Alternative splicing is highly variable among Daphnia pulex lineages in response to acute copper exposure.

BACKGROUND: Despite being one of the primary mechanisms of gene expression regulation in eukaryotes, alternative splicing is often overlooked in ecotoxicogenomic studies. The process of alternative splicing facilitates the production of multiple mRNA isoforms from a single gene thereby greatly increasing the diversity of the transcriptome and proteome. This process can be important in enabling the organism to cope with stressful conditions. Accurate identification of splice sites using RNA sequencing requires alignment to independent exonic positions within the genome, presenting bioinformatic challenges, particularly when using short read data. Although technological advances allow for the detection of splicing patterns on a genome-wide scale, very little is known about the extent of intraspecies variation in splicing patterns, particularly in response to environmental stressors. In this study, we used RNA-sequencing to study the molecular responses to acute copper exposure in three lineages of Daphnia pulex by focusing on the contribution of alternative splicing in addition to gene expression responses. RESULTS: By comparing the overall gene expression and splicing patterns among all 15 copper-exposed samples and 6 controls, we identified 588 differentially expressed (DE) genes and 16 differentially spliced (DS) genes. Most of the DS genes (13) were not found to be DE, suggesting unique transcriptional regulation in response to copper that went unnoticed with conventional DE analysis. To understand the influence of genetic background on gene expression and alternative splicing responses to Cu, each of the three lineages was analyzed separately. In contrast to the overall analysis, each lineage had a higher proportion of unique DS genes than DE genes suggesting that genetic background has a larger influence on DS than on DE. Gene Ontology analysis revealed that some pathways involved in stress response were jointly regulated by DS and DE genes while others were regulated by only transcription or only splicing. CONCLUSIONS: Our findings suggest an important role for alternative splicing in shaping transcriptome diversity in response to metal exposure in Daphnia, highlighting the importance of integrating splicing analyses with gene expression surveys to characterize molecular pathways in evolutionary and environmental studies.

Impact of antagonistic crustaceans on the population of Aedes aegypti L. larvae under laboratory conditions.

BACKGROUND & OBJECTIVES: Dengue and chikungunya are two mosquito-borne viral diseases transmitted by Aedes mosquito species and are a great public health concern in India. The present study was aimed to check the influence of antagonistic crustaceans, especially Mesocyclops aspericornis and Daphnia magna on Ae. aegypti L. mosquito population. METHOD: Variable ratios of these crustaceans (Aedes: Mesocyclops: Daphnia) against Ae. aegypti larvae were tested by putting them in plastic beakers having dechlorinated water along with yeast stock solution provided as food, and kept in BOD incubator at a temperature of 26 ±1°C. RESULTS: Out of all tested concentrations, 1:1:3 where the number of D. magna was thrice the number of Mesocyclops and Aedes; larvae showed a significant delay of 5-6 days in the developmental period. Maximum reduction in the emergence of females was recorded in the ratio 1:1:3, i.e. only 6.5 ± 0.47 females emerged when Daphnia used thrice the number of Aedes larvae. Body size of both males and females emerged from treated sets was found to be significantly reduced. The longevity of adults was also reduced from 8-17 days to 5-8 days in the case of males and from 14-26 days to 5-9 days in females. INTERPRETATION & CONCLUSION: Among variable ratios tested under laboratory conditions, 1:1:2 and 1:1:3 ratios were found to be the effective ratios that greatly reduced the development duration, survivorship of larvae, and the number of larvae emerging into adulthood. Thus, antagonistic crustaceans specifically Mesocyclops and Daphnia can be used as biocontrol agents for the sustainable control of container breeding mosquitoes.

The evolution of reproductive isolation in Daphnia.

