Biosynthesis of Long-Chain Polyunsaturated Fatty Acids in Marine Gammarids: Molecular Cloning and Functional Characterisation of Three Fatty Acyl Elongases.

Long-chain (C20-24) polyunsaturated fatty acids (LC-PUFAs) are essential nutrients that are mostly produced in marine ecosystems. Previous studies suggested that gammarids have some capacity to endogenously produce LC-PUFAs. This study aimed to investigate the repertoire and functions of elongation of very long-chain fatty acid (Elovl) proteins in gammarids. Our results show that gammarids have, at least, three distinct elovl genes with putative roles in LC-PUFA biosynthesis. Phylogenetics allowed us to classify two elongases as Elovl4 and Elovl6, as they were bona fide orthologues of vertebrate Elovl4 and Elovl6. Moreover, a third elongase was named as "Elovl1/7-like" since it grouped closely to the Elovl1 and Elovl7 found in vertebrates. Molecular analysis of the deduced protein sequences indicated that the gammarid Elovl4 and Elovl1/7-like were indeed polyunsaturated fatty acid (PUFA) elongases, whereas Elovl6 had molecular features typically found in non-PUFA elongases. This was partly confirmed in the functional assays performed on the marine gammarid Echinogammarus marinus Elovl, which showed that both Elovl4 and Elovl1/7-like elongated PUFA substrates ranging from C18 to C22. E. marinus Elovl6 was only able to elongate C18 PUFA substrates, suggesting that this enzyme does not play major roles in the LC-PUFA biosynthesis of gammarids.

Origin of the natural variation in the storage of dietary carotenoids in freshwater amphipod crustaceans.

Carotenoids are diverse lipophilic natural pigments which are stored in variable amounts by animals. Given the multiple biological functions of carotenoids, such variation may have strong implications in evolutionary biology. Crustaceans such as Gammarus amphipods store large amounts of these pigments and inter-population variation occurs. While differences in parasite selective pressure have been proposed to explain this variation, the contribution of other factors such as genetic differences in the gammarid ability to assimilate and/or store pigments, and the environmental availability of carotenoids cannot be dismissed. This study investigates the relative contributions of the gammarid genotype and of the environmental availability of carotenoids in the natural variability in carotenoid storage. It further explores the link of this natural variability in carotenoid storage with major crustacean immune parameters. We addressed these aspects using the cryptic diversity in the amphipod crustacean Gammarus fossarum and a diet supplementation protocol in the laboratory. Our results suggest that natural variation in G. fossarum storage of dietary carotenoids results from both the availability of the pigments in the environment and the genetically-based ability of the gammarids to assimilate and/or store them, which is associated to levels of stimulation of cellular immune defences. While our results may support the hypothesis that carotenoids storage in this crustacean may evolve in response to parasitic pressure, a better understanding of the specific roles of this large pigment storage in the crustacean physiology is needed.

Polysaccharide hydrolase of the hadal zone amphipods Hirondellea gigas.

Hirondellea species are common inhabitants in the hadal region deeper than 7,000 m. We found that Hirondellea gigas thrived in the Challenger Deep possessed polysaccharide hydrolases as digestive enzymes. To obtain various enzymes of other H. gigas, we captured amphipods from the Japan Trench, and Izu-Ogasawara (Bonin) Trench. A phylogenetic analysis based on the cytochrome oxidase I gene showed close relationships among amphipods, despite the geographic distance between the localities. However, several differences in enzymatic properties were observed in these H. gigas specimens. We also carried out RNA sequencing of H. gigas from the Izu-Ogasawara Trench. The cellulase gene of H. gigas was highly homologous to cellobiohydrolase of Glucosyl Hydrolase family 7 (GH7). On the other hand, enzymatic properties of H. gigas's cellulase were different from those of typical GH7 cellobiohydrolase. Thus, these results indicate that hadal-zone amphipod can be good candidates as the new enzyme resource.

Seasonal sensitivity of Gammarus pulex towards the pyrethroid cypermethrin.

The aquatic toxicity of insecticides like the pyrethroids have been discussed intensively over the recent years especially in relation to risk assessment and how seasonality may or may not affect the sensitivity of non-target organisms. To address this issue, the crustacean Gammarus pulex was collected once a month for 16 months and acclimated to 10 °C for four days before being exposed to a 90 min pulse of cypermethrin. In vitro cytochrome P450 activity, total lipid content, total protein content, and dry weight were measured in male and female gammarids from each sampling date and used along with the water temperature as variables for sensitivity prediction by Partial Least Squares (PLS) regression models. The 24 h EC50-values varied more than 30 fold across the sampling period from 0.21 ±â€¯0.05 µg L-1 (April 2015) to 6.60 ±â€¯3.46 µg L-1 (October 2015), indicating seasonal variances in the acute sensitivity of G. pulex towards cypermethrin. After 168 h of recovery this difference in EC50-values was reduced to seven-fold. In both male and female gammarids seasonal patterns were observed in the total lipid content and in vitro CYP P450 activity, which peaked in spring and fall, respectively. The current study shows the importance of reporting time of organism collection and experimental execution for risk assessment of pyrethroids as season is important for the acute sensitivity of G. pulex. We suggest prolonged acclimation times of sampled macroinvertebrates to constant laboratory conditions in order to even out possible seasonal differences in sensitivity.

Biochemical responses of Gammarus pulex to malachite green solutions decolorized by Coriolus versicolor as a biosorbent under batch adsorption conditions optimized with response surface methodology.

The current study was aimed to investigate the detoxifying and antioxidant enzyme response of Gammarus pulex exposed to malachite green (MG) after decolorization by Coriolus versicolor. Response surface methodology (RSM) was utilized to optimize the decolorization conditions of MG synthetic solutions by C. versicolor. Glutathione (GSH), malondialdehyde (MDA) levels and glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), cytochrome P450 1A1 (CYP1A1) activities in G. pulex exposed to undecolorized (A1) and decolorized (A2) MG synthetic solution during 24 and 96 h were tested by using ELISA method. SOD and GPX enzyme activity was increased after decolorization (p > 0.05). CAT enzyme activity was increased in A2 group during 24 h (p > 0.05) but decreased during 96 h (p < 0.05). GSH levels were increased in A2 group during 24 and 96 h (p < 0.05). GST, CYP1A1 enzyme activity and MDA levels were decreased after decolorization during 96 h (p < 0.05). In this study, GSH levels, CAT, GST and CYP1A1 activities in G. pulex approved the capability of C. versicolor in MG decolorization, optimized with RSM.

Effects of endosulfan, thiamethoxam, and indoxacarb in combination with atrazine on multi-biomarkers in Gammarus kischineffensis.

Studies addressing the toxicity of pesticides towards non-target organisms focus on the median lethal concentration and biochemical response of individual pesticides. However, when determining environmental risks, it is important to test the combined effects of pesticides, such as insecticides and herbicides, which are frequently used together in agricultural areas. Here we aimed to investigate the toxic effects of the combined use of the herbicide atrazine and the insecticides, endosulfan, indoxacarb, and thiamethoxam on Gammarus kischineffensis. To do this, we tested the activities of oxidative stress, detoxification, and neurotoxicity biomarkers. Compared to atrazine alone, we detected higher glutathione-S-transferase, catalase and superoxide dismutase activities (oxidative stress biomarkers) when atrazine was combined with either endosulfan or indoxacarb. However, higher IBR values were determined in organisms where pesticide mixtures were used according to individual use. Based on these results, mixtures of atrazine and other pesticides may cause synergistic effects and may be evidence of increased toxicity and oxidative stress.

Toxicological effects of fungicide mixtures on the amphipod Austrochiltonia subtenuis.

Approaches to assess the toxicity of mixtures often use predictive models with acute mortality as an endpoint at relatively high concentrations. However, these approaches do not reflect realistic situations where organisms could be exposed to chemical mixtures over long periods at low concentrations at which no significant mortalities occur. The present study investigated chronic effects of 2 common fungicides, Filan® (active ingredient [a.i]) boscalid) and Systhane™ (a.i. myclobutanil), on the amphipod Austrochiltonia subtenuis at environmentally relevant concentrations under laboratory conditions. Sexually mature amphipods were exposed singly and in combination to Filan (1, 10, and 40 µg a.i./L) and Systhane (3 µg a.i./L) over 28 d. Survival, growth, a wide range of reproduction endpoints, and glutathione-S-transferase (GST) activity were measured at the end of the experiment. Both fungicides had significant independent effects on male growth, sex ratio, and juvenile size. Filan mainly affected female growth and the number of embryos per gravid female, whereas Systhane mainly affected the time for females to become gravid. The combined effects of these fungicides on numbers of gravid females and juveniles were antagonistic, causing a 61% reduction in the number of gravid females and a 77% reduction in the number of juveniles produced at the highest concentrations (40 µg a.i./L of boscalid and 3 µg a.i./L of myclobutanil) compared with the controls. There were no significant effects on survival or GST activity. The present study demonstrated that the effects of mixtures were endpoint dependent and that using a variety of endpoints should be considered for a comprehensive understanding of mixture effects. Also, chronic studies are more informative than acute studies for environmentally relevant fungicide concentrations. Environ Toxicol Chem 2017;36:2651-2659. © 2017 SETAC.

Examining impacts of current-use pesticides in Southern Ontario using in situ exposures of the amphipod Hyalella azteca.

In situ exposures with Hyalella azteca were used to assess impacts of current-use pesticides in Southern Ontario, Canada. Exposures were conducted over 2 growing seasons within areas of high pesticide use: 1 site on Prudhomme Creek and 3 sites on Twenty Mile Creek. Three sites on Spencer Creek, an area of low pesticide use, were added in the second season. Surface water samples were collected every 2 wk to 3 wk and analyzed for a suite of pesticides. Hyalella were exposed in situ for 1 wk every 4 wk to 6 wk, and survival and acetylcholinesterase (AChE) activity were measured. Pesticides in surface waters reflected seasonal use patterns: lower concentrations in spring and fall and higher concentrations during summer months. Organophosphate insecticides (chlorpyrifos, azinphos methyl, diazinon) and acid herbicides (2,4-dichlorophenoxyacetic acid [2,4-D], mecoprop) were routinely detected in Prudhomme Creek, whereas neutral herbicides (atrazine, metolachlor) dominated the pesticide signature of Twenty Mile Creek. Spencer Creek contained fewer pesticides, which were measured at lower concentrations. In situ effects also followed seasonal patterns: higher survival and AChE activity in spring and fall, and lower survival and AChE activity during summer months. The highest toxicity was observed at Prudhomme Creek and was primarily associated with organophosphates. The present study demonstrated that current-use pesticides in Southern Ontario were linked to in situ effects and identified sites of concern requiring further investigation.

Changes in antitoxic defense systems of the freshwater amphipod Gammarus pulex exposed to BDE-47 and BDE-99.

