Concomitant investigation of crustacean amphipods lipidome and metabolome during the molting cycle by Zeno SWATH data-independent acquisition coupled with electron activated dissociation and machine learning.

BACKGROUND: DIA (Data-Independent Acquisition) is a powerful technique in Liquid Chromatography coupled with high-resolution tandem Mass Spectrometry (LC-MS/MS) initially developed for proteomics studies and recently emerging in metabolomics and lipidomics. It provides a comprehensive and unbiased coverage of molecules with improved reproducibility and quantitative accuracy compared to Data-Dependent Acquisition (DDA). Combined with the Zeno trap and Electron-Activated Dissociation (EAD), DIA enhances data quality and structural elucidation compared to conventional fragmentation under CID. These tools were applied to study the lipidome and metabolome of the freshwater amphipod Gammarus fossarum, successfully discriminating stages and highlighting significant biological features. Despite being underused, DIA, along with the Zeno trap and EAD, holds great potential for advancing research in the omics field. RESULTS: DIA combined with the Zeno trap enhances detection reproducibility compared to conventional DDA, improving fragmentation spectra quality and putative identifications. LC coupled with Zeno-SWATH-DIA methods were used to characterize molecular changes in reproductive cycle of female gammarids. Multivariate data analysis including Principal Component Analysis and Partial Least Square Discriminant Analysis successfully identified significant features. EAD fragmentation helped to identify unknown features and to confirm their molecular structure using fragmentation spectra database annotation or machine learning. EAD database matching accurately annotated five glycerophospholipids, including the position of double bonds on fatty acid chain moieties. SIRIUS database predicted structures of unknown features based on experimental fragmentation spectra to compensate for database incompleteness. SIGNIFICANCE: Reproducible detection of features and confident identification of putative compounds are pivotal stages within analytical pipelines. The DIA approach combined with Zeno pulsing enhances detection sensitivity and targeted fragmentation with EAD in positive polarity provides orthogonal fragmentation information. In our study, Zeno-DIA and EAD thereby facilitated a comprehensive and insightful exploration of pertinent biological molecules associated with the reproductive cycle of gammarids. The developed methodology holds great promises for identifying informative biomarkers on the health status of an environmental sentinel species.

The trematode Podocotyle atomon modulates biochemical responses of Gammarus locusta to thermal stress but not its feeding rate or survival.

Although parasitism is one of the most common species interactions in nature, the role of parasites in their hosts' thermal tolerance is often neglected. This study examined the ability of the trematode Podocotyle atomon to modulate the feeding and stress response of Gammarus locusta towards temperature. To accomplish this, infected and uninfected females and males of Gammarus locusta were exposed to temperatures (2, 6, 10, 14, 18, 22, 26, 30 °C) for six days. Shredding (change in food biomass) and defecation rates (as complementary measure to shredding rate) were measured as proxies for feeding activity. Lipid and glycogen concentrations (energy reserves), catalase (oxidative stress indicator), and phenoloxidase (an immunological response in invertebrates) were additionally measured. Gammarid survival was optimal at 10 °C as estimated by the linear model and was unaffected by trematode infection. Both temperature and sex influenced the direction of infection effect on phenoloxidase. Infected females presented lower phenoloxidase activity than uninfected females at 14 and 18 °C, while males remained unaffected by infection. Catalase activity increased at warmer temperatures for infected males and uninfected females. Higher activity of this enzyme at colder temperatures occurred only for infected females. Infection decreased lipid content in gammarids by 14 %. Infected males had significantly less glycogen than uninfected, while infected females showed the opposite trend. The largest infection effects were observed for catalase and phenoloxidase activity. An exacerbation of catalase activity in infected males at warmer temperatures might indicate (in the long-term) unsustainable, overwhelming, and perhaps lethal conditions in a warming sea. A decrease in phenoloxidase activity in infected females at warmer temperatures might indicate a reduction in the potential for fighting opportunistic infections. Results highlight the relevance of parasites and host sex in organismal homeostasis and provide useful insights into the organismal stability of a widespread amphipod in a warming sea.

A new species of Gammarus (Crustacea, Amphipoda, Gammaridae) from South Korea.

