Ecotoxicity Evaluation of Pristine and Indolicidin-coated Silver Nanoparticles in Aquatic and Terrestrial Ecosystem.

BACKGROUND: Metallic nanoparticles (NPs) are highly exploited in manufacturing and medical processes in a broad spectrum of industrial applications and in the academic sectors. Several studies have suggested that many metallic nanomaterials including those derived by silver (Ag) are entering the ecosystem to cause significant toxic consequences in cell culture and animal models. However, ecotoxicity studies are still receiving limited attention when designing functionalized and non.-functionalized AgNPs. OBJECTIVE: This study aimed to investigate different ecotoxicological profiles of AgNPs, which were analyzed in two different states: in pristine form uncoated AgNPs and coated AgNPs with the antimicrobial peptide indolicidin. These two types of AgNPs are exploited for a set of different tests using Daphnia magna and Raphidocelis subcapitata, which are representatives of two different levels of the aquatic trophic chain, and seeds of Lepidium sativum, Cucumis sativus and Lactuca sativa. RESULTS: Ecotoxicological studies showed that the most sensitive organism to AgNPs was crustacean D. magna, followed by R. subcapitata and plant seeds, while AgNPs coated with indolicidin (IndAgNPs) showed a dose-dependent decreased toxicity for all three. CONCLUSION: The obtained results demonstrate that high ecotoxicity induced by AgNPs is strongly dependent on the surface chemistry, thus the presence of the antimicrobial peptide. This finding opens new avenues to design and fabricate the next generation of metallic nanoparticles to ensure the biosafety and risk of using engineered nanoparticles in consumer products.

Multiplanar full-field blur correction method for infrared microscopy imaging.

We propose a 3D full-field focusing method for microscopic mid-wave infrared (MWIR) imagery. The method is based on the experimental estimation of a confined volumetric vision microscope point spread function. The technique employs our well-known constant-range-based nonuniformity correction algorithm as a preprocessing step and then an iteration in the z-axis Fourier-based deconvolution. The technique's ability to compensate for localized blur is demonstrated using two different real MWIR microscopic video sequences, captured from two microscopic living organisms using a Janos-Sofradir MWIR microscopy setup. The performance of the proposed algorithm is assessed on real and simulated noisy infrared data by computing the root-mean-square error and the roughness Laplacian pattern indexes, which are specifically developed for the present work.

Mitochondria-Anchored Colorimetric and Ratiometric Fluorescent Chemosensor for Visualizing Cysteine/Homocysteine in Living Cells and Daphnia magna Model.

Cysteine (Cys) and homocysteine (Hcy) are essential for maintaining the cellular redox homeostasis and play critical roles in pathological and physiological processes. The development of Cys/Hcy-specific responsive fluorescent probes that are independent of the surrounding environment, equipment, and abundant endogenous GSH is critical to accurately investigate the roles of Cys/Hcy in living biological systems. In this work, a novel ratiometric and mitochondria-anchored fluorescence chemosensor, PYR, was constructed on the basis of 4-methylphenol-substituted pyronin fluorophore. The probe exhibited ratiometric fluorescence emission (F540 nm/F620 nm) for the detection of Cys/Hcy with high selectivity, sensitivity (Cys, 22 nM; Hcy, 23 nM), rapid response (Cys, 5 min), and a merit enhancement of ratio fluorescent signal (Cys, 163-fold; Hcy, 125-fold). The probe showed excellent membrane permeability and was applied to visualize mitochondrial biothiols in living cells under H2O2-induced redox imbalance, kidney tissues with a penetration depth of 100 µm, and Daphnia magna model for the first time. The results demonstrate that PYR will provide a promising platform for the diagnosis of thiol-related diseases.

Mitotic activity patterns and cytoskeletal changes throughout the progression of diapause developmental program in Daphnia.

BACKGROUND: Diapause is a form of dormancy that is genetically predetermined to allow animals to overcome harsh environmental conditions. It is induced by predictive environmental cues bringing cellular activity levels into a state of suspended animation. Entering diapause requires organismal, molecular and cellular adaptation to severely reduced energy flows. Cells must therefore have evolved strategies that prepare them for periods with limited metabolic resources. However, changes that occur on the (sub-)cellular level have not been thoroughly described. RESULTS: We investigated mitotic activity and we monitored cytoskeletal network changes in successive stages of diapausing and non-diapausing Daphnia magna embryos using (immuno-)fluorescent labeling. We find that embryos destined to diapause show a delayed and 2.5x slower mitotic activity in comparison to continuously developing embryos. Development is halted when D. magna embryos reach ~ 3500 cells, whereupon mitotic activity is absent and cytoskeletal components are severely reduced, rendering diapause cells compact and condensed. CONCLUSION: In the initiation phase of diapause, the slower cell division rate points to prolonged interphase duration, preparing the cells for diapause maintenance. During diapause, cytoskeletal depletion and cellular condensation may be a means to save energy resources. Our data provide insights into the sub-cellular change of diapause in Daphnia.

The morphology of the male reproductive system, spermatogenesis and the spermatozoon of Daphnia magna (Crustacea: Branchiopoda).

This study analyses the histological and cellular morphology of the testis and sperm development in the male Daphnia magna Straus 1820. Due to the rarity of males and predominately parthenogenetic lifecycle of Daphnia, there has been limited detailed information on males in contrast to the well-studied female. Using light and electron microscopy approaches, we describe the morphology of the testis during the progression from an immature to mature testis. The testis has an encasing muscular mesh sheath outside the basal lamina, beneath which is a thin somatic epithelial cell layer. Internal to the epithelium are the spermatogonial stem cells and subsequent syncytial clusters of the germ cells as they progress through spermatogenesis; spermatozoa occupy the entire testis in sexually mature D. magna. We describe the structure of developing and mature spermatozoa; mature spermatozoa are non-flagellated, ovoid in shape with plasmalemma filapodia and are encased in an extracellular capsule.

Effect of subcellular distribution on nC60 uptake and transfer efficiency from Scenedesmus obliquus to Daphnia magna.

The potential uptake and trophic transfer ability of nanoparticles (NPs) in aquatic organisms have not been well understood yet. There has been an increasing awareness of the subcellular fate of NPs in organisms, but how the subcellular distribution of NPs subsequently affects the trophic transfer to predator remains to be answered. In the present study, the food chain from Scenedesmus obliquus to Daphnia magna was established to simulate the trophic transfer of fullerene aqueous suspension (nC60). The nC60 contaminated algae were separated into three fractions: cell wall (CW), cell organelle (CO), and cell membrane (CM) fractions, and we investigated the nC60 uptake amounts and trophic transfer efficiency to the predator through dietary exposure to algae or algal subcellular fractions. The nC60 distribution in CW fraction of S. obliquus was the highest, following by CO and CM fractions. nC60 uptake amounts in D. magna were found to be mainly relative to the NPs' distribution in CW fraction and daphnia uptake ability from CW fraction, whereas the nC60 trophic transfer efficiency (TE) were mainly in accordance with the transfer ability of NPs from the CO fraction. CW fed group possessed the highest uptake amount, followed by CO and CM fed groups, but the presence of humic acid (HA) significantly decreased the nC60 uptake from CW fed group. The CO fed groups acquired high TE values for nC60, while CM fed groups had low TE values. Moreover, even though CW fed group had a high TE value; it decreased significantly with the presence of HA. This study contributes to the understanding of fullerene NPs' dietary exposure to aquatic organisms, suggesting that NPs in different food forms are not necessarily equally trophically available to the predator.

Luminescent Nanocellulose Platform: From Controlled Graft Block Copolymerization to Biomarker Sensing.

A strategy is devised for the conversion of cellulose nanofibrils (CNF) into fluorescently labeled probes involving the synthesis of CNF-based macroinitiators that initiate radical polymerization of methyl acrylate and acrylic acid N-hydroxysuccinimide ester producing a graft block copolymer modified CNF. Finally, a luminescent probe (Lucifer yellow derivative) was labeled onto the modified CNF through an amidation reaction. The surface modification steps were verified with solid-state (13)C nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy. Fluorescence correlation spectroscopy (FCS) confirmed the successful labeling of the CNF; the CNF have a hydrodynamic radius of about 700 nm with an average number of dye molecules per fibril of at least 6600. The modified CNF was also imaged with confocal laser scanning microscopy. Luminescent CNF proved to be viable biomarkers and allow for fluorescence-based optical detection of CNF uptake and distribution in organisms such as crustaceans. The luminescent CNF were exposed to live juvenile daphnids and microscopy analysis revealed the presence of the luminescent CNF all over D. magna's alimentary canal tissues without any toxicity effect leading to the death of the specimen.

Neuroanatomy of the optic ganglia and central brain of the water flea Daphnia magna (Crustacea, Cladocera).

We reveal the neuroanatomy of the optic ganglia and central brain in the water flea Daphnia magna by use of classical neuroanatomical techniques such as semi-thin sectioning and neuronal backfilling, as well as immunohistochemical markers for synapsins, various neuropeptides and the neurotransmitter histamine. We provide structural details of distinct neuropiles, tracts and commissures, many of which were previously undescribed. We analyse morphological details of most neuron types, which allow for unravelling the connectivities between various substructural parts of the optic ganglia and the central brain and of ascending and descending connections with the ventral nerve cord. We identify 5 allatostatin-A-like, 13 FMRFamide-like and 5 tachykinin-like neuropeptidergic neuron types and 6 histamine-immunoreactive neuron types. In addition, novel aspects of several known pigment-dispersing hormone-immunoreactive neurons are re-examined. We analyse primary and putative secondary olfactory pathways and neuronal elements of the water flea central complex, which displays both insect- and decapod crustacean-like features, such as the protocerebral bridge, central body and lateral accessory lobes. Phylogenetic aspects based upon structural comparisons are discussed as well as functional implications envisaging more specific future analyses of ecotoxicological and endocrine disrupting environmental chemicals.

Formation and structure of the ephippium (resting egg case) in relation to molting and egg laying in the water flea Daphnia pulex De Geer (Cladocera: Daphniidae).

Resting eggs produced by daphnid species in response to environmental deterioration play an important role in colonizing new habitats or in re-establishing extinct populations. Females lay resting eggs into the space within the dorsal part of their carapace and form an egg case called the ephippium to protect them. Previous studies mainly reported the morphology of the completely formed ephippium and/or the forming ephippium of an uncertain stage. To understand ephippium formation and to clarify key transitions in the formation of resting eggs, we examined the structure and formation of the ephippium in the water flea Daphnia pulex De Geer (Cladocera: Daphniidae) by stereomicroscopy, histology, and scanning electron microscopy. The females used in this study produced resting eggs by obligate parthenogenesis. We divided ephippium formation into four stages based on two molts and a single ovulation, as follows. Stage I begins 13 min after molting in adult females that do not ovulate. In Stage II, immediately after the first molt, a protuberance appears beneath the neck region and the carapace begins to thicken. In Stage III, the resting eggs ovulate and the carapace in the area of the forming ephippium becomes much thicker than the normal carapace and accumulates dark pigmentation. In Stage IV, following the second molt, the female sheds the ephippium with the enclosed resting eggs and forms a new carapace. These stages will provide a useful reference for future studies on resting egg formation.

[Morphology and age variation of Daphnia galeata Sars (Cladocera: Daphniidae) in two adjacent water bodies of the Kola Peninsula].

The morphology and age variation of Daphnia galeata Sars (Cladocera: Daphniidae) in two adjacent water bodies of the Kola Peninsula are studied. The reliability of characters usually used for diagnosis of adult individuals of the given species was verified, and their formation in the postembryonic period was traced. If the identification of D. galeata from Lake Bolshoi Vudvyar caused no doubts, the population from Lake Malyi Vudvyar was atypical with respect to several morphological characters. We concluded that plastic characters, such as body shape, as well as the shape of the dorsal keel and helmet on the head are unreliable for distinguishing closely related species of water fleas. At the same time, some small-size characters, for instance, the relative depth of the medial crest on the posterior side of the head, allow us to diagnose this species with confidence.

Morphology of the Daphnia nervous system: a comparative study on Daphnia pulex, Daphnia lumholtzi, and Daphnia longicephala.

The freshwater crustacean Daphnia is well known for its phenotypic plasticity, in which environmental cues are perceived by the nervous system and transformed into phenotypic adaptations beneficial under current conditions. Critical knowledge regarding the distribution and localization of neuronal antigens or neurotransmitters and differentially expressed proteins is sparse. Here, we applied immunohistochemical and histological-staining methods in combination with confocal laser scanning microscopy on whole mount preparations in Daphnia pulex, Daphnia longicephala, and Daphnialumholtzi. We document the nervous system, elements of the sensory system, and cell clusters with secretory characteristics in the Daphnia head. This is the first report on the nervous system of the species D.longicephala and D.lumholtzi. The methods established in this investigation will help to foster research on specific neuronal mechanisms in this rapidly advancing model system of life science research.

Oxidative and photochemical processes for the removal of galaxolide and tonalide from wastewater.

Synthetic musks have been reported in wastewaters at concentrations as high as tens of micrograms per litre. The two most significant polycyclic musk fragrance compounds are 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g)-2-benzopyran (HHCB, trade name galaxolide®) and 7-acetyl-1,1,3,4,4,6-hexamethyltetrahydronaphthalene (AHTN, trade name tonalide®). We report the result of several irradiation and advanced oxidation processes carried out on samples of the effluent of a wastewater treatment plant located in Alcalá de Henares, Madrid. Wastewater samples were pre-ozonated and spiked with 500 ng/L of tonalide or galaxolide in order to obtain final concentrations in the same order as the raw effluent. The treatments assayed were ozonation with and without the addition of hydrogen peroxide (O3, O3/H2O2), ultraviolet (254 nm low pressure mercury lamp) and xenon-arc visible light irradiation alone and in combination with ozone (UV, O3/UV, Xe, O3/Xe) and visible light photocatalytic oxidation using a Ce-doped titanium dioxide photocatalyst performed under continuous oxygen or ozone gas bubbling (O2/Xe/Ce-TiO2, O3/Xe/Ce-TiO2). In all cases, samples taken at different contact times up to 15 min were analyzed. An analytical method based on stir bar sorptive extraction (SBSE), followed by comprehensive two-dimensional gas chromatography (SBSE-GC × GC-TOF-MS), was used for the automatic searching and evaluation of the synthetic musks and other nonpolar or semipolar contaminants in the wastewater samples. In all cases tonalide was more easily removed than galaxolide. The best results for the latter (more than 75% removal after 5 min on stream) were obtained from ozonation (O3) and visible light photocatalytic ozonation (O3/Xe/Ce-TiO2). A significant removal of both pollutants (∼60% after 15 min) was also obtained during visible light photocatalysis (O2/Xe/Ce-TiO2). UV radiation was able to deplete tonalide (+90%) after 15 min but only reduced the concentration of galaxolide to about half of its initial concentration. The toxicity of treated samples decreased for O3/UV and O3/Ce-TiO2, but increased during irradiation processes UV, Xe and Xe/Ce-TiO2. Ozone treatments tend to decrease toxicity up to a certain dosage, from which point the presence of toxic transformation products has adverse effects on aquatic microorganisms.

Abortive meiosis in the oogenesis of parthenogenetic Daphnia pulex.

Most daphnid species adopt parthenogenesis and sexual reproduction differentially in response to varied environmental cues, resulting in the production of diploid progenies in both cases. Previous studies have reportedly suggested that daphnids produce their parthenogenetic eggs via apomixis; the nuclear division of mature oocytes should be an equational division similar to somatic mitosis. However, it seems premature to conclude that this has been unequivocally established in any daphnids. Therefore, the objective of our research was to precisely reveal the process and mechanism of parthenogenetic oogenesis and maintenance of diploidy in Daphnia pulex through histology, karyology, and immunohistochemistry. We found that, when a parthenogenetic egg entered the first meiosis, division was arrested in the early first anaphase. Then, two half-bivalents, which were dismembered from each bivalent, moved back to the equatorial plate and assembled to form a diploid equatorial plate. Finally, the sister chromatids were separated and moved to opposite poles in the same manner as the second meiotic division followed by the extrusion of one extremely small daughter cell (resembling a polar body). These results suggest that parthenogenetic D. pulex do not adopt typical apomixis. We hypothesize that D. pulex switches reproductive mode depending on whether the egg is fertilized or not.

Quick assessment of cytotoxins effect on Daphnia magna using in vivo fluorescence microscopy.

A novel approach to contaminant toxicity screening is proposed. The use of fluorescent microscopy with fluorescent dyes allows for assessing intoxication of Daphnia magna tissues, at various stages of exposure, to contaminants present in water. As shown, D. magna may not only be used as a test species in toxicity tests based on its lethality, but due to its translucency and application of fluorescent probes, separate steps of its intoxication and dying can be visualized. Using a variety of fluorescent probes, the present study also contributes to a better understanding of the toxicity mechanisms.

Intracellular conversion of environmental nitrate and nitrite to nitric oxide with resulting developmental toxicity to the crustacean Daphnia magna.

BACKGROUND: Nitrate and nitrite (jointly referred to herein as NO(x)) are ubiquitous environmental contaminants to which aquatic organisms are at particularly high risk of exposure. We tested the hypothesis that NO(x) undergo intracellular conversion to the potent signaling molecule nitric oxide resulting in the disruption of endocrine-regulated processes. METHODOLOGY/PRINCIPAL FINDINGS: These experiments were performed with insect cells (Drosophila S2) and whole organisms Daphnia magna. We first evaluated the ability of cells to convert nitrate (NO(3)(-)) and nitrite (NO(2)(-)) to nitric oxide using amperometric real-time nitric oxide detection. Both NO(3)(-) and NO(2)(-) were converted to nitric oxide in a substrate concentration-dependent manner. Further, nitric oxide trapping and fluorescent visualization studies revealed that perinatal daphnids readily convert NO(2)(-) to nitric oxide. Next, daphnids were continuously exposed to concentrations of the nitric oxide-donor sodium nitroprusside (positive control) and to concentrations of NO(3)(-) and NO(2)(-). All three compounds interfered with normal embryo development and reduced daphnid fecundity. Developmental abnormalities were characteristic of those elicited by compounds that interfere with ecdysteroid signaling. However, no compelling evidence was generated to indicate that nitric oxide reduced ecdysteroid titers. CONCLUSIONS/SIGNIFICANCE: Results demonstrate that nitrite elicits developmental and reproductive toxicity at environmentally relevant concentrations due likely to its intracellular conversion to nitric oxide.

Genome-wide analysis of tandem repeats in Daphnia pulex--a comparative approach.

BACKGROUND: DNA tandem repeats (TRs) are not just popular molecular markers, but are also important genomic elements from an evolutionary and functional perspective. For various genomes, the densities of short TR types were shown to differ strongly among different taxa and genomic regions. In this study we analysed the TR characteristics in the genomes of Daphnia pulex and 11 other eukaryotic species. Characteristics of TRs in different genomic regions and among different strands are compared in details for D. pulex and the two model insects Apis mellifera and Drosophila melanogaster. RESULTS: Profound differences in TR characteristics were found among all 12 genomes compared in this study. In D. pulex, the genomic density of TRs was low compared to the arthropod species D. melanogaster and A. mellifera. For these three species, very few common features in repeat type usage, density distribution, and length characteristics were observed in the genomes and in different genomic regions. In introns and coding regions an unexpectedly high strandedness was observed for several repeat motifs. In D. pulex, the density of TRs was highest in introns, a rare feature in animals. In coding regions, the density of TRs with unit sizes 7-50 bp were more than three times as high as for 1-6 bp repeats. CONCLUSIONS: TRs in the genome of D. pulex show several notable features, which distinguish it from the other genomes. Altogether, the highly non-random distribution of TRs among genomes, genomic regions and even among different DNA-stands raises many questions concerning their functional and evolutionary importance. The high density of TRs with a unit size longer than 6 bp found in non-coding and coding regions underpins the importance to include longer TR units in comparative analyses.

Methods for meiotic chromosome preparation, immunofluorescence, and fluorescence in situ hybridization in Daphnia pulex.

The genus Daphnia has an intriguing reproductive mode of cyclical parthenogenesis. This reproductive mode has been studied for centuries, but cytogenetic information is lacking due to technical limitations of classical methods. We have developed methods for the preparation and examination of meiotic chromosomes of Daphnia pulex from oocytes and spermatocytes. Oocyte chromosome preparations are obtained by isolating individual oocytes after the release of yolk granules from the ovary using pressure and capillary action. Spermatocyte chromosomes are prepared using a conventional squash method. Cryosectioning is an easy and fast way to prepare sections. We also illustrate the application of immunofluorescence staining against alpha tubulin, as well as fluorescence in situ hybridization (FISH) using the intergenic spacer of ribosomal DNA or single-copy cosmid clones.

Viral transgenesis of embryonic cell cultures from the freshwater microcrustacean Daphnia.

The aim of this study was to determine whether a recombinant vesicular stomatitis virus (VSV) vector encoding a transgene could be used to infect and express a foreign gene in embryonic primary cell cultures derived from the freshwater microcrustacean Daphnia, the most widely used ecotoxicological model organism. To facilitate the evaluation of gene transfer, a reproducible method for establishing primary cultures from Daphnia embryonic tissues was developed. Within 24 hr after infection, transgene expression could be detected in cell culture. VSV was found to replicate in the cells with no apparent cytopathic effect. Here we report the first evidence of gene transfer and foreign gene expression in cultures of Daphnia embryonic cells using a recombinant viral vector.

Exploring embryonic germ line development in the water flea, Daphnia magna, by zinc-finger-containing VASA as a marker.

VASA is an ATP-dependent RNA helicase belonging to the DEAD-box family that, in many organisms, is specifically expressed in germ line cells throughout the life cycle, making it a powerful molecular marker to study germ line development. To obtain further information on germ line development in crustaceans, we cloned VASA cDNAs from three branchiopod species: water fleas Daphnia magna and Moina macrocopa, and brine shrimp Artemia franciscana. RNA helicase domains in branchiopod VASA were highly conserved among arthropod classes. However, N-terminal RNA-binding domains in branchiopod VASA were highly diverged and, unlike other arthropod VASA reported so far, possessed repeats of retroviral-type zinc finger (CCHC) motifs. Raising specific antibodies against Daphnia VASA revealed that the primordial germ cells (PGCs) in this organism segregate at a very early cleavage stage of embryogenesis in parthenogenetic and sexual eggs. Clusters of PGCs then start to migrate inside the embryo and finally settle at both sides of the intestine, the site of future gonad development. RNA analyses suggested that maternally supplied vasa mRNA was responsible for early VASA expression, while zygotic expression started during blastodermal stage of development.