The Effects of Repeated Administration of the Micellar Complex of Methylprednisolone on the Locomotor Activity of a Terrestrial Snails.

We studied the effects of repeated injections of methylprednisolone and its micellar complex with block-copolymer on locomotor activity of a terrestrial snail. It was shown that methylprednisolone solution injected into the hemolymph of the animal produced a direct effect on the muscle system of the animal as soon as 1 h after administration: it slowed down snail locomotion and reduced contractile activity of the foot muscles. The micellar complex of methylprednisolone with block-copolymer prevented this effect during the first 2 days of injection and negatively affected locomotion only in 2 days after injection, the decrease in locomotion in this case was not accompanied by a decrease in contractile activity of the foot muscle.

Metabolic effects of epinephrine on the crab Neohelice granulata.

Although widely known for their involvement in the control of carbohydrate and lipid metabolism of vertebrates, the participation of catecholamines (CAs) in the metabolism of invertebrates is less understood. This study was designed to identify the physiological role of Epinephrine (E) in the intermediary metabolism of the burrowing crab Neohelice granulata and how E regulates the metabolism in crabs fed with a high-carbohydrate (HC) or a high-protein (HP) diet. To answer these questions, we evaluated in vivo the effects of E injections on glucose and triglycerides in the hemolymph and tissue glycogen levels of crabs fed with HC or HP diet. An in vitro investigation was carried out to assess the direct effects of E on glycogenolysis, lipolysis and glycolysis pathways in the hepatopancreas, mandibular muscle and anterior and posterior gills of this crab. E injections increased glucose and did not affect triglycerides levels in the hemolymph of either group of crabs, and E decreased glycogen in the hepatopancreas and mandibular muscle only in HP crabs, suggesting that these effects may be mediated by the crustacean hyperglycemic hormone (CHH). When the tissues were incubated with different concentrations of E, the concentration of glucose released to the medium decreased in the hepatopancreas and posterior gills, while glucose oxidation increased in the posterior gills of HP crabs. Incubation with E did not alter any parameter in tissues of HC crabs. These effects suggest that E may be involved in the metabolic response to osmotic stress.

Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins.

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.

Effects of glyphosate and polyoxyethylenamine on growth and energetic reserves in the freshwater crayfish Cherax quadricarinatus (Decapoda, Parastacidae).

Freshwater crayfish Cherax quadricarinatus have a high commercial value and are cultured in farms where they are potentially exposed to pesticides. Therefore, we examined the sublethal effects of a 50-day exposure to glyphosate acid and polyoxyethylenamine (POEA), both alone and in a 3:1 mixture, on the growth and energetic reserves in muscle, hepatopancreas and hemolymph of growing juvenile crayfish. Exposure to two different glyphosate and POEA mixtures caused lower somatic growth and decreased muscle protein levels. These effects, caused by both compounds interacting in the mixture, could also be synergistic because they were expressed even at the lowest concentration. The decrease in protein levels could be related to the greater use of other energy reserves. This hypothesis is supported by the decrease in muscle glycogen stores due to glyphosate exposure and the decrease in lipid reserves associated with exposure to POEA.

Impacts of microplastic fibres on the marine mussel, Mytilus galloprovinciallis.

Tumble dryer lint has been employed as a surrogate for synthetic and processed (microplastic) fibres discharged to the environment from laundering activities and exposed to marine mussels (Mytilus galloprovinciallis) in controlled experiments for a period of 7 d. A range of biological responses at different levels of organisation were subsequently determined, with copper employed concurrently as a positive control. Physiological changes were assessed from measurements of clearance rate, histopathological effects were evaluated from abnormalities in (or injuries to) gill and digestive gland tissues, and genetic damage was determined by measuring DNA strand breaks using the comet assay. With increasing lint concentration (over the range 56-180 mg L-1) we observed a reduction in mean clearance rate, increasing extents of abnormality in both gills (e.g. deciliation and hypertrophy) and digestive gland (e.g. atrophy and necrosis), and an increase in damage to DNA. The precise causes of these effects are unclear but likely arise from both the fibrous material itself and from chemicals (e.g. additives and metals) that are mobilised from the polymers into seawater or the digestive tract. The latter assertion is consistent with an observed increase in the release of certain trace elements (e.g. zinc) into the exposure medium with increasing lint concentration. Although microfibre concentrations we employed are significantly greater than those typically encountered in the environment, the results indicate the potential for this type of material to exert a range of adverse effects on exposed marine animals.

Effects of exposure to waterborne polystyrene microspheres on lipid metabolism in the hepatopancreas of juvenile redclaw crayfish, Cherax quadricarinatus.

Previous research has identified microplastics as new environmental pollutants that are widely distributed in a variety of environments, including aquaculture environments. However, the potential hazard of microplastics to aquaculture animals, especially toward lipid metabolism involved with the survival and growth of aquatic animal, has not yet been investigated. In the present study, redclaw crayfish (Cherax quadricarinatus) were exposed to different concentrations of 200 nm-sized polystyrene microspheres (0, 0.5, and 5 mg/L) for 21 days, to investigate the effects of microplastics on lipid metabolism. After ingestion, the microplastics were distributed in the intestines and hepatopancreas, and appeared to inhibit the growth of Cherax quadricarinatus. Subsequently, the lipid levels in the hepatopancreas and hemolymph was detected, and found that after 21 days of exposure, the lipid content and free fatty acids in the hepatopancreas and hemolymph decreased significantly, and total cholesterol and triglycerides levels increased significantly in the hemolymph. This might have been caused by insufficient intake of exogenous fat. A significant decrease in lipase activity also supported this view. The activity of lipoprotein lipase related to lipolysis in the hepatopancreas increased significantly, while the activity of fatty acid synthase related to fat synthesis increased, and the activity of acetyl-CoA carboxylase decreased. These results indicated disturbed lipid metabolism in the hepatopancreas. The significant increase of lipid transport-related low-density lipoprotein indicated that the lipolytic capacity was higher than the lipid synthesis capacity. The expression levels of fatty acid metabolism-related genes FAD6 and FABP decreased significantly, indicating that the fatty acid utilization ability of hepatopancreas cells was inhibited, which was consistent with the results of enzyme activities. Thus, microplastics represent a potential hazard to redclaw crayfish, at least on lipid metabolism. This study provided basic data on the ecotoxicological effects of microplastics on crustaceans.

Exposure of bay scallop Argopecten irradians to micro-polystyrene: Bioaccumulation and toxicity.

Marine microplastic pollution poses a threat to aquatic organisms, including bivalves. In this study, we investigated the accumulation of microplastics and their elicited antioxidant stress response in the bay scallop Argopecten irradians. Scallops were exposed to 1 µm diameter micro-polystyrene (MP) beads at 10, 100, and 1000 beads/mL concentrations for a 7 day period. Bead presence in the digestive diverticula and defense responses in the digestive diverticula and hemolymph were measured at 1, 3, 5, and 7 days. The activity and expression of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and H2O2 in the digestive diverticula and/or hemolymph of scallops increased with microplastic concentration and exposure duration. These results suggest that microplastics can accumulate in the digestive diverticula of A. irradians, and that exposure to microplastics induces oxidative stress in bivalves. It is likely that exposure to high concentrations of micro- or nano-sized plastic particles could potentially have adverse effects in bivalves.

A 1H-NMR Based Study on Hemolymph Metabolomics in Eri Silkworm after Oral Administration of 1-Deoxynojirimycin.