A new species of freshwater gammarid, Gammarussomaemulensis sp. nov., was collected from Somaemuldo Island, South Korea. This new species is morphologically characterised by the absence of calceoli in antenna 2, absence of anteroproximal setae on bases in pereopods 6 and 7, outer ramus in uropod 3 with plumose setae on both margins, and a small terminal article of the outer ramus, which is shorter than adjacent spines. A description of the new species and morphological differences from related species are provided in the text. The new species was also compared to related species using partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Genetic distances of COI sequences between the new species and related species, consisting of 21.5-26.3% difference, support Gammarussomaemulensis sp. nov. as a valid species. Additionally, a key to identifying Gammarus species in South Korea is provided.

Marine amphipods (Parhyale hawaiensis) as an alternative feed for the lined seahorse (Hippocampus erectus, Perri 1810): nutritional value and feeding trial.

Finding new alternatives to traditional live preys such as Artemia and rotifers, which do not always promote optimal fish growth and survival, is required for the successful aquaculture of highly specialized predatory species, including seahorses. The present study assessed the nutritional value of an interesting marine amphipod (Parhyale hawaiensis), and evaluates through a feeding trial its potential use as a natural prey for 10-months lined seahorse, Hippocampus erectus. P. hawaiensis showed high levels of valuable lipids (20.4-26.7% on dry matter basis) and polyunsaturated fatty acids (PUFAs) ( 26.4-41% of total FAs), including the long-chain PUFAs (LC-PUFAs) arachidonic acid (ARA) (2.9-7.7%), eicosapentaenoic acid (EPA) (4.3-6.5%) and docosahexaenoic acid (DHA) (2.1-6.2%). A comparison between wild-captured and cultured amphipods revealed a significant improvement of the amphipod FA profile in terms of DHA%, total omega-3 (n3) FAs and n3/n6 ratio when employing both a conventional amphipod culture based on a commercial shrimp diet, and, to a lesser extent, a large (3,500 L) biofloc system. Seahorses fed with frozen/wild amphipods, either singly or in combination with Artemia enriched with Super Selco® (INVE Aquaculture, Belgium) for 57 days, substantially improved seahorse growth and FA profiles in terms of ARA, EPA and DHA%, including indices associated to marine sources, such as Σn3 and n3/n6, compared to a diet based solely on enriched Artemia. These results support the use of marine amphipods as an alternative food organism for juvenile H. erectus and suggest a potential use for general marine aquaculture.

The complete mitogenome of freshwater gammarid Grandidierella taihuensis (Crustacea: Amphipoda).

The freshwater gammarid Grandidierella taihuensis is an important composition of benthic community. In this study, the complete mitogenome sequences of G. taihuensis are determined using next-generation and PacBio long-read sequencing. The mitogenome of G. taihuensis is 15,099 bp in size, and consisted of 13 protein-coding genes, two ribosomal RNA genes, 22 tRNA genes, and a putative control region. Gene arrangement is as same as that of G. rubroantennata. The base composition of the entire mitogenome showed a conspicuous A + T bias of 69.4%. The mitogenome data produced in this study provides a useful resource for future evolutionary and ecological studies.

Temperature, not salinity, drives impact of an emerging invasive species.

Biological invasions are a growing ecological and socioeconomic problem worldwide. While robust predictions of impactful future invaders are urgently needed, understandings of invader impacts have been challenged by context-dependencies. In aquatic systems in particular, future climate change could alter the impacts of invasive non-native species. Widespread warming coupled with sea freshening may exacerbate ecological impacts of invaders in marine environments, compromising ecosystem structure, function and stability. We examined how multiple abiotic changes affect the potential ecological impact of an emerging invasive non-native species from the Ponto-Caspian region - a notorious origin hotspot for invaders, characterised by high salinity and temperature variation. Using a comparative functional response (feeding rates across prey densities) approach, the potential ecological impacts of the gammarid Pontogammarus maeoticus towards native chironomid prey were examined across a range of current and future temperature (18, 22 °C) and salinity (14, 10, 6, 2 ppt) regimes in a factorial design. Feeding rates of P. maeoticus on prey significantly increased with temperature (by 60%), but were not significantly affected by salinity regime. Gammarids displayed significant Type II functional responses, with attack rates not significantly affected by warming across all salinities. Handling times were, however, shortened by warming, and thus maximum feeding rates significantly increased, irrespective of salinity regime. Functