Significance of the study The output helps guide people to determine the toxicity of the different sources of water

and gives idea to these ordinary people on how to help lessen the toxicities of the water. The research helps encourage scientists to study more on some of the possible uses of Daphnia magna and how it helps the environment.

BioAssay A bioassay is a single step within a microarray experiment. There are 3 types of bioassays. A physical bioassay correspond to wet-lab microarray experimental step. A measured bioassays corresponds to a situation after feature extraction has been performed. A derived bioassay corresponds to data processing experimental steps. A physical bioassay is created by applying some amount of some biomaterial to a microarray. Bioassay treatment events (e.g., wash, apply blocking agent etc.) transform physical bioassays into new physical bioassays. A particular type of bioassay treatment is image acquisition. Measured bioassays can have corresponding MeasuredBioAssayData objects (raw data). Derived bioassays are obtained by data transformations, they are linked by BioAssayMap objects.
Bioassay (commonly used shorthand for biological assay), or biological standardization is a type of scientific experiment. Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants. Both are procedures by which the potency or the nature of a substance is estimated by studying its effects on living matter. Bioassay is a procedure for the determination of the concentration of a particular constitution of a mixture.

Use[edit]
Bioassays are procedures that can determine the concentration of purity or biological activity of a substance such as vitamin, hormone, and plant growth factor. While measuring the effect on an organism, tissue cells, enzymes or the receptor is preparing to be compared to a standard preparation. Bioassays may be qualitative or quantitative. Qualitative bioassays are used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity. An example of a qualitative bioassay includes Arnold Adolph Berthold's famous experiment on castrated chickens. This analysis found that by removing the testes of a chicken, it would not develop into a rooster because the endocrine signals necessary for this process were not available. Quantitative bioassays involve estimation of the concentration or potency of a substance by measurement of the biological response that it produces. Quantitative bioassays are typically analyzed using the methods of biostatistics.

Definition[edit]

"The determination of the relative strength of a substance (as a drug) by comparing its effect on a test organism with that of a standard preparation." ....

Purpose[edit]
1. Measurement of the pharmacological activity of new or chemically undefined substances 2. Investigation of the function of endogenous mediators 3. Determination of the side-effect profile, including the degree of drug toxicity 4. Measurement of the concentration of known substances (alternatives to the use of whole animals have made this use obsolete) 5. Assessing the amount of pollutants being released by a particular source, such as wastewater or urban runoff. 6. Determining the specificity of certain enzymes to certain substrates.

Chapter 2Introduction to Daphnia Biology

This chapter provides an overview of the biology of waterfleas of the genus Daphnia. It describes basic aspects of individual physiology and nutrition, including some remarks about immunity. It summarizes the typical life cycle and development of Daphnia. The modes of reproduction and the induction of resting egg production in cyclic and obligate parthenogenetic forms are discussed. Finally, population biological aspects, such as behavioral ecology, habitat preferences, population genetics, and population dynamics, are introduced. Go to:

Introduction
The following introduction summarizes the key aspects of the natural history of the genus Daphnia. It is far from exhaustive. Because much of the biology has been studied over the past 250 years and is considered to be common knowledge, I give no references to specific studies in most cases. The interested reader may consult Freyer (1991), Kstner (1993), Lampert and Sommer (1999), and Peters and De Bernardi (1987), from which much of the information was taken. A search on the Internet using the term "Daphnia" or "Cladocera" will also bring up a large body of information. Of particular interest is the Cladocera Web page of the University of Guelph in Canada. The reader familiar with Daphnia may skip this chapter and move on to Chapter 3. Daphnia are planktonic crustaceans that belong to the Phyllopoda (sometimes called Branchiopoda), which are characterized by flattened leaf-like legs used to produce a water current for the filtering apparatus. Within the branchiopods, Daphnia belong to theCladocera, whose bodies are enclosed by an uncalcified shell (Figures 2.1 and 2.2), known as the carapace. It has a double wall, between which hemolymph flows and which is part of the body cavity. The carapace is largely made of chitin, a polysaccharide. Cladocera have up to 10 pairs of appendages, which are (from front to back): antennules, antennae (the second antennae, used for swimming); maxillae; and mandibles; followed by 5 (as in Daphnia) or 6 limbs on the trunk. The limbs form an apparatus for feeding and respiration. At the end of the abdomen is a pair of claws. The body length of Cladocera ranges from less than 0.5 mm to more than 6 mm. Males are distinguished from females by their smaller size, larger antennules, modified postabdomen, and first legs, which are armed with a hook used in clasping.

Figure 2.1 The functional anatomy of Daphnia .

Figure 2.2 Ventral view of Daphnia Adult females. On the left, a photograph of Daphnia longispinafrom a rock pool population in southern Finland. On the right (more...) The genus Daphnia includes more than 100 known species of freshwater plankton organisms found around the world (see Figures 2.3, 2.4, and 2.5 for three European representatives of the genus). They inhabit most types of standing freshwater except for extreme habitats, such as hot springs. All age classes are good swimmers and are mostly pelagic, i.e., found in the open water. They live as filter feeders, but some species may frequently be seen clinging to substrates such as water plants or even browsing over the bottom sediments of shallow ponds. Adults range from less than 1 mm to 5 mm in size, with the smaller species typically found in ponds or lakes with fish predation. The ecology of the genus Daphnia may be better known than the ecology of any other group of organisms.

Figure 2.3 Daphnia magna . Adult female with a clutch of freshly laid parthenogenetic eggs in its brood chamber. The female is from a laboratory culture of a clone (more...)

Figure 2.4 Daphnia cucullata . Adult female with one embryo in her brood chamber. This female comes from a laboratory culture of a clone originally isolated from (more...)

Figure 2.5 Daphnia longispina . Adult female with three eggs in the brood chamber. This female was isolated from a rock pool in southern Finland close to Tvrminne. (more...) Go to:

Physiology, Metabolism, and Immunity

Daphnia feed on small, suspended particles in the water. They are suspension feeders (filter feeders). The food is gathered with the help of a filtering apparatus, consisting of the phylopods, which are flattened leaf-like legs that produce a water current. As the current flows anterior to posterior, the Daphnia collect particles that are transferred into the food groove by special setae. Although the feeding apparatus is so efficient that even bacteria can be collected, the food is usually made up of planktonic algae. Green algae are among the best food, and most laboratory experiments are done with either Scenedesmus or Chlamydomonas, both of which are easy to culture in monoclonal chemostats. Daphnia usually consume particles from around 1 m up to 50 m, althoug h particles of up to 70 m in diameter may be found in the gut content of large individuals. The dynamics of food uptake follow a functional response type 1. Below a certain food concentration (the incipient limiting level), the food uptake from the water (feeding rate) is proportional to the food concentration, and the filtering rate (amount of water filtered per unit time) is maximal. Above this level, the feeding rate is constant because the filtering rate decreases with increasing food concentration in the water. For parasites that enter the host with the food particles, infection rates depend on the food concentration in the water. Highest infection rates are expected when filtering rates are maximal. The gut is more or less tubular with three parts: the esophagus, the midgut, and the hindgut. There are two small digestive ceca(diverticula) that are easily seen in the head section of the midgut (Figures 2.1 and 2.6). The midgut is lined with an epithelium and bears microvilli. Peristaltic contractions of the gut wall pass food through the gut, but a peritrophic membrane contains the food and prevents it from entering the ceca. Epithelial cells do not phagocytose particles but absorb molecules. The pH is 6 to 6.8 in the anterior part of the midgut and 6.6 to 7.2 in the posterior part. Food is expelled from the hindgut by peristaltic movement but also requires the pressure of more recently acquired food particles. The color of Daphnia adapts to the food that is predominant in their diet. Daphnia feeding on green algae will be transparent with a tint of green or yellow, whereas those feeding on bacteria will be white or salmon-pink. Well-fed animals are more strongly colored than starved animals.

Figure 2.6 Gut of Daphnia magna . Gut dissected from a female. On the left, the paired intestinal ceca can be seen. The gut ends at the right side. The esophagus (more...) Daphnia have an open blood circulation. The heart is located dorsally and anterior from the brood chamber. At 20C, it beats about 200 times per minute, slowing down at lower temperatures. Blood cells are easily visible through the transparent body as they flow rapidly through the body cavity. To support oxygen transport, Daphnia have the extracellular respiratory protein hemoglobin (Hb), a multi-subunit, multi-domain macromolecule. There are at least four Hb genes. Daphnia tend to develop more Hb to increase oxygen uptake from the water. In response to environmental changes (oxygen concentration, temperature), the Hb concentration varies up to about 20-fold. Oxy-hemoglobin, the form that is loaded with oxygen, is red and gives the transparent animals a reddish appearance (Figure 2.7). Because certain parasites also cause the hemolymph to become red, one cannot easily determine the cause of the red color from sight alone. However, low oxygen usually affects an entire population, coloring all animals reddish, whereas parasites usually infect only a portion of the population.

Figure 2.7 Two Daphnia magna with contrasting hemolymph color due to haemoglobin (Hb).. These two adult females were taken from two adjacent rock pool populations (more...) Daphnia have the usual osmoregulatory problems of freshwater animals, i.e., too much water and too few solutes. They are able to absorb ions with chloride-absorbing glands. The shell gland (maxillary gland; Figure 2.1) may have a role in excretion and/or osmoregulation. The nervous system is characterized by the cerebral ganglion, which is located close to the gut and near the eye. Juvenile and adult Daphnia have one large compound eye, whereas embryos show two brownish eye spots that fuse during the last part of the development. The compound eye helps to orient the animal while swimming. A small structure called a naupliar eye is located between the mouth and the compound eye on top of the cerebral ganglion. Many invertebrates, including crustaceans, have a well-developed innate immune system that includes melanization by activation of the prophenoloxidase (proPO) activating system, a clotting process, phagocytosis, encapsulation of foreign material, antimicrobial action, and cell agglutination (Sderhall 1999). Of these mechanisms, only the proPO system (Mucklow and Ebert 2003) and phagocytosis (Metchnikoff 1884) have been identified in Daphnia, although there is no reason to doubt that the other mechanisms work as well. The proPO system is not only a defense system against parasites but is also involved in wound healing of the cuticle (Figure 2.8). The enzyme involved in melanin formation, PO, has been detected in the blood of many arthropods, including D. magna. Melanin is a brown pigment that is also used for carapace pigmentation in some Daphnia, particularly Daphnia in the high Arctic, where the dark pigment protects them from uninterrupted solar radiation (repair of UV damage is only possible in the dark). Acquired immunity is thought to be absent in invertebrates; however, transmission of strain-specific immunity from mothers to offspring has recently been suggested for D. magna (Little et al. 2003).

Figure 2.8 Wound healing in Daphnia magna . An injury in the carapace heals within a few hours to days. Wound healing involves a melanization reaction that stains the wound dark. Here a (more...) Go to:

Life Cycle and Development

The life cycle of Daphnia during the growth season is characterized by its asexual mode of reproduction (apomixis) (Figure 2.9). A female produces a clutch of parthenogenetic (amictic) eggs after every adult molt (if feeding conditions permit). Figures 2.3 to 2.5show females with parthenogenetic eggs. The eggs are placed in the brood chamber, which is located dorsally beneath thecarapace and which is closed by the abdominal processes (Figures 2.1 and 2.10). Development of eggs is direct (immediate). At 20C, the embryos hatch from the eggs after about 1 day but remain in the brood chamber for further development (Figure 2.11). After about 3 days in the brood chamber, the young Daphnia are released by the mother through ventral flexion of the post-abdomen. The newborn look more or less like the adult Daphnia, except that the brood chamber is not yet developed (Figure 2.12). In most species, a juvenile Daphnia passes through four to six juvenile instars before it becomes primipare, i.e., produces eggs for the first time. The age at which the first eggs are deposited into the brood chamber is around 510 days at 20C, but this may take longer under poor feeding conditions. An adult female may produce a clutch of eggs every 3 to 4 days until her death. In the laboratory, females may live for more than 2 months, with a higher age being reached under poorer feeding conditions. Clutch sizes vary among species, from 1 to 2 eggs in small species such as D. cucullata (Figure 2.4) to more than 100 in large species such asD. magna (Figure 2.3).

Figure 2.9 Life cycle of a cyclic parthenogenetic Daphnia . This diagram depicts the sexual and the asexual (parthenogenetic) life cycle of a Daphnia. During the parthenogenetic cycle,(more...)

Figure 2.10 Egg laying by Daphnia magna . This series of pictures, taken within a 15-minute time span, shows the process of placing eggs into the brood chamber. The eggs are initially(more...)

Figure 2.11 Development of Daphnia embryos. This sequence shows the development of parthenogenetic eggs and embryos raised in a culture dish. The top left picture (more...)

Figure 2.12 Newborn Daphnia magna . A parthenogenetic offspring of D. magna within the first 12 hours after release from the brood chamber. Although in a typical growth season Daphnia produce diploid (2N) eggs that develop directly and without a resting phase, a different type of egg is produced for resting (Figure 2.9). These resting eggs are encapsulated in a protective, saddle-like structure called anephippium (Figures 2.13 and 2.14), which is usually strongly melanized and contains 2 large eggs, 1 from each ovary. It is not uncommon, however, to

find ephippia with only 1 egg, or none at all. The ephippium is cast off at the next molt. In most cases, these eggs are produced sexually, but obligate parthenogenetic Daphnia, which are typically found in the northern parts of America and Eurasia and are an excellent example of geographic parthenogenesis (best described for D. pulex), may produce diploid resting eggs asexually as well. In the more common sexual Daphnia (with cyclic parthenogenesis), resting egg production follows the asexual production of diploid males, which are needed to fertilize the haploid eggs (Figures 2.15 and 2.16). A sexual clone can produce males and females and is capable of self-fertilization. For fertilization, males copulate with females (sometimes two males can be observed on the same female). Fertilization is internal and happens between molting and deposition of eggs into the ephippium. Sperm are either tailless (Figure 2.17) or may contain short, pseudopodia-like extensions. The induction of sexuality seems to be triggered by a complex set of stimuli, the most important possibly being those that go hand-in-hand with a highDaphnia density, e.g., increased competition and reduced food availability. Abiotic factors alone, such as decreased day length and lowered temperature, also seem to play a minor role. Daphnia in intermittent populations (such as ponds that are likely to dry up during part of the year) have a much higher tendency to produce resting eggs than Daphnia in permanent populations, e.g., in large lakes.

Figure 2.13 Daphnia magna carrying a resting egg.. The ephippium is easily recognized by its dark color. It usually contains two eggs, which are haploid and require (more...)

Figure 2.14 Daphnia longispina carrying a resting egg.. The ephippium is not yet fully developed. Its wall is still transparent, and the two eggs are visible. These (more...)

Figure 2.15 Male Daphnia magna . A male D. magna from a laboratory population of a clone isolated from a rock pool in southern Finland. Photo by Dita B. Vizoso, Fribourg (more...)

Figure 2.16 Scanning electron microscopic of an adult male Daphnia magna . A male D. magnafrom a laboratory population of a clone isolated from a rock pool in southern Finland. Photo by (more...)

Figure 2.17 Sperm of Daphnia magna . Sperm in D. magna are tailless, rod-shaped, and up to 9 m in length. Other Daphnia species have sperm with short pseudopodia (more...) The ephippia from the females are released with the shed carapace during molting and sink to the bottom or float with the help of small gas chambers. They may disperse with the wind or with animals (e.g., attached to the feathers of waterfowl) or may drift with the water. Floating ephippia may be driven by winds to form large piles of resting eggs in wind-protected parts of ponds and lakes. Depending on the habitat, resting eggs may endure unfavorable seasons (e.g., winter colds, periods of low bioproductivity, summer droughts), and hatching is induced by external stimuli, such as an appropriate photoperiod, light, rising temperatures, or simply the presence of water in a previously dry pond. From resting eggs, only females hatch, which usually produce parthenogenetic eggs themselves but may directly produce resting stages under conditions of a very short growing season. Go to:

Habitat
Daphnia populations can be found in a range of water bodies, from huge lakes down to very small temporary pools, such as rock pools (Figures 2.18 and 2.19) and vernal pools (seasonally flooded depressions). Often they are the dominant zooplanktor and form, as such, an essential part of the food web in lakes and ponds. In many lakes, Daphnia are the predominant food for planktivorous fish, at least at times. As a consequence, the Daphnia species distribution and life history are closely linked with the occurrence ofpredators. Typically, Daphnia species found in lakes with planktivorous fish are smaller and more transparent than species found in fishless water bodies. Large species such as D. magna and D. pulex usually cannot survive under intensive fish predation, whereas small species such as D. galeata, D. cucullata, and D. hyalina are usually not found in fishless water bodies. A number of invertebrates are known to prey on Daphnia, the best investigated probably being the larvae of the phantom midge Chaoborus and the water boatman Notonecta and related genera. Although visually-hunting fish usually show a preference for larger prey items, invertebrates may prefer smaller prey or even a specific size class. These differences in size-specific mortality rates are believed to be a key factor in the evolution of Daphnia body size. High juvenile mortality caused by Chaoborus has been suggested as the cause for evolution of larger sizes at birth and to phenotypically plastic adjustments of birth size and growth rates. In contrast, predation by fish has led to smaller sizes and earlier maturation age.

Figure 2.18 Rock pools with Daphnia populations on four islands in the Tvrminne archipelago in southern Finland. These pools are part of a metapopulation (more...)

Figure 2.19 Dry rock pool with Daphnia magna populations on an island in the Tvrminne archipelago in southern Finland. The upper photo shows a dry pool (more...) It has been proposed that in water bodies without predation, the composition of Daphnia species is influenced by size-dependent competition, with larger species out-competing smaller species. There is, however, some debate about the efficiency of size-dependent processes in the absence of predation. The water quality of Daphnia habitats can vary widely. A pH between 6.5 and 9.5 is acceptable for most species, with the optimum being between 7.2 and 8.5. Salinity should usually be below 5% of seawater (about 1.5 grams of sea salt per liter), but some species can tolerate much higher salinities, such as. D. magna, which can be found in up to 20% seawater. Go to:

Behavioral Ecology
The English name for Daphnia, waterflea, originates from the jumping-like behavior they exhibit while swimming. This behavior stems from the beating of the large antennae, which they use to direct themselves through the water. The rapid downbeat produces a quick upward movement, whereas the relatively high density of the animals creates a sinking. Motionless Daphnia sink rapidly to the ground. A second well-known behavior of Daphnia is diel vertical migration, in which they migrate toward upper levels of the water body during nighttime and then back downward during the early morning and daytime. This behavior probably developed as a predator avoidance strategy. During daylight, the Daphnia hide from fish that hunt visually by moving to darker depths, whereas during nighttime, they take advantage of the richer food (planktonic algae) in the well-illuminated upper water levels. Inverse diel vertical migration has been described as a strategy to escape other predators that migrate themselves. Part of the behavioral repertoire of Daphnia that is key to diel vertical migration is phototaxis. Clones of D. magna vary strongly in their phototactic behavior. Phototactic-positive genotypes spend much time in the upper water level, whereas phototactic-negative genotypes spend most of their time close to the bottom sediments. Phototactic behavior is also influenced by the presence of fish. If Daphnia sense that fish are present in the water, they behave more phototactically negative than they would otherwise.

Daphnia are also known to migrate toward or away from the banks of ponds or lakes. Again, the most likely explanation for this behavior is predator avoidance. Certain species of Daphnia, e.g., D. magna, can be observed clinging occasionally to plants or other substrate. Furthermore, they may browse over the surface substrates to pick up small particles. This behavior is more apparent when food is limited and seems to enrich the diet. The stirring movement of the Daphnia brings small particles into suspension, which are then ingested by filter feeding. Go to:

Evolutionary Genetics
Following the pioneering work of P.D.N. Hebert, the population genetics of Daphnia have been intensively studied around the world. This study was facilitated by allozyme electrophoresis (more recently also microsatellite loci), which revealed a fair degree of enzyme polymorphism in many populations and opened the door for many studies on migration and gene flow (e.g., population divergence, isolation by distance, F-statistics), hybridization (many Daphnia species tend to form hybrids), inbreeding (small pools may be colonized by a few clones that subsequently inbreed), and clonal selection (clone frequency changes across the summer season have been frequently observed). It also helped explain phylogenetic relationships among species (later refined with DNA data). Because of their clonal reproduction, Daphnia present a superb tool for quantitative genetic studies, which can enhance our understanding of their evolutionary ecology. Within- and between-clone comparisons can demonstrate genetic variation for various traits within and between populations, thus helping to reconstruct the evolutionary history of a population. For nearly every trait that has been investigated, genetic variation has been reported. Examples include age and size at maturity, size at birth, aging, reaction norms for life history traits, vertical migration, phototactic behavior, fish escape behavior, production of defense spines and helmets, resistance against parasites, immune response, competitive ability, Malthusian growth rate, carrying capacity under stable conditions, and many more. Furthermore, complex matrices of genetic covariances among traits have shown that, in a first approximation, most traits share some covariance with other traits, indicating that an evolutionary change of many traits is constrained by the evolution of other traits. For those Daphnia species that have been karyologically investigated, between 20 and 24 chromosomes have been counted (2N) (Zaffagnini 1987). With the announcement that the genome sequence of D. pulex will be produced in 2005, the genetic study of Daphnia is about to enter a new phase. With Daphnia, one of the first organisms with a wellknown ecology will be sequenced, which is a milestone in the field of ecological genetics. Other molecular tools have been developed in parallel, not only for D. pulex but also for D. magna(e.g., microarrays, expressed sequence tags (ESTs)). Go to:

Population Dynamics
Daphnia populations vary strongly in density throughout the growing season. They typically go through pronounced cycles, with densities varying by more than seven orders of magnitude within a single season. A number of studies have refuted the belief that cycles are largely influenced by abiotic conditions; now it is commonly believed that abiotic conditions play a role only in limiting the growing season, not as a factor shaping the population dynamics during the growing season. In many populations, density peaks are observed two or even three times per year, and it is not uncommon for populations to

disappear entirely during unfavorable seasons. Although it is difficult to make generalizations about Daphnia population dynamics, a few points are apparent. In most habitats, Daphnia have low density or completely disappear during part of the year, usually the cold or the dry season. Recruitment in the following growing season is from resting eggs and/or from surviving females. There is rapid population increase in the early season (exponential growth), with doubling times of a few days (down to 3 days at temperatures of 20C and above). Growth is eventually slowed down by density-dependent competition, usually because of food shortage; however, predators may contribute as well. During this part of the season, parasites seem to play little role in affecting population numbers. The peak inDaphnia density usually follows a peak in algae density and may be followed by the clear-water phase in which the Daphniaeffectively remove most of the phytoplankton from the water. The resulting food shortage leads to a rapid decline in Daphniadensity. In large eutrophic lakes in temperate regions, phytoplankton and Daphnia may go through two density cycles (a spring and a summer peak), whereas in nutrient-poor lakes, only one peak may occur in midseason. In small water bodies such as rock pools and vernal pools, the dynamics may look very different, depending on the expected length of the growing season. In pools with a very short growing season (e.g., Arctic and desert pools), Daphnia populations may produce resting eggs after only one or two generations, which curtails the exponential growth phase early. In longer-lasting small pools, populations may go through several population cycles within one season. Factors that increase population growth rate (e.g., eutrophication) or that amplify the response to high population density (e.g., sexual reproduction) increase the likelihood of more cycles occurring. Daphnia parasites are most commonly observed after the first peak in population density. However, it is not clear whether parasites influence the population dynamics of Daphnia in natural populations. Parasites are an attractive ecological force for Daphniapopulation regulation because transmission is often density dependent, and they are found in nearly every Daphnia population investigated thus far. Experimental epidemiology with various zooplankton parasites has shown that parasites not only suppress host density but also may bring host populations to extinction (Ebert et al. 2000). Thus, it seems likely that the dynamics of naturalDaphnia populations are influenced by parasites as well. Go to:

References
1. Ebert D, Lipsitch M, Mangin KL. The effect of parasites on host population density and extinction: Experimental epidemiology with Daphnia and six microparasites. Am Nat. 2000;156:459477. 2. Freyer G. Functional morphology and the adaptive radiation of the Daphniidae (Branchiopoda: Anomopoda). Philos Trans R Soc. 1991;331:199. 3. Kstner. 1993. Lehrbuch der speziellen Zoologie, Band 1, Teil 4. Heidelberg, Germany, Elsevier GmbH, Spektrum Akademischer Verlag. 4. Kkenthal W, Matthes E. 1944. Leitfaden fr das Zoologische Praktikum. Jena, Germany, Gustac Fischer Verlag. 5. Lampert W, Sommer U. 1999. Limnokologie, 2. edition. Stuttgart, Thieme Verlag. 6. Little TJ, O'Connor B, Colegrave N, Watt K, Read AF. Maternal transfer of strain-specific immunity in an invertebrate. Curr Biol. 2003;13:489492. [PubMed] 7. Metchnikoff E. Ueber eine Sprosspilzkrankheit der Daphniden. Beitrag zur Lehre ber den Kampf der Phagocyten gegen Krankheitserreger. Virchows Arch Pathol Anat Physiol. 1884;96:177193. 8. Mucklow PT, Ebert D. The physiology of immunity in the water flea Daphnia magna: Environmental and genetic aspects of phenoloxidase activity. Physiol Biochem Zool. 2003;76:836842. [PubMed] 9. Peters RH, De Bernardi R. 1987. Daphnia. Memorie Dell 'Istituto Italiano di Idrobiologia. p. 502.

10. Sderhall K. Editorial. Special issue. Invert Immun. 1999;23:263266. [PubMed] 11. Zaffagnini F. Reproduction in Daphnia. Mem Ist Ital Idrobiol. 1987;45:245284.

Daphnids
Ordering Information: We currently culture Ceriodaphnia dubia and Daphnia magna. Less than 24-hour age organisms are available for immediate shipment. Starter cultures with mixed ages are also available. Please see our Products page for information about daphnid food. Current Price List (PDF) Faxable Order Form (PDF)

Scientific Name: Ceriodaphnia dubia Common Name: Water flea

Physical description:Ceriodaphnia dubia are very small, never larger than one millimeter in length. When in good health, they appear to be a brownish-orange color. There is a large, dark spot on the anterior end of every Ceriodaphnia, this is the compound eye. Males and females can be distinguished from each other by their shape and size. Males are smaller and less rotund than are the females; the males are almost triangular in shape. Movement is achieved by a powerful set of second antennae. Because of this, movement is generally vertical and jerky.

Ecology: The water flea, Ceriodaphnia dubia, occurs in littoral areas lakes, ponds, and marshes throughout most of the world. Uses in Aquatic Toxicology: Ceriodaphnia dubia are an EPA recommended freshwater invertebrate used in both acute and chronic toxicity testing. In acute toxicity testing, Ceriodaphnia are used at <24 hours old and survival rates are recorded. In chronic toxicity testing, Ceriodaphnia are used at <24 hours old and all neonates must have been released within 8 hours of each other. In chronic tests, survival and reproduction are recorded. Other Uses: Live food source in freshwater larviculture and in the ornamental fish industry.

Scientific Name: Daphnia magna Common Name: Water flea

Photo: (2005) Are We Underestimating Species Extinction Risk? PLoS Biol 3(7): e253

Physical description: Daphnia magna are similar toCeriodaphnia dubia but larger, usually five to six millimeters in length. Ecology: Daphnia magna is principally a lake dweller and is restricted to waters in northern and western North America. Daphnia magna reproduce only by cyclic parthenogenesis in which the males contribute to the genetic makeup of the young during the sexual stage of reproduction.

Uses in Aquatic Toxicology:Daphnia magna are used in freshwater acute toxicity testing at <24 hours old and survival rates are recorded. Other Uses: Live food source in freshwater larviculture and in the ornamental fish industry.

Plan The initial variable to be considered will be the effect that caffeine and temperature has on the heart beat and if it has a direct or indirect effect on the daphnia's lifespan. If there is enough time to consider a second variable however, the effects of a temperature or alcohol on the daphnia's body system will also be considered alongside the initial variable of caffeine. These will then be slotted into the same graph which will show us how the figure correspond to the results. In order to conduct a true experiment the following questions must be considered and answered and only then can acurate conclusion be drawn. ;How variable is the heart rate in an individual Daphnia? ;How does the drug caffeine affect the daphnia's heart beat? ;At what concentration is the heart rate slowest? The fastest? ;What indication in the graph is the optimum concentration for heart rate of Daphnia? ;What is the significance of the shape of the graph line when the data is plotted? ; ;What would happen if you increased the temperature in 5C increments (if time is available)? ;What would happen if alcohol was added into the solution and why would this affect the daphnia's heart rate and general behavior. To fully understand the way the daphnia works one must examine its body and the uses for the different parts of it:- (fig.1) [1] - Second antenna, used for swimming and sensing the environment[2] - eye controlled by muscles with nerve connections to the brain[3] - legs used for gathering food stabilizing the animal as it slowly sinks[4] - An intestine where ground up food particles are digested[5] - A brood pouch for incubating young that hatch from large yolk filled eggs [6] - A protective outer shell [7] - A heartthat pushes clear circulatory fluid around the body[8] - Undigested material is eliminated out the anus. As it is now seen that the daphnia incorporates its food through the use of its legs it could explain why the drugs such as alcohol or caffeine take somewhat longer to enter

its brain and alter its bodily functions-also its body acts as a form of a semi-permeable barrier of which small molecules (such as caffeine 'C8H10O2N4H20') can penetrate the walls of its internal organs, this is why it penetrates the whole of the daphnia's body at once (see 'prediction' for information relating to this fact). If temperature is to be used as a chosen variable, it must be considered that daphnia are cold-blooded animals, they do not thermoregulate. This means that their body temperature is the same as the water they are floating in. The chemical reactions that occur in the cells of Daphnia are dependent on certain enzymes, or proteins, to help the reactions proceed. As you increased the temperature of the water, the metabolism of the Daphnia increased as well, because chemical reactions occur faster at higher temperatures. This means that the heart rate will speed up in order to provide oxygen to the cells as the metabolism increases. However, at 40 degrees Celsius, the enzymes break down, and the chemical reactions can no longer occur, so metabolism stops and the Daphnia dies. In its natural environment, Daphnia does not ever experience temperatures of 40 degrees, so this is unlikely to occur. However, they do experience changes in temperature in their natural environment, and their metabolism does increase or decrease as the temperature changes. Method I have initially decided to see how the drug caffeine affects the heartbeat and lifespan of a daphnia, A daphnia will be extracted from a pond nearby, so the condition of the daphnia will not be altered too drastically during transport. These daphnia will be allowed to adjust to their new surroundings and then be taken out through the use of a pipette and placed in a petri dish containing the relevant amount of caffeine/alcohol concentrate solution (aq). This daphnia will be left in the solution for a minimum time of one minute before any sort of results are taken (for this experiment one and a half minutes were used) this is to allow the daphnia to adjust to its new surroundings and its semi-permeable barrier to in-secrete the surrounding solution, although when doing this, it must be remembered that if left too long, the daphnia will have adjusted itself and the heartbeat will begin to regulate again. Once the period of one minute thirty seconds has elapsed, a 'clicker counter' will be used to count the number of beats of the daphnia's heart in one minute (obviously a correctly focussed microscope will be necessary to see the daphnia's heart and how it beats) for problems relating to this method please see 'evaluation'. The concentration of alcohol/caffeine will then be varied to see how this

affects the regulation the daphnia's heartbeat. (the daphnia's heart is illustrated in the following diagram fig.2). In an ideal lab condition the plan would be such as this, although the time period and equipment are limited so this method will be followed as close as is possible:1. Prior to conducting the bioassay, check the Daphnia to ensure the culture is healthy. 2.select test organisms. Although you may use mixed age populations for bioassays, it is better to use only young individuals in order to minimize biological differences among the test organisms. Because the appearance of resting eggs indicates a poor culture environment, do not use Daphnia with resting eggs. To obtain a good supply of young Daphnia, begin 24 hours in advance by removing females bearing embryos from the stock culture and placing them in 400-mL beakers containing 300 ml of spring or stream water and the appropriate amount of food. Five beakers, each containing 10 adults, usually will supply enough young individuals for one toxicity test. When you are ready to begin the bioassay, choose young (small) Daphnia from these cultures. 3. Introduce the same number of neonates (at least 10) into each test vessel and control using a plastic, disposable pipette with a 5-mm diameter. Be sure to release the young below the surface to avoid killing them by trapping air under their carapaces. 4. Prepare a table that records the percent concentration, the time, and the total number of dead at each time interval. As close to one hour as possible, but prior to the end of the period, record the number dead at each concentration. Remember Daphnia molt to grow, so neonates will molt as they develop. Therefore, do not count the molt castings, which appear as clear shells of the Daphnia on the bottom of each cup. Remove dead Daphnia and molt castings at each monitoring interval. 5. Ideally it is best to check again in 4 hours and again after 24 hours. If schedules do not permit (such as ours having to be completed in a one hour 45 minute time period), Must not feed animals during tests. Steps 4-6 will take about 15 minutes of two consecutive class periods. 6. At the end of the bioassay, test the water to determine the pH, hardness, and dissolved oxygen content. Count and record how many Daphnia in each dish have died, then analyze the data.. Apparatus