We aimed to investigate whether 1-deoxynojirimycin (DNJ) modulates glycometabolism and has toxicity in Eri silkworm (Samia cynthia ricini, Saturniidae). In this paper, hemolymph metabolites were used to explore metabolic changes after oral administration of DNJ or mulberry latex and to characterize the biological function of DNJ at the metabolic and systemic levels. Hemolymph samples were collected from fourth-instar larvae of Eri silkworm and ex-vivo high-resolution 1H nuclear magnetic resonance (NMR) spectra were acquired from the collected hemolymph samples. Then the obtained spectra were analyzed by principal component analysis (PCA) and independent-samples t-test. Metabolic pattern recognition analysis of hemolymph samples indicated that the groups of 0.25% DNJ, latex, and the mixture of 0.5% DNJ and latex (1:1) were significantly different from the control group. Moreover, compared to the control group, the groups of 0.25% DNJ, latex, and the mixture of 0.5% DNJ and latex (1:1) showed the decreased levels of citrate, succinate, fumarate, malate, and glutamine in hemolymph, the groups of 0.25% DNJ and the mixture of 0.5% DNJ and latex (1:1) showed the increased levels of trehalose and lactate. In addition, mulberry leaves exude latex was highly toxic to Eri silkworm because rich unidentified high-molecular-weight factor (s) acted as toxic substances. In our results, latex caused 20 deaths among 50 fourth-instar larvae of Eri silkmoth, but DNJ or the mixture did not caused death. All these results suggest that DNJ has a positive impact on the reverse glycometabolism by modulating glycometabolism and inhibiting glucogenesis and energy metabolism. DNJ is a secure substance as a single-ingredient antidiabetic medicine due to its nontoxicity and bioactivity.

In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus.

Considerable evidence indicates that methyl farnesoate (MF) production by the crustacean mandibular organs is negatively regulated by neuropeptides from the sinus gland (SG) in the eyestalk. In the crab Cancer pagurus, two neuropeptides (MO-IH-1 and -2) have been isolated from the SG that inhibit MF synthesis by mandibular organs of female crabs in vitro. To test their activity in vivo, we treated eyestalk-ablated male crabs with SG extracts (SGEs) or MO-IH-1 and -2. SGEs reduced haemolymph levels of MF by 60-80%, while MO-IH-1 and -2 had little effect. Protease treatment of SGEs destroyed the in vivo activity, suggesting that the extract contains an additional peptide responsible for the in vivo activity. When separated by reversed-phase high performance liquid chromatography (HPLC), the in vivo activity eluted in fractions prior to MO-IH-1 and -2. When mandibular organs were removed from animals previously treated in vivo with these active fractions, they had reduced levels of MF synthesis and activity of farnesoic acid O-methyl transferase compared with mandibular organs from animals treated with saline. Together, these results indicate that the regulation of the crustacean mandibular organ is complex and may involve several SG compounds. Some of these compounds (i.e., MO-IH-1 and -2) act directly on the tissue while others affect the mandibular organ indirectly.

Characterization of hemolytic and cytotoxic Gallysins: a relationship with arylphorins.

Gallysin-1, an inducible effector protein in the protective response of Galleria mellonella larvae is a 75 kDa component of hemolytically active material (HAM) isolated from immune cell-free hemolymph. The sequence of the first 20 N-terminal amino acids of the antibacterial protein Gallysin-1 is identical to the predicted sequence of the first 20 amino acids of the Galleria arylphorin Lhp76 (larval hemolymph protein 76). A murine monoclonal antibody to the 20 amino acid N-terminal peptide of Gallysin-1 (GYPQYHYDVETRKLDPSLVN) provides additional evidence for a link between Gallysin-1 and Lhp76, and is used to characterize HAM further. HAM, initially characterized as a mixture of two proteins, Gallysin-1 and a 69 kDa component is now identified as a 450-500 kDa heteromultimer, designated Gallysin. In vivo levels of Gallysin rise during the effector phase of an induced immune response. The monoclonal antibody inhibits the hemolytic activity of Gallysin. In addition to a hemolytic activity for mammalian erythrocytes, Gallysin possesses a cytotoxic activity for the human tumor cell line, K562. Lipopolysaccharides (LPS) and a Pseudomonas aeruginosa vaccine induce a cytotoxic activity which reaches its maximum levels in the hemolymph early (2 hours post-vaccination) in the protective response. The partially purified cytotoxic material (Cyt-M) obtained from cell-free hemolymph collected 2 hours after vaccination has hemolytic activity and shows structural similarities to Gallysin and Lhp76. The previously established role of Gallysin-1 as an effector protein in the protective response of Galleria mellonella indicates that arylphorins may play a role in insect immune responses.

Cryoprotective role of polyols independent of the increase in supercooling capacity in diapausing adults of Pyrrhocoris apterus (Heteroptera: Insecta).

Diapausing cold-acclimated adults of the bug Pyrrhocoris apterus accumulate four 'winter' polyols, ribitol, sorbitol, mannitol and arabinitol, in total concentrations of up to 100 mM. The accumulation started only when the temperatures dropped below a threshold of +5 degrees C in laboratory acclimated insects. The supercooling capacity (SCP) was not affected by polyol accumulation and remained constant at approximately -17 degrees C. Cold hardiness, measured as survival time (Lt50) at -15 degrees C, increased from approximately 1 day to approximately 1 week in parallel with polyol accumulation. There was a tight correlation (r=0.98) between the concentration of 'winter' polyols in haemolymph and Lt50(-15). When a mixture of ribitol and sorbitol was injected into the haemolymph of the bugs acclimated to +5 degrees C, the concentration of polyols increased from 2.5 to 83.1 mM in haemolymph, or from 0.07 to 6.61 microg/mg of fresh weight in the whole body, the SCP remained unchanged and survival after exposure to -14 degrees C for 3 days increased approximately three-fold in comparison to untreated controls. Such results were interpreted as evidence for the cryoprotective role of accumulated polyols independent of the depression of SCP. Acclimation protocol using thermoperiod, mimicking daily temperature oscillations, resulted in moderately lower SCP, higher sum of polyols accumulated and significantly longer Lt50(-15) than at acclimation protocol with constant temperatures.

Silver nanowire exposure results in internalization and toxicity to Daphnia magna.

Nanowires (NWs), high-aspect-ratio nanomaterials, are increasingly used in technological materials and consumer products and may have toxicological characteristics distinct from nanoparticles. We carried out a comprehensive evaluation of the physicochemical stability of four silver nanowires (AgNWs) of two sizes and coatings and their toxicity to Daphnia magna . Inorganic aluminum-doped silica coatings were less effective than organic poly(vinyl pyrrolidone) coatings at preventing silver oxidation or Ag(+) release and underwent a significant morphological transformation within 1 h following addition to low ionic strength Daphnia growth media. All AgNWs were highly toxic to D. magna but less toxic than ionic silver. Toxicity varied as a function of AgNW dimension, coating, and solution chemistry. Ag(+) release in the media could not account for observed AgNW toxicity. Single-particle inductively coupled plasma mass spectrometry distinguished and quantified dissolved and nanoparticulate silver in microliter-scale volumes of Daphnia magna hemolymph with a limit of detection of approximately 10 ppb. The silver levels within the hemolymph of Daphnia exposed to both Ag(+) and AgNW met or exceeded the initial concentration in the growth medium, indicating effective accumulation during filter feeding. Silver-rich particles were the predominant form of silver in hemolymph following exposure to both AgNWs and Ag(+). Scanning electron microscopy imaging of dried hemolymph found both AgNWs and silver precipitates that were not present in the AgNW stock or the growth medium. Both organic and inorganic coatings on the AgNW were transformed during ingestion or absorption. Pathway, gene ontology, and clustering analyses of gene expression response indicated effects of AgNWs distinct from ionic silver on Daphnia magna .

Titanium dioxide nanoparticles relieve biochemical dysfunctions of fifth-instar larvae of silkworms following exposure to phoxim insecticide.

Phoxim insecticide is widely used in agriculture, which is toxic to insect pests and nontarget organisms. The phoxim poisoning is hard to prevent for silkworms. TiO(2) NPs have been widely applied in whitening, brightening foods, toothpaste or sunscreens, and orally-administered drugs. However, whether TiO(2) NPs can increase resistance of silkworm to phoxim poisoning has not been reported. The results demonstrated that added TiO(2) NPs significantly decreased reduction of protein, glucose and pyruvate contents, lactate dehydrogenase, succinate dehydrogenase and malate dehydrogenase activities, and attenuated increases of free amino acids, urea, uric acid and lactate levels, activities of protease, alanine aminotransferase and aspartate aminotransferase in the hemolymph of silkworms caused by phoxim exposure. From the present study, it is clearly evident that added TiO(2) NPs may relieve toxic impacts of phoxim insecticide on silkworm metabolism, which in turn may result in an increase in silk yield.