and Wlodkowic, 2018b ). Consequently, a limited number of studies combine micro/millifluidics with ecotoxicology, and most address unicellular organisms such as bacteria, algae or protozoa. Such devices can detect growth, cell viability, bioluminescence, movement, and electrochemical changes to understand the mechanisms of toxicity for those model organisms ( Kim J. et al., 2017 ; Zhang et al., 2017 ; Altintas et al., 2018 ). For multicellular organisms, some ecotoxicity studies were reported, for example, with: rotifers ( Brachionus calycifloru s) ( Cartlidge and Wlodkowic, 2018 ), Crustacea ( Artemia sp.) ( Huang et al., 2015 ) , Allorchestes copressa ( Cartlidge et al., 2017 ) and Daphnia magna ( Huang et al., 2017 ; Tabatabaei Anaraki et al., 2018 ) , nematode ( Caenorhabditis elegans ) ( Kim J. et al., 2017 ; Zhang et al., 2021 ; Aubry et al., 2022 ) and fish ( Danio rerio ) models ( Yang et al., 2016 ; Khalili and Rezai, 2019 ).

Traditional ecotoxicology assays evaluate the survival, reproduction, or growth rate at a specific time point (hours/days) and estimate the chemical median lethality (LC 50 ) or effective concentrations (EC 50 ). Exploring microfluidics technology, it is possible to refine the analysed toxicological parameters and assess different preliminary responses and/or obtain real-time mortality rates. For example, behavioural parameters are often more sensitive than physiological, developmental or reproductive endpoints ( Melvin and Wilson, 2013 ). Therefore, microfluidics technology can increase data analysis and collection by caging test specimens in miniaturized devices allowing the observation of mobility and/or swimming alteration ( Bai et al., 2018 ). Another limitation for classical assays is the caging and manipulation of organisms for imaging, or measurements that need the animal to stay still, such as optical imaging, size and heartbeat measurements. For example, for C. elegans model microfluidic devices allowed immobilization by the restriction in thin microfluidic channels ( Chokshi et al., 2009 ; Kim J. et al., 2017 ). Microfabricated devices for precise and controllable rotation of organisms, for analysis of specific body parts, imaging or injection of substances, were already fabricated for C. elegans ( Pan et al., 2021 ) and zebrafish models ( Zhang et al., 2017 ).

As for Daphnia , there is a complete open avenue for innovation using microfluidics and lab-on-chip technologies because there are just three reports in the literature ( Figure 4 ). Tabatabaei Anaraki et al. (2018) , developed a low-volume flow system that allows D. magna in vivo analysis under nuclear magnetic resonance (NMR) testing using a 5 mm NMR tube; inside the tube are two capillary tubes for injection and suction of fluids (sample, media and/or algae injection), allowing the exposure of the living organism to low volumes of chemicals for in vivo metabolomic studies ( Tabatabaei Anaraki et al., 2018 ). Huang et al. (2015) , automated the Daphtox kit-F with a microfluidic technology by developing a microchip, consisting of a toxicity chamber with a fluid inlet and outlet and loading chamber, for caging D. magna. The system was connected with a high-definition time resolved video data analysis to monitor Daphnia behaviour when exposed to CuCl 2 as a model toxicant ( Huang et al., 2015 ). The was further improved ( Huang et al., 2017 ) to include an array of 24 cuboid test chambers, grouped in eight clusters of three chambers, each chamber having its own specimen loading port and interconnected chambers in a cluster have a shared inlet and outlet for media and sample injection. The device was connected with a time-resolved video microscopy and software to track and analyse D. magna locomotory responses towards pollutants (i.e., CuCl 2 , potassium dichromate, xanthine alkaloids (caffeine), ethanol and dimethyl sulfoxide) ( Huang et al., 2017 ). Interestingly, these results showed that the milli-fluidic device (Daphtox II) presented an EC 50 equivalent to the conventional multi-well plate acute toxicity assay but can assess multiple behavioural endpoints and provide a more sensitive test with higher automation than the conventional multi-well test.

(A) Low-volume flow system for D. magna in vivo NMR ( Tabatabaei Anaraki et al., 2018 ); (B) microfluidic chip for D. magna toxicity testing ( Huang et al., 2015 ); (C) Photograph of the system setup consisting of a millifluidic chip-based array, fluid actuation and optical detection modules ( Huang et al., 2017 ). Reproduced with permission from SCRIP and SPIE, respectively.

Despite the enormous benefits that can be achieved by microfluidics applications in nanotoxicity assessment, there are still few studies addressing this subject. With the Daphnia model, to the best of our knowledge, there are only two toxicity studies with chemicals ( Huang et al., 2015 ; Huang et al., 2017 ) and no reports involving NMs, daphnids and microfluidics at the moment. This knowledge gap has been initially addressed by Seitz et al. (2013) when studying the toxicity of TiO 2 NMs towards D. magna ( Seitz et al., 2013 ), who showed that in semi-static experiments the initial concentration of TiO 2 decreased approximately 95% while in flow-through the concentration of TiO 2 remained constant in the water column throughout the test duration, with a concurrent decrease in particle agglomeration and sedimentation, as determined by Dynamic Light Scattering. The flow-through conditions also showed lower toxicity of TiO 2 NMs suggesting that agglomeration may play an important role in the toxicity profile of NMs (see Section 2.1 ). In addition, the NM sample is usually limited and expensive, and microfluidics devices can be useful not only in this sense (saving sample and producing less residues) but also in improving test conditions, increasing flow control, decreasing evaporation, controlling media oxygenation, temperature, etc. The fabrication of devices may require specific laboratory facilities and can be laborious, limiting mass production of devices for application in ecotoxicology currently ( Campana and Wlodkowic, 2018a ). However, this technology can obtain measurements and information that traditional assays are not able such as toxicity dynamics events, in situ analysis and real-time monitoring. Additionally, all the data generated can be automatically collected using specific software analysis and computational tools, supporting the implementation of big data and machine learning methods in nanoecotoxicology, especially, when considering Daphnia as a key organism model in nanosafety regulation.

8 Key recommendations and future directions

Daphnia have been well established as an important model organism for ecotoxicity testing due to their role in the ecosystem, rapid parthenogenetic reproduction, their responsiveness to xenobiotics and environmental stressors and the range of endpoints available to access for toxicity testing. In addition, their use in toxicity testing is compliant with the principles of reduction, replacement, and refinement (NC3Rs) of traditional animal testing makes them an ideal model organism to explore and develop methods for to evaluate emerging contaminants and concerns. Their historic use in chemical testing made them an ideal species to evaluate NM and MP toxicity in freshwater ecosystems, and as highlighted several advances have already been established in the field of NM and MP ecotoxicity assessments. The importance of characterisation of particles has been well established for NM, and lessons and best practice can be taken into MP research to further advance this field, including methods to quantify uptake and techniques to characterise particle surface which are have been identified as important aspects of ecotoxicity studies.

When evaluating the use of Daphnia as an ecotoxicology model to determine biological and environmental impacts of NMs and MPs, several developments in terms of both methodology and understanding emerged that offer enormous promise for the future. Highlights include the complete sequencing and elucidating the Daphnia genome which, when paired with the parthenogenetic reproductive pathways in Daphnia, enables detailed genetic responses to be determined and subsequent changes to offspring to be ascertained ( Section 5 ). Although further research is required, the capacity to compare common genes and pathways across species, by comparing genes shared by common descent among species, and the biochemical pathways that link differential transcriptomes offers meaningful insights into the relationship between model species and environmental health exposure, which can help to identify areas for development in NM risk assessment and support the development of New Approach Methodologies and Next-Generation Risk assessment approaches that rely far less on mammalian in vivo testing ( Ellis and Lynch, 2020 ). In addition to transcriptomics of single organisms, application of omics techniques can further support the development of AOPs for Daphnia populations and whole ecosystems in response to exposure to NM and MPs, the use of proteomics, metabolomics, lipidomics can help to link the responses to MIEs ( Section 6 ).

Additionally, new methodologies such as microfluidic and lab-on-chip technologies have been identified as areas ripe for development in NMs ecotoxicology. The use of microfluidics can enable real time monitoring of physico-chemical properties of the NM in addition to the toxicity response of the organism. The potential for NM to agglomerate and sediment in test systems can be overcome by the optimised flow conditions that can be established in the microfluidic systems compared to the tradition static test system set up ( Section 8 ). Furthermore, data collection during this application is automatic, enabling machine learning methods and big data computations approached to evaluate the changes and response in due course, utilising harmonised and curated datasets produced according to the FAIR data principles ( Wilkinson et al., 2016 ) . While developments of in silico models for NMs toxicity to Daphnia have been limited to date, and focus primarily on predicting acute toxicity ( Varsou et al., 2021 ), exciting progress in terms of models for assessing impacts from mixtures of different types of NMs ( Zhang et al., 2022 ) and on use of machine learning from images of daphnids exposure to NMs and assessment of changes in tail length, lipid deposits and other phenotypical characteristics from paired multi-generational studies comparing continuous versus parent-only NMs exposure ( Karatzas et al., 2020 ) suggest that computational modelling is a very promising direction for the future.

There are also key areas that have been highlighted where adaptation or further development of approaches and methodologies would be beneficial to strengthen the capacity to evaluate the biological and environmental effects of NMs and MPs. Firstly, through ongoing efforts to adapt test guideline and toxicity study designs to take into consideration the surface characterisation and changes of NM and MPs that result from the exposure medium/conditions and the local environment. This has been demonstrated to have significant impacts on the chemical and biological signalling of the particles that can influence the subsequent interaction. Furthermore, the testing of particles that have been ‘aged’ in the biological/environmental test medium can lead to substantial changes to the observed toxicity response in both acute and chronic exposure. A balance between comparable/reproducible results and environmentally realistic exposure scenarios would address these challenges going forwards ( Section 2 ), and a strong focus on knowledge transfer for NMs to MPs researchers is essential in order to prevent re-invention of knowledge.

Whilst developing the complexity of the testing conditions for particles, increasing the scale of exposure timeframes can also lead to significant changes to the observed toxicity. Multigenerational assessments to date have highlighted that the offspring of the exposed parents most often have increased sensitivity (and therefore observed toxicity) compared to the initially exposed parent. This suggests that there could be significant detrimental impacts to natural populations based on the assumption that the toxicity response of all daphnids would be within the range observed in initial acute (48-h short term exposures) and even chronic (21-day reproductive exposures) test windows when not considering the impact of subsequent generations ( Section 3 ). Application of machine learning approaches might enable assessment of the impact of different media compositions and thus different underlying Daphnia fitness conditions, as well as the role of additional stressors, such as competition for food or climate change.

As particle uptake does not follow the octanal-water partition coefficient (log Kow) principles, the qualification of uptake of NMs and MPs is an important aspect of ecotoxicity studies to determine an accurate dose-response relationship, and to enable Toxicokinetic-Toxicodynamic (TK-TD) modelling which can link life history traits observed as a result of exposure to changes in the population dynamics in the ecosystem. There are several methods currently available, including TEM imaging, ICP-MS quantification of metal and metal doped particles and the use of fluorescence for stained particles, however the method used depends on the material of the particle ( Section 4 ). However, there are limitations to the use of fluorescence, such as the leaching of fluorescence dye or the impedance of these methods based on the NM properties which can cause interference with assay read-outs including autofluorescence. As a result, further work into accurate methods for determining the internalised concentration of particles would be beneficial to make this a valuable source of data for machine learning.

Funding Statement

This work was financially supported by the EU H2020 projects NanoCommons (731032), NanoSolveIT (Grant Agreement no. 814572) and RiskGONE (Grant Agreement no. 814425). SS acknowledges funding for her PhD studies from Malaysian Government MARA (Majlis Amanah Rakyat). DM. thanks the Sao Paulo Research Foundation (FAPESP) for the visiting research fellowship at GEES/UoB (2018/25140-3) and the National Council for Scientific and Technological Development (CNPq) for the productivity research fellowship and the FAPESP-UoB research grant.

Author contributions

KR and IL conceptualised the paper; KR, L-JAE, HHD, SS, MTM, GHS, ZG, DSTM, and IL wrote sections of the manuscript; KR, L-JAE, DSTM, and IL edited the final draft of the manuscript; DSTM and IL secured funding. All authors read and approved the submitted version.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Abbreviations

AOPs, adverse outcome pathways; CAT, catalase; CB, carbon black; CNT, carbon nanotube; DEB, Dynamic Energy Budgets; EPS, extracellular polymeric substances; FAIR, Findable, Accessible, Interoperable, Re-usable; GPx, glutathione peroxidase; GST, glutathione-s-transferase; KEs, key events; MDA, malondialdehyde; MIE, molecular initiating event; MOA, mechanism/mode of action; MP(s), microplastic(s); NM, nanomaterial(s); NOM, natural organic matter; QD, quantum dot; ROS, reactive oxygen species; SOD, Superoxide dismutase; TEM, Transmission electron microscopy; TK-TD, Toxicokinetic-Toxicodynamic.

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Change in growth and prey utilization for a native salmonid following invasion by an omnivorous minnow in an oligotrophic reservoir

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Aquatic invasive species can affect food web structure, native fish growth, and production, depending on the traits of the invasive species and the pre-invasion conditions of the ecosystem. Thermal tolerances and behavioral traits can further influence differential exploitation of resources shared between native and invasive species. An unauthorized introduction of redside shiner ( Richardsonius balteatus ) into reservoirs in the Upper Skagit River, Washington, USA caused concern of potential competition, decreased production, and recruitment of rainbow trout ( Oncorhynchus mykiss ). We combined bioenergetics modeling and stable isotope analysis with field data to quantify consumption demand of native and invasive fishes and related consumption to the availability of key zooplankton prey. Per capita consumption on  Daphnia  by redside shiner was low; however, their high abundance imposed considerable demand on prey resources in Ross Lake. Although monthly consumption demand by the fish community was less than 50% of the monthly production and biomass of  Daphnia  in Ross Lake, the current  Daphnia  densities and growth of rainbow trout were considerably lower than before the invasion. These reductions correspond to lower annual consumption of  Daphnia . Our study provides insight on mechanisms that influence food web impacts of an invasive omnivore in cold-water reservoirs.