Polybrominated diphenyl ethers (PBDEs) are emerging pollutants widely distributed in aquatic environment. Although the bioaccumulation of this compound has been well studied, few studies have investigated their impacts on antitoxic systems of invertebrates, considering both genders. Here, we have evaluated the effects of BDE-47 and BDE-99 congeners on the antitoxic defence systems of the freshwater amphipod Gammarus pulex, and especially on the multixenobiotic resistance (MXR) activity, as well as the activities of two antitoxic enzymes, the glutathione-S-transferase (GST) and the glutathione peroxidases (GPx). Results revealed that BDE-47 and BDE-99 have inhibited the MXR activity whatever the gammarid gender, which could lead to a reduction of the pollutant elimination from the organism. In addition, a gender-biased response and a congener-biased effect on the antitoxic enzymes activities were observed. Indeed, both BDE congener exposures increased the GST activity in males, whereas in females, only BDE-99 congener modified this activity by decreasing it. On the contrary, BDE exposures did not impact the GPx activity in females, while in males BDE-99 has increased it. Results of the present study highlight that a PBDE exposure at 0.1 µg L(-1) modify antitoxic enzymes activities differently according to gender, which could lead to a change in G. pulex sensitivity on the long term. Finally, this work confirms the ecotoxicological implication of gender in the pollutant toxicity assessment, in order to evaluate impact on populations.

Effects of two PBDE congeners on the moulting enzymes of the freshwater amphipod Gammarus pulex.

Polybrominated diphenyl ethers (PBDEs) are abundant in aquatic environment. However, only few studies have investigated their impacts on freshwater invertebrates. This work aimed to study the effects of BDE-47 and BDE-99 congeners on the chitobiase and chitinolytic enzymes activities of the freshwater amphipod Gammarus pulex, according to gender, PBDE concentration and time of exposure. In addition, the bioaccumulation of BDE-47 and BDE-99 were measured. Results revealed that females have bioaccumulated more PBDE than males, and BDE-99 was more accumulated than BDE-47. PBDE exposures for 96 h have caused chitobiase and chitinolytic enzymes inhibition. This study not only indicate the importance of taking into account various confounding factors (gender, congeners, concentration) to understand PBDE effects, but underline also disruptions of molting enzymes activities. These disturbances suggest effects on the gammarid development and reproduction, and consequently effects on the gammarid population, and on a larger scale, a dysfunction of the ecosystem.

Influence of molting and starvation on digestive enzyme activities and energy storage in Gammarus fossarum.

Among the many biological responses studied in ecotoxicology, energy-based biomarkers such as digestive enzyme activities and energy reserves appear to be useful predictive tools for detecting physiological disturbances in organisms. However, the use of these biological responses as biomarkers could be limited by the effects of confounding factors (biotic and abiotic) and physiological processes, such as the reproductive cycle. Thus, the optimal use of these biomarkers will be facilitated by understanding the effects of these factors on the energy metabolism of the sentinel species being studied. We considered abiotic factors (temperature and conductivity) in a previous study, whereas the present study investigated the effects of gender, the female reproductive stage, and food availability on the digestive enzyme activities and energy storage of Gammarus fossarum. The results indicated that, during the female reproductive cycle, the activities of digestive enzymes (amylase, cellulase, and trypsin) decreased significantly, whereas the levels of reserves (proteins, lipids, and sugar) increased until the last premolt stage. Restricted food diets only led to decreased amylase activities in both sexes. Food starvation also induced a decrease in the energy outcomes in females, whereas there were no effects in males. In general, the biochemical (digestive enzyme activities) and physiological (energy reserves) responses were more stable in males than in females. These results support the use of males fed ad libitum to limit the effects of confounding factors when using these energy biomarkers in Gammarus fossarum during biomonitoring programs.

Cholinesterase activity on Echinogammarus meridionalis (Pinkster) and Atyaephyra desmarestii (Millet): characterisation and in vivo effects of copper and zinc.

Metals are released into freshwater ecosystems from natural and anthropogenic sources, compromising their structural and functional equilibrium. As early warning tools, cholinesterases (ChEs) are usually used to assess the effects of organophosphate and carbamate pesticides, but are also known to be inhibited by metals. The objectives of this work were to characterise the activity of ChE present in the amphipod Echinogammarus meridionalis and the shrimp Atyaephyra desmarestii and to evaluate the in vivo effects of the metals copper and zinc in their ChE activity. To achieve this, firstly the activity of ChE forms were characterised using different in vitro assays with substrates and selective inhibitors. Then, the in vivo effects of 48 h exposures to increasing concentrations of copper and zinc on ChE activity were determined. The ChE form present in both species was acetylcholinesterase (AChE) since both revealed preference for the acetylthiocholine iodide substrate, total inhibition with eserine, the inhibitor of ChEs, and with 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, the specific inhibitor of AChE, and presented insensitivity to iso-OMPA, a specific inhibitor of butyrylcholinesterase. The activity of ChEs was inhibited by zinc exposures in the amphipod species, but was not affected by copper. Exposure to copper and zinc did not affect ChEs activity in the shrimp at the concentrations tested. This work is a relevant contribution as foundation for the use of AChE in freshwater crustaceans in further studies including biomonitoring campaigns in different contamination scenarios.

Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum.

The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms' digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms' health and the chemical quality of aquatic ecosystems.

Exposure to contaminants exacerbates oxidative stress in amphipod Monoporeia affinis subjected to fluctuating hypoxia.

Fitness and survival of an organism depend on its ability to mount a successful stress response when challenged by exposure to damaging agents. We hypothesized that co-exposure to contaminants may exacerbate oxidative stress in hypoxia-challenged benthic animals compromising their ability to recover upon reoxygenation. This was tested using the amphipod Monoporeia affinis exposed to hypoxia followed by reoxygenation in sediments collected in polluted and pristine areas. In both sediment types, oxygen radical absorbance capacity (ORAC) and antioxidant enzyme activities [superoxide dismutase (SOD) and catalase (CAT)] increased during hypoxia, suggesting that M. affinis has a strategy of preparation for oxidative stress that facilitates recovery after a hypoxic episode. Exposure to contaminants altered this anticipatory response as indicated by higher baselines of ORAC and SOD during hypoxia and no response upon reoxygenation. This coincided with significantly elevated oxidative damage evidenced by a marked reduction in glutathione redox status (ratio of reduced GSH/oxidized GSSG) and an increase in lipid peroxidation (TBARS levels). Moreover, RNA:DNA ratio, a proxy for protein synthetic activity, decreased in concert with increased TBARS, indicating a linkage between oxidative damage and fitness. Finally, inhibited acetylcholinesterase (AChE) activity in animals exposed to contaminated sediments suggested a neurotoxic impact, whereas significant correlations between AChE and oxidative biomarkers may indicate connections with redox state regulation. The oxidative responses in pristine sediments suggested a typical scenario of ROS production and removal, with no apparent oxidative damage. By contrast, co-exposure to contaminants caused greater increase in antioxidants, lipid peroxidation, and slowed recovery from hypoxia as indicated by CAT, GSH/GSSG, TBARS and AChE responses. These results support the hypothesized potential of xenobiotics to hamper ability of animals to cope with fluctuating hypoxia. They also emphasize the importance of understanding interactions between antioxidant responses to different stressors and physiological mechanisms of oxidative damage.

Optical diagnostic test of stress conditions of aquatic organisms.

Global climate change has become a dire reality and its impact is expected to rise dramatically in the near future. Combined with the day-to-day human activities the climatic changes heavily affect the environment. In particular, a global temperature increase accompanied by a number of anthropogenic chemicals falling within the freshwater ecosystem results in a dramatic enhancement of the overall stress for most aquatic organisms. This leads to a significant shift in the species inventory and potential breakdown of the water ecosystem with severe consequences for local economies and water supply. In order to understand and predict the influence of climatic changes on the physiological and biochemical processes that take place in living aquatic organisms we explore the application of optical spectroscopy for monitoring and quantitative assessment of antioxidant enzymes activity in benthic amphipods of Lake Baikal. We demonstrate that the changes of the enzymes activity in Baikal amphipods undergoing thermal and/or hypoxia stress can be observed and documented by UV and optical spectroscopy both in vivo and in vitro.

Biochemical response of amphipods (Gammarid: Paramorea) in a sediment laboratory exposure from Ushuaia Bay, Beagle Channel.

A coastal system (Ushuaia Bay, Argentina) impacted by anthropogenic activities was studied by the response of local amphipods (Parmorea sp., Gammaridae) to the exposure of coastal sediments in a laboratory assay. Four coastal areas with different loadings of contaminants and one considered as reference were studied. Organic matter, carbohydrates, proteins and heavy metals were measured in sediment samples. Organisms were exposed to sediments for seven days and catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE) and lipid peroxidation (LPO) were measured afterward. Amphipods exhibited an activation of GST and inhibition of AChE in most impacted areas. Principal Component Analysis (PCA) was conducted in order to associate the biological responses with sediment metal concentration and its eutrophicated status. Levels of Cd and Cr were associated with the inhibition of AChE and with the enhancement of GST. CAT and LPO were enhanced in most areas, but no link was found with the contaminants studied by PCA, suggesting that other parameters present in sediments not included in the PCA affect the amphipods. The most impacted area corresponds to Nautical Club station, with a highly eutrophicated status and high content of metals, where amphipods after the exposure were affected in a biochemical level.

Sub-littoral and supra-littoral amphipods respond differently to acute thermal stress.

Thermal tolerance was determined in two closely related amphipod species from contrasting environments (sub-littoral and supra-littoral zones of the sea) using HSP expression and the activity of antioxidant enzymes. The levels of HSP70 and small HSPs present in untreated control animals were higher in the supra-littoral Orchestia gammarellus than in the sub-littoral Gammarus oceanicus. Under the acute thermal stress, HSP levels increased less strongly in O. gammarellus than in G. oceanicus. Activities of antioxidant enzymes peroxidase, catalase and glutathione S-transferase, were more pronounced in the supra-littoral O. gammarellus then in the sub-littoral G. oceanicus. We conclude that the environmental temperature regime modifies key cellular defense mechanisms in amphipods. Higher levels of constitutive HSP synthesis and higher levels of antioxidant enzymes in the supra-littoral species likely reflects adaptation to this highly thermally variable environment.

Biological invasion and parasitism: invaders do not suffer from physiological alterations of the acanthocephalan Pomphorhynchus laevis.

Biological invasions expose parasites to new invasive hosts in addition to their local hosts. However, local parasites are often less successful in infecting and exploiting their new hosts. This may have major consequences for the competitive ability of hosts, and finally on the fate of the parasite-host community. In Burgundy (Eastern France), the acanthocephalan parasite, Pomphorhynchus laevis, infects 2 amphipod species living in sympatry: the native Gammarus pulex and the invasive Gammarus roeseli. While P. laevis affects the behaviour and the immunity of G. pulex, G. roeseli seems unaffected by the infection. In this study, we examined in detail the ability of the parasite to affect the immune system and resource storage of both gammarid species. We found that the infection was associated with a general decrease of the prophenoloxidase activity, haemocyte density, resistance to an artificial bacterial infection and level of sugar reserves in G. pulex, but not in G. roeseli. These results demonstrate a differential ability of P. laevis to exploit its local and its invasive gammarid hosts. Potential mechanisms of these differential physiological alterations and their potential consequences on the coexistence of both gammarid species in sympatry are discussed.

Different natural organic matter isolates cause similar stress response patterns in the freshwater amphipod, Gammarus pulex.

BACKGROUND, AIM, AND SCOPE: Dissolved humic substances (HSs) are exogenous stressors to aquatic plants and animals which activate a variety of transcriptional and biochemical reactions or block photosynthesis. While there are consistent indications which structures may lead to reduced photosynthetic activity, there is much less clear information available on which HS structures or building blocks act as stressors in animals. Consequently, this work was designed to comparatively study the impact of natural organic matter (NOMs) from different sources on major anti-stress mechanisms in one single animal. We utilized major antioxidant responses and relative expression levels of stress proteins (small HSPs and HSP70) and expected that different HSs provoke different response patterns. MATERIALS AND METHODS: We tested the freshwater amphipod Gammarus pulex which was collected from several shallow creeks in Northern Germany. All specimens were maintained in aerated 5-L tanks with filtered water from their natural environment at 10 degrees C with prior acclimation. Animals were fed ad libitum with a commercial preparation once every second day. The exposure water was exchanged with the same frequency. NOMs were isolated from three different sources: two from small brown-water lakes in Northern Germany by reverse osmosis and the third one as an aqueous extract from a black layer of a Brazilian sandbar soil (State of Rio de Janeiro). The rationale was to apply NOMs of contrasting quality. Chemical fingerprint features of the NOMs were taken by high-performance size exclusion chromatography. As stress parameters in the animals, the activities of peroxidase and catalase were recorded quantitatively, and stress proteins, HSP70, as well as small alpha-crystalline HSPs were analyzed semiquantitatively. RESULTS: The three NOMs clearly differed in molecular masses, humic substance contents, the moieties of polysaccharides, and low-molecular-weight substances. With the exception of one short-term response, the peroxidase activity increased after 3 to 12 h exposure, whereas the catalase activity did not show any significant modulation. With one exception, the stress protein expression increased after 30 min exposure in a biphasic pattern, and the sHSPs responded less strongly than HSP70. DISCUSSION: Although the quality of the exposed NOMs differs significantly, a rather uniform response pattern appears in the animals. Obviously, the contrasting contents of HSs and polysaccharides did not affect the anti-stress response of the exposed gammarids which is in contrast to previous lifespan studies with Caenorhabditis elegans. Furthermore, all NOM sources led to increased contents of both HSP70 and sHSPs. To the best of our knowledge, this is the first protein study to show that also small HSPs are expressed when the animals are exposed against humic material. CONCLUSIONS: Since the response patterns of the exposed gammarids, in contrast to the initial hypothesis, are rather uniform and since HSs are parts of life on Earth, we furthermore presume that they may have been a primordial exogenous trigger for the development of anti-stress systems in exposed organisms. RECOMMENDATIONS AND PERSPECTIVES: Effect studies of chemical stresses on organisms should consider exposure to both natural triggers and xenobiotic compounds in low concentrations--in order to prospectively differentiate between these triggers and, subsequently, classify them.

Direct evidence of the cyclooxygenase pathway of prostaglandin synthesis in arthropods: genetic and biochemical characterization of two crustacean cyclooxygenases.

Prostaglandins, well-known lipid mediators in vertebrate animals, have also shown to play certain regulatory roles in insects and other arthropods acting on reproduction, immune system and ion transport. However, knowledge of their biosynthetic pathways in arthropods is lacking. In the present study, we report the cloning and expression of cyclooxygenase (COX) from amphipod crustaceans Gammarus spp and Caprella spp. The amphipod COX proteins contain key residues shown to be important for cyclooxygenase and peroxidase activities. Differently from all other known cyclooxygenases the N-terminal signal sequence of amphipod enzymes is not cleaved during protein expression in mammalian cells. The C-terminus of amphipod COX is shorter than that of mammalian isoforms and lacks the KDEL(STEL)-type endoplasmic reticulum retention/retrieval signal. Despite that, amphipod COX proteins are N-glycosylated and locate similarly to the vertebrate COX on the endoplasmic reticulum and nuclear envelope. Both amphipod COX mRNAs encode functional cyclooxygenases that catalyze the transformation of arachidonic acid into prostaglandins. Using bioinformatic analysis we identified a COX-like gene from the human body louse Pediculus humanus corporis genome that encodes a protein with about 30% sequence identity with human COX-1 and COX-2. Although the COX gene is known to be absent from genomes of Drosophila sp., Aedes aegypti, Bombyx mori, and other insects, our studies establish the existence of the COX gene in certain lineages within the insect world.