Equipment dissecting microscope water chemistry test kits (pH, hardness, dissolved oxygen) Light (from lamp) petri dishes salt solution (NaCl) clicker counters Materials 5mm diameter pipette or eye dropper Alcohol Caffeine microscope slide 1 ml pipette with .01 divisions thermometer spring water or unpolluted stream water Daphnia magna culture, mixed age Wipes or other tissues food: possibilities include Roti-Rich dried yeast, or unicellular algae such as Selenastrum Diagram

Daphnia Size - There is a big size difference in the Daphniidae, depending on the species. Newly hatched Moina are slightly larger than newly hatched brine shrimp, and twice as big as average adult rotifers, but newly hatched Daphnia are twice as big as Moina, and may not be suitable for some of the smaller fish fry because of their size. Life cycle of Daphnia - The daphnia has both sexual and asexual phases. In most environments, the population consists entirely of females that reproduce asexually. Under optimum conditions, a female may produce more than 100 eggs per

brood, repeating every 3 days. A female may have as many as 25 broods in its lifetime, but the average is about 6. The female will start to reproduce at about 4 days old with a brood size of 4 to 22 eggs. Under adverse conditions, males are produced, and sexual reproduction begins. The result is the laying of resting eggs, just like the brine shrimp. Factors that can trigger this are a lack of food, low oxygen supply, a high population density, or low temperatures. Nutritional Value - The nutritional content of Daphnia varies with age, and what its been eating. The protein content is usually around 50% of dry weight. Quite the opposite from Artemia, adults normally have a higher fat content than juveniles, about 20-27% for adults, and 4-6% for juveniles. Some species have been reported to have protein contents exceeding 70%. Live Moina are about 95% water, 4% protein, 0.54% fat, 0.67% carbohydrates, and 0.15% Ash. The fatty acid composition of food is important to the survival and growth of fish fry. Omega-3 highly saturated fatty acids are essential for many species of fish. Moina cultured on bakers yeast are high in monoenoic fatty acids. By using what is called w-yeast (yeast enriched with cuttlefish oil), Moina will contain very high levels of Omega-3 fatty acids. Moina can take up lipids very easily from the emulsion, but there is a side effect to this, apparently it also slows productivity, so this emulsion should only be fed to a batch separate from the main growout colony. Commercial formulas are available in pet supply houses for the enrichment of Artemia, Rotifer, and Daphnia cultures. Physical Requirements Salinity - Daphnia are typically freshwater organisms, but, some are found in slightly brackish water. Some species have been observed in salinities up to 4 ppt, and salinities of 1.5 to 3.0 ppt are common in pond cultures in the orient.

Oxygen - Daphnia are generally tolerant of poor water quality, and dissolved oxygen varies from almost zero to supersaturation. Like the Brine Shrimp, their ability to survive in an oxygen poor environment is in their ability to synthesize hemoglobin. The production of hemoglobin may be promoted by high temperatures, and a high population. Also, like brine shrimp, Daphnia are not tolerant of fine air bubbles. A slow aeration is needed with Daphnia as a large bubble column will strip the Daphnia out and kill them. pH and ammonia - A pH between 6.5 and 9.5 is acceptable. High ammonia levels, with high pH will drastically reduce reproduction, but will not affect the actual health of the animals themselves. So it seems that on the small scale that we require, monitoring of pH and ammonia is not critical to success. Dissolved minerals - In contrast to their tolerance of low oxygen, Daphnia are very sensitive to disturbances of the ionic composition of their environment. They become immobile and eventually die with the addition of salts like sodium, potassium, magnesium, and calcium. Low concentrations of phosphorus (less than 0.5 ppm) will stimulate reproduction, but concentrations higher than 1.0 are lethal to the young. Daphnia magna are quite resistant to phosphorus and can withstand concentrations as high as 5-7 ppm. Daphnia are not affected by the addition of nitrogen in fertilizers for the promotion of algae growth. As with any aquarium venture, the water used should be treated with aeration or de-chlor to remove chlorine before the culture is started. Concentrations of only 0.01 ppm copper will result in reduced movement in Daphnia. They are extremely sensitive to metal ions like copper and zinc, pesticides, detergents, bleaches and other dissolved toxins. Municipal and well water may be contaminated enough to kill the culture. The best source of water is filtered stream or lake water, rain water collected

for low air polluted areas, or, use the water from your aquarium water changes. Temperature - Daphnia have a wide tolerance to temperature. The optimum temperature for Daphnia Magna is 18-22 deg C (64-72 F) Moina withstand extremes even more, resisting daily variations of 5-31 deg C (41-88 F) ;their optimum being 24-31 deg C (75-88 F). The higher temperature tolerance of Moina make this species a better choice where temperatures may rise above the comfort levels for Magna at certain times of the year.

Prediction To create a reasonable prediction, it must first be known how daphnia react in their surroundings this background knowledge will undoubtedly help when attempting to make conclusions and/or predictions relating to the daphnia's bodily systems and reactions. Daphnia typically live 40-56 days. This varies according to species and environmental conditions. Each brood typically holds 6-10 eggs, which turn into embryos and are released within a few days. Juveniles reach sexual maturity in 6 to 10 days. Daphnia is a small Crustacean - barely visible with the naked eye. It lives in water and has large antennae in comparison with the rest of its body. These are used to make (jumpy) movements - hence the name "water flea". Daphnia's seem to serve two purposes: (1) they are very popular fish foodespecially because it is quite easy to keep them in culture, they reproduce at high rates and they keep the aquarium water clean (2) aschanges in the heart rate might suggest a chemical compound has some physiological effect, and - more importantly - Daphnia magna is used to measure the toxicity of a chemical compound in water (LD50 measurements). From a sciencepoint of view, Daphnia is not intensively studied. In essence, a few ATPase subunits and ribosomal RNA genes are sequenced. Also under investigation is the nature of this species unusual pigmentation. daphnia are translucent and are usually amber or devoid of color. These dune daphnia, when found in their natural environment, possess a striking melanic pigmentation. It has been

hypothesized that the melanism serves as a photoprotection device, but it is possible that it also serves as a defensive protection from the predatory copeopods which also inhabit these ponds. In a study in which melanic forms of daphnia were found in arctic ponds a strong correlation was found between whether or not the pond was surrounded by vegetation and the extent to which melanic forms were present. It was found that in all cases, melanic forms were present in ponds which were devoid of surrounding vegetation. It was hypothesized that organic nutrient runoff from local fauna filters out harmful ultraviolet radiation within the first few inches of water. Ponds
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Daphnia Bioassay for Detecting Toxins and Endocrine Disrupters in Aquatic Samples
Wisconsin Alumni Research Foundation (University of Wisconsin)

posted on 04/25/2008 request more info ShareThis LICENSE ONLINE

Cladoceran zooplankton, such as Daphnia, are widely used to test for the presence of aquatic toxins. Typically, toxicity tests using Daphnia measure changes in survivorship and fecundity.This invention provides a Daphnia reproductive bioassay for detecting the presence of toxic substances in aqueous samples and for screening the substances as endocrine disrupters. In the assay, a test sample is contacted with adult, oviporous Daphnia clones under conditions that stimulate sexual reproduction and the production of males. Next, several biological parameters are quantified, including survivorship, numbers of male and female offspring, number of resting eggs, number of abnormal offspring, and nutritional status of the offspring. This information is then compared with data from a control sample to determine if toxic substances and endocrine disrupters are present in the test sample.

ADVANTAGES

Provides ecologically meaningful test information to evaluate environmental toxicity Fast and relatively easy to perform Consistent within and between laboratories Highly accurate and sensitive Useful for determining concentrations of chemical substances that have either a toxic effect or an endocrine disrupting effect Uses more informative and sensitive parameters than current Daphnia toxicity tests

May be used to evaluate toxic effects of individual chemical formulations in product testing, as required by the FDA May be used for pesticide registration, as required by the USDA May be used to screen natural waters or effluents from industry or municipalities for toxic potential, as required by various EPA programs

By Christine Najdowski

Introduction
As human population centers grow to cover more and more of the planet, watersheds are increasingly affected by the presence of buildings, roadways, and parking lots. A watershed is "the area of land that catches rain and snow and drains or seeps into a marsh, stream, river, lake or groundwater." (EPA, 2006b.) This project asks the question: "Can run-off from parking lots be toxic to organisms in nearby ponds and streams?" You will learn how to conduct a bioassay to determine if sediments from a parking lot are toxic for aquatic organisms. As the name suggests, a bioassay uses living organisms as the "detector" for an experimental procedure. When doing environmental testing for toxins, the bioassay is typically a viability assay. You count how many organisms are present at the beginning of the experiment, expose the organisms to different concentrations of the suspsected toxin, and count the number of organisms that remain viable. It is critically important to maintain a control population of the organisms, so that you don't mistake the naturally-occuring death rate of the organisms as the effect of toxins. The organism that you will use for testing water toxicity is Daphnia magna. Daphnia magna (common name "water fleas") are tiny freshwater crustaceans. They are filter feeders, and can survive in culture by eating algae, bacteria, or yeast.

Figure 1. Photomicrograph of Daphnia, the common water flea.

If you make a graph of the percentage of Daphnia that remain viable (y-axis) vs. the concentration of the compound tested (x-axis), you can determine the LC50value for the compound. This is the concentration at which 50% of the population remains viable. It is one useful measure for comparing the relative toxicities of compounds.

Variations
Are sediments from other locations (e.g., a playground, or a sidewalk) toxic to Daphnia? You could also use this bioassay technique to test water from a local pond or stream. Another idea would be to collect run-off water from your lawn at different time points after application of fertilizer. To collect the run-off water, dig a hole so that you can put a plastic collection container with its opening just below the soil surface level. Sample water at one day, three days and one week after application of fertilizer. Since Daphnia are transparent, it is possible to monitor their heart rate under a microscope. This makes pharmacological bioassays a possibility. See the Science Buddies project Caffeine and Heart Rate: A Pharmacological Study Using Daphnia magna. Here are some additional Science Buddies projects that involve testing environmental samples with aquatic bioassays: There's Something Fishy About That Fertilizer, Acid Rain and Aquatic Life, and Heavy Metals and Aquatic Environments. J Environ Sci Health B. 2001 Jan;36(1):67-74.

Chronic toxicity tests with Daphnia magna for examination of river water quality.
Sakai M. Source
Yokohama Environmental Research Institute, Takigashira, Isogo, Japan.

Abstract
Chronic toxicity tests with Daphnia magna were applied for examination of river water quality. Water was sampled from the Maioka River in Yokohama City on May 14, 20, and 27, 1999, and used for the test after solid-phase extraction. The chronic test was carried out according to the OECD method. The duration was 21 days and the total number of live offspring produced per parent animal was counted. The results of the tests showed, survival rates of 100% using river water sampled on May 14 and 20 and the total numbers of live offspring produced per parent animal did not differ from the control. However, the survival rate of the sample collected on May 27 was 0% and the pesticides, fenitrothion, and thiobencarb were detected in the water. In addition to the river water samples, reconstituted water (Elendt M7) with additions of fenitrothion and thiobencarb was prepared to investigate mortality. When the reconstituted water with thiobencarb was applied to the test, the total number of live offspring produced per parent animal did not differ from the control. In contrast, when reconstituted water with fenitrothion was applied to the test, most parents were alive, but the total number of live offspring produced per parent animal was apparently different. The results of the above tests indicate that D. magna was affected not only by fenitrothion in the river water collected on May 27, but also by other factors that were not clarified in this study.