BACKGROUND: The process by which populations evolve to become new species involves the emergence of various reproductive isolating barriers (RIB). Despite major advancements in understanding this complex process, very little is known about the order in which RIBs evolve or their relative contribution to the total restriction of gene flow during various stages of speciation. This is mainly due to the difficulties of studying reproductive isolation during the early stages of species formation. This study examines ecological and non-ecological RIB within and between Daphnia pulex and Daphnia pulicaria, two recently diverged species that inhabit distinct habitats and exhibit an unusual level of intraspecific genetic subdivision. RESULTS: We find that while ecological prezygotic barriers are close to completion, none of the non-ecological barriers can restrict gene flow between D. pulex and D. pulicaria completely when acting alone. Surprisingly, we also identified high levels of postzygotic reproductive isolation in 'conspecific' interpopulation crosses of D. pulex. CONCLUSIONS: While the ecological prezygotic barriers are prevalent during the mature stages of speciation, non-ecological barriers likely dominated the early stages of speciation. This finding indicates the importance of studying the very early stages of speciation and suggests the contribution of postzygotic isolation in initiating the process of speciation.

Genetic resistance and specificity in sister taxa of Daphnia: insights from the range of host susceptibilities.

BACKGROUND: Host genetic diversity can affect various aspects of host-parasite interactions, including individual-level effects on parasite infectivity, production of transmission stages and virulence, as well as population-level effects that reduce disease spread and prevalence, and buffer against widespread epidemics. However, a key aspect of this diversity, the genetic variation in host susceptibility, has often been neglected in interpreting empirical data and in theoretical studies. Daphnia similis naturally coexists with its competitor Daphnia magna and is more resistant to the endoparasitic microsporidium Hamiltosporidium tvaerminnensis, as suggested by a previous survey of waterbodies, which detected this parasite in D. magna, but not in D. similis. However, under laboratory conditions D. similis was sometimes found to be susceptible. We therefore asked if there is genetic variation for disease trait expression, and if the genetic variation in disease traits in D. similis is different from that of D. magna. METHODS: We exposed ten clones of D. similis and ten clones of D. magna to three isolates of H. tvaerminnensis, and measured infection rates, parasite-induced host mortality and parasite spore production. RESULTS: The two Daphnia species differ in the range and variation of their susceptibilities. The parasite produced on average two-fold more spores when growing in D. magna clones than in D. similis clones. CONCLUSIONS: We confirm that D. similis is indeed much more resistant than D. magna and suggest that this could create a dilution effect in habitats where both species coexist.

Unexpected endemism in the Daphnia longispina complex (Crustacea: Cladocera) in Southern Siberia.

The biological significance of regional cladoceran morphotypes in the montane regions of the central Palearctic remains poorly understood. In the Holarctic Daphnia longispina complex (Cladocera: Daphniidae), several variants, lineages and species have been proposed as endemic for Southern Siberia. Daphnia turbinata Sars, for example, named after its unusual head shape, is known only from Southern Siberia. Here we sequence DNA of Daphnia from three mitochondrial genes (12S rRNA, 16S rRNA, and NADH dehydrogenase subunit 2, ND2) from 57 localities in Russia and Mongolia (the majority being from Southern Siberia) and place them in evolutionary context with existing data. Our aim was to examine regional endemism of the Daphnia longispina complex in Southern Siberian; to improve the phylogenetic understanding with improved taxonomic and regional sampling, and to better understand the influence of Pleistocene glaciation on the biogeography of these lineages. At least three lineages showed genetic evidence for endemism in Southern Siberia. There was strong support for D. turbinata as a sister lineage to to D. longispina/D. dentifera. Another endemic, Siberian D. cf. longispina, is a sister group to the longispina group in general. Within D. longispina s. str. there was an endemic Siberian clade with a western range boundary near the Yenisei River Basin. Gene flow estimates among populations (based on FST values) were very low for clades of D. longispina on a regional (the original 12S dataset), and on a pan-Eurasian (the extended 12S dataset) scale. Negative values of Fu's FS and Tajima's D tests prevailed for the species examined with significant values found for two D. longispina clades, D. dentifera, D. galeata and D. cristata. Our results support the notion that Southern Siberia is an important biogeographic region for cladocerans as it contained unexpected diversity of endemics (such as D. turbinata, D. cf. longispina and lineages of D. umbra and D. longsipina s.str.) and from being the geographic meeting place of expanding postglacial lineages from eastern and western refugia.

A fossil-calibrated phylogenomic analysis of Daphnia and the Daphniidae.

In the post-genomic era, much of phylogenetic analyses still relies on mitochondrial DNA, either alone or in combination with few nuclear genes. Although this approach often makes it possible to construct well-supported trees, it is limited because mtDNA describes the history of a single locus, and nuclear phylogenies based on a few loci may be biased, leading to inaccurate tree topologies and biased estimations of species divergence time. In this study, we perform a phylogenomic analysis of the Daphniidae family (Crustacea: Branchiopoda: Anomopoda) including some of the most frequently studied model organisms (Daphnia magna and D. pulex) whose phylogenetic relationships have been based primarily on an assessment of a few mtDNA genes. Using high-throughput sequencing, we were able to assemble 38 whole mitochondrial genomes and draft nuclear genomes for 18 species, including at least one species for each known genus of the family Daphniidae. Here we present phylogenies based on 636 nuclear single-copy genes shared among all sampled taxa and based on whole mtDNA genomes. The phylogenies we obtained were highly supported and showed some discrepancies between nuclear and mtDNA based trees at deeper nodes. We also identified a new candidate sister lineage of Daphnia magna. Our time-calibrated genomic trees, which we constructed using both fossil records and substitution rates, yielded very different estimates of branching event times compared to those based on mtDNA. By providing multi-locus, fossil-calibrated trees of the Daphniidae, our study contributes to an improved phylogenetic framework for ecological and evolutionary studies that use water fleas as a model system.

Contrasting endemism in pond-dwelling cyclic parthenogens: the Daphnia curvirostris species group (Crustacea: Cladocera).

Pond-dwelling cyclic parthenogens are often proposed to be highly vagile. However, the Holarctic biogeography of parthenogens has been hampered by very limited sampling in the eastern Palearctic. Here we examine the geographic boundaries, diversity, and connectivity across the Palearctic for the Daphnia curvirostris complex (Cladocera: Daphniidae). Nuclear (HSP90) and mitochondrial (ND2) sequence data supported the existence of five main clades (most of which corresponded to presumptive species) with one eastern Palearctic clade being novel to this study (the average mitochondrial genetic divergence from known species was 19.2%). D. curvirostris s.s. was geographically widespread in the Palearctic, with a population genetic signature consistent with postglacial expansion. The Eastern Palearctic had local nine endemic species and/or subclades (other Holarctic regions lacked more than one endemic subclade). Even though several endemic species appeared to have survived Pleistocene glaciation in the eastern Palearctic, much of the Palearctic has been recolonized by D. curvirostris s.str. from a Western Palearctic refugium. A disjunct population in Mexico also shared its haplotypes with D. curvirostris s.str., consistent with a recent introduction. The only apparently endemic North American lineage was detected in a thermally disturbed pond system in northwestern Alaska. Our results for pond-dwelling cyclic parthenogens further support the hypothesis that the Eastern Palearctic is a diversity hotspot for freshwater invertebrates.

Lineage diversity and reproductive modes of the Daphnia pulex group in Chinese lakes and reservoirs.

Recent studies of the distribution and diversity of freshwater zooplankton have indicated that the previously understudied Eastern Palearctic region is an important biogeographic hotspot. Here, we explored the lineage diversity and reproductive modes of the Daphnia pulex species group across China. Members of this group are often keystone species of standing water bodies and are frequently used as a model system for ecological, evolutionary and, more recently, genomic studies. We found members of the D. pulex group in seven of seventy-six Chinese water bodies examined. We analyzed their phylogenetic position using mitochondrial markers, and explored the genetic structure of six populations using microsatellite markers. Mitochondrial DNA analysis suggested the presence of two distinct species complexes in China: the D. pulex complex that has a global distribution, and an apparently endemic Eastern Palearctic D. mitsukuri complex. Microsatellite analyses of six populations suggested that three of these reproduced by cyclical parthenogenesis, as evidenced by high clonal diversity and the absence of deviations from the Hardy-Weinberg equilibrium. In contrast, three other populations showed remarkably low diversity of multilocus genotypes. This suggests an obligate parthenogenetic reproductive mode, which was confirmed in one of the populations by comparison of genotypes of Daphnia adults and dormant embryos. All presumably obligate parthenogenetic clones were heterozygous at the majority of microsatellite loci, suggesting their hybrid origin. This was further supported by analyses of a small GTPase nuclear gene (rab4), as two alleles within single individuals belonged to different clades. Interestingly, one putatively obligate parthenogenetic clone carried three distinct alleles suggesting higher ploidy and potential gene flow between the D. pulicaria and D. mitsukuri complexes. Our data show that the expansion of the D. pulex complex in the Eastern Palearctic was associated with widespread hybridization.

Rediscovery after Almost 120 Years: Morphological and Genetic Evidence Supporting the Validity of Daphnia mitsukuri (Crustacea: Cladocera).

We examined the morphology of Daphnia individuals maintained in our laboratory for several years, originally collected in Lake Inbanuma, Chiba, Japan. We determined partial sequences of the mitochondrial cytochrome c oxidase subunit I and 12S rRNA genes from specimens in the cultured material. These animals are morphologically similar to D. obtusa Kurz, 1874 , but genetically distinct from this species. Our detailed observation shows that the morphological characteristics in the female and male individuals of our material are highly congruent with those of D. mitsukuri Ishikawa, 1896 , which has not been identified positively for more than 120 years since its original description, with its taxonomic identity having been questioned for almost 90 years. Based on our morphological and genetic data, we conclude that D. mitsukuri should be regarded as a taxonomically valid species. A search among public DNA sequence databases suggests D. mitsukuri is also distributed in China, although these Chinese sequences have been labeled as 'Daphnia pulex', representing misidentification.

Conserved Transcription Factors Steer Growth-Related Genomic Programs in Daphnia.

Ecological genomics aims to understand the functional association between environmental gradients and the genes underlying adaptive traits. Many genes that are identified by genome-wide screening in ecologically relevant species lack functional annotations. Although gene functions can be inferred from sequence homology, such approaches have limited power. Here, we introduce ecological regulatory genomics by presenting an ontology-free gene prioritization method. Specifically, our method combines transcriptome profiling with high-throughput cis-regulatory sequence analysis in the water fleas Daphnia pulex and Daphnia magna. It screens coexpressed genes for overrepresented DNA motifs that serve as transcription factor binding sites, thereby providing insight into conserved transcription factors and gene regulatory networks shaping the expression profile. We first validated our method, called Daphnia-cisTarget, on a D. pulex heat shock data set, which revealed a network driven by the heat shock factor. Next, we performed RNA-Seq in D. magna exposed to the cyanobacterium Microcystis aeruginosa. Daphnia-cisTarget identified coregulated gene networks that associate with the moulting cycle and potentially regulate life history changes in growth rate and age at maturity. These networks are predicted to be regulated by evolutionary conserved transcription factors such as the homologues of Drosophila Shavenbaby and Grainyhead, nuclear receptors, and a GATA family member. In conclusion, our approach allows prioritising candidate genes in Daphnia without bias towards prior knowledge about functional gene annotation and represents an important step towards exploring the molecular mechanisms of ecological responses in organisms with poorly annotated genomes.

Diversification of mitochondrial genome of Daphnia galeata (Cladocera, Crustacea): Comparison with phylogenetic consideration of the complete sequences of clones isolated from five lakes in Japan.

To characterize genetic diversity and gene flow among Daphnia galeata populations, the complete nucleotide (nt) sequences of the mitochondrial (mt) DNAs of D. galeata clones isolated from five lakes in Japan (Lakes Shirakaba, Suwa, Kizaki, Kasumigaura, and Biwa) were determined. Comparison of non-synonymous (amino acid altering) substitution rates with synonymous substitution rates of D. galeata mt protein-coding genes demonstrated that ATPase8 and COI genes were the most and least susceptible, respectively, to the evolutional forces selecting the aa substitutions. Several non-synonymous substitutions were found in ATPase8 and ATPase6 even in the comparison that no synonymous substitution was found. Comparison of the total number of nt variations among the mt DNAs suggested the phylogenetic relationship ((((Shirakaba/Suwa, Kizaki), Kasumigaura), Biwa), D. pulex). Maximum-likelihood analysis using the total nt sequences of mt protein-coding genes confirmed this relationship with bootstrap values higher than 98%. All the mtDNAs of the analyzed Japanese D. galeata clones contained a control region of essentially the same structure that is distinct from those of the previously reported European Daphnia species of the D. longispina complex. The two control regions of different structures spread among mtDNAs of the Japanese and European Daphnia species, respectively, probably after the divergence of the Japanese D. galeata under different selection pressures associated with their habitats.

Revision of the Old World Daphnia (Ctenodaphnia) similis group Cladocera: Daphniidae).

Species of the genus Daphnia O.F. Müller, 1785 (Cladocera: Daphniidae) have become very important models in evolutionary biology research. Previous morphological and genetic evidence suggests that numerous closely related "species groups" exist within the subgenus Daphnia (Ctenodaphnia) Dybowski & Grochowski, 1895, containing both described and undescribed species. The Daphnia similis group is among these species groups. The aim of the present paper is to revise the taxonomy of the Daphnia (Ctenodaphnia) similis group in the Old World with both morphological and genetic evidence (based on mitochondrial COI and 12S rRNA genes). We found that there are at least four species in the Old World D. similis species group: D. similis Claus, 1876; D. sinensis Gu, Xu, Li, Dumont et Han, 2013; D. similoides Hudec, 1991 and D. inopinata sp. nov. These four taxa of the similis-group, confused previously with D. similis, have different distributional ranges in the Old World, from extremely wide, spanning several biogegraphic regions (as D. sinensis), to regional endemics (D. similoides) and even species known so far from a single locality (D. inopinata sp. nov.). The Daphnia similis group provides another example in the cladocerans whereby the study of males yields more valuable characters for taxonomy than the study of parthenogenetic females.

A critical review of the current taxonomy of the genus Daphnia O. F. Müller, 1785 (Anomopoda, Cladocera).

The genus Daphnia O. F. Müller, 1785 (Anomopoda, Cladocera) has become a model genus for ecological, toxicological, and evolutionary studies. At the same time, Daphnia is a well-known example of a genus with a confused taxonomy. This review aims to identify the main sources of confusion (natural and human-caused) for the current taxonomy of Daphnia. A complete check-list of all species-group names of Daphnia was built using the available literature sources. I graphed the total number of taxa and number of valid taxa described during each year, and estimated a curve of accumulation of formal taxa from year to year. Four main periods in the history of Daphnia taxonomy were recognized, with the latest decades showing an increased rate of valid taxa to more than 50%. Overall, only 24 % of 361 known taxa of species rank are valid. A failure to find real, sometimes fine-scale diagnostic characters, led to a tradition of pseudo-taxonomy based mainly on the body shape and other phenotypically plastic characters, e.g. associated with defensive structures. I recommend revising the suite of taxonomically informative characters by emphasizing the anatomical details of males and thoracic limbs of both males and females. Such a revision of global scale, should be conducted in close coordination with phylogenetic studies. But this revision is impossible without accurate redescriptions of all previously described taxa, and a continuous step-by-step resolving of taxonomic problems in each species group. 

Arginine kinase in the cladoceran Daphnia magna: cDNA sequencing and expression is associated with resistance to toxic Microcystis.

Nutrient loading derived from anthropogenic activities into lakes have increased the frequency, severity and duration of toxic cyanobacterial blooms around the world. Although herbivorous zooplankton are generally considered to be unable to control toxic cyanobacteria, populations of some zooplankton, including Daphnia, have been shown to locally adapt to toxic cyanobacteria and suppress cyanobacterial bloom formation. However, little is known about the physiology of zooplankton behind this phenomenon. One possible explanation is that some zooplankton may induce more tolerance by elevating energy production, thereby adding more energy allocation to detoxification expenditure. It is assumed that arginine kinase (AK) serves as a core in temporal and spatial adenosine triphosphate (ATP) buffering in cells with high fluctuating energy requirements. To test this hypothesis, we studied the energetic response of a single Daphnia magna clone exposed to a toxic strain of Microcystis aeruginosa, PCC7806. Arginine kinase of D. magna (Dm-AK) was successfully cloned. An ATP-gua PtransN domain which was described as a guanidine substrate specificity domain and an ATP-gua Ptrans domain which was responsible for binding ATP were both identified in the Dm-AK. Phylogenetic analysis of AKs in a range of arthropod taxa suggested that Dm-AK was as dissimilar to other crustaceans as it was to insects. Dm-AK transcript level and ATP content in the presence of M. aeruginosa were significantly lower than those in the control diet containing only the nutritious chlorophyte, Scenedesmus obliquus, whereas the two parameters in the neonates whose mothers had been previously exposed to M. aeruginosa were significantly higher than those of mothers fed with pure S. obliquus. These findings suggest that Dm-AK might play an essential role in the coupling of energy production and utilization and the tolerance of D. magna to toxic cyanobacteria.

Lifetime response of contemporary versus resurrected Daphnia galeata Sars (Crustacea, Cladocera) to Cu(II) chronic exposure.

Resurrecting legacy lineages of organisms from sediment cores of known geological age allows us to understand how environmental change can cause selection pressures that constrain the variation of populations over time. We quantified the lifetime tolerance and effects of Cu(II) exposure on Daphnia galeata in a polluted subalpine lake by comparing extant individuals with those resurrected from ephippia extracted from ca. 30-years-old sediments. Laboratory experiments were conducted using two Cu(II) concentrations, 40 and 10 µg L(-1), corresponding to the levels recorded in the lake, during chemical recovery, when Daphnia first re-appeared and succeeded. Contemporary Daphnia were unable to survive after the 10th day at either of the Cu(II) concentrations, and were unable to successfully reproduce. Daphnia cohorts from the past performed better in low Cu(II) concentrations than in copper-free, control conditions. The copper-adapted, tolerant Daphnia strains grew faster under non-toxic conditions, but were unable to survive new pollution events.

Investigation of differences in sensitivity between 3 strains of Daphnia magna (crustacean Cladocera) exposed to malathion (organophosphorous pesticide).

Acute and chronic ecotoxic effects of organophosphorous insecticide malathion (Fyfanon 50 EC 500 g L(-1)) were investigated on three strains of Daphnia magna. The nominal effective concentrations immobilizing 50% (EC50) of Daphnia after 24 and 48 h were 0.53 and 0.36 µg L(-1), 0.70 and 0.44 µg L(-1), and 0.75 and 0.46 µg L(-1) for the strains 1, 2, and 3 respectively. There was an increase in malathion ecotoxicity with time of exposure as confirmed by chronic studies. In fact, after 21 days of exposure, significant effects on survival (lowest observed effect concentration or LOECs) were recorded at malathion concentrations of 22, 220 and 230 ng L(-1) for strains 1, 2 and 3 respectively. Other endpoints were also examined, including reproduction (with different parameters), body length and embryo toxicity. ICs10 and ICs20 were calculated for these different parameters. ICs10 ranged from 4.7 to more than 220 ng L(-1) for the three tested strains. The most sensitive parameter was the number of neonates per adult (ICs10 = 4.7 and 10.8 ng L(-1) for strains 1 and 2 respectively) or the number of broods (IC10 = 10 ng L(-1) for strain 3). Moreover, an increase in embryo development abnormalities was observed at the two highest tested malathion concentrations. Abnormalities comprised undeveloped second antennae, curved or unextended shell spines, and curved post-abdomen spines in live neonates. Results suggest that malathion could act as an endocrine disruptor in D. magna as it interferes with development. It also induces a significant decrease in acetylcholinesterase (AChE) activities for the three strains. Both strains 2 and 3 seemed more sensitive (LOECs = 60 ng L(-1)) than strain 1 (LOEC = 120 ng L(-1)). Our results suggest that the AChE activity response can also be used as a biomarker of inter-strain (or inter-clonal) susceptibility (i.e. strain (or clone)-specific response).

Copy number of the transposon, Pokey, in rDNA is positively correlated with rDNA copy number in Daphnia obtuse [corrected].

Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼ 87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species.

Seasonal dynamics of Daphnia laevis Birge, 1878 ephippia in a tropical lake with a description of a new methodology for in situ evaluation.

The effect of dormancy in zooplankton populations is still unknown, largely because of the lack of methods to estimate hatching and production of the dormant stages. This study aimed to compare the production and hatching rates of ephippia of Daphnia laevis between thermal stratification and mixing periods in Jacaré Lake (Middle Rio Doce, Minas Gerais, Brazil). For this, we collected ephippia on the sediment with core sampler and we created a device called the "Ephippial Collector". There was a significant difference in ephippia hatching in situ between stratification and mixing periods (Pearson's Chi-squared test p <0.001), being higher in the second one. Significant differences in the hatching rates between periods was observed in the laboratory only for ephippia collected with Ephippial Collectors (Pearson's Chi-squared test p <0.001), being higher during the mixing period (∼ 8%). The core sample allows the collection of a certain fraction of the sediment that may contain a mixture of ephippia produced in different periods, i.e., may contain old and not viable ephippia, which masks the hatching rate. Thus, seasonality in hatching rates of ephippia was reported only by Ephippial Collectors. The higher hatching rate observed during the mixing period in the lake suggests that individuals hatched from ephippia may contribute to the increase in the population of D. laevis in the water column at this time.

Functional genomics of acclimation and adaptation in response to thermal stress in Daphnia.

BACKGROUND: Gene expression regulation is one of the fundamental mechanisms of phenotypic plasticity and is expected to respond to selection in conditions favoring phenotypic response. The observation that many organisms increase their stress tolerance after acclimation to moderate levels of stress is an example of plasticity which has been long hypothesized to be based on adaptive changes in gene expression. We report genome-wide patterns of gene expression in two heat-tolerant and two heat-sensitive parthenogenetic clones of the zooplankton crustacean Daphnia pulex exposed for three generations to either optimal (18°C) or substressful (28°C) temperature. RESULTS: A large number of genes responded to temperature and many demonstrated a significant genotype-by-environment (GxE) interaction. Among genes with a significant GxE there were approximately equally frequent instances of canalization, i.e. stronger plasticity in heat-sensitive than in heat-tolerant clones, and of enhancement of plasticity along the evolutionary vector toward heat tolerance. The strongest response observed is the across-the-board down-regulation of a variety of genes occurring in heat-tolerant, but not in heat-sensitive clones. This response is particularly obvious among genes involved in core metabolic pathways and those responsible for transcription, translation and DNA repair. CONCLUSIONS: The observed down-regulation of metabolism, consistent with previous findings in yeast and Drosophila, may reflect a general compensatory stress response. The associated down-regulation of DNA repair pathways potentially creates a trade-off between short-term benefits of survival at high temperature and long-term costs of accelerated mutation accumulation.

Characterization of newly gained introns in Daphnia populations.

As one of the few known species in an active phase of intron proliferation, the microcrustacean Daphnia pulex is an especially attractive system for interrogating the gain and loss of introns in natural populations. In this study, we used a comparative population-genomic approach to identify and characterize 90 recently gained introns in this species. Molecular clock analyses indicate that these introns arose between 3.9 × 10(5) and 1.45 × 10(4) years ago, with a spike in intron proliferation approximately 5.2 × 10(4) to 1.22 × 10(5) years ago. Parallel gains at homologous positions contribute to 47.8% (43/90) of discovered new introns. A disproportionally large number of new introns were found in historically isolated populations in Oregon. Nonetheless, derived, intron-bearing alleles were also identified in a wide range of geographic locations, suggesting intron gain and, to a lesser degree, intron loss are important sources of genetic variation in natural populations of Daphnia. A majority (55/90 or 61.1%) of the identified neointrons have associated internal direct repeats with lengths and compositions that are unlikely to occur by chance, suggesting repeated bouts of staggered double-strand breaks (DSBs) during their evolution. Accordingly, internal, staggered DSBs may contribute to a passive trend toward increased length and sequence diversity in nascent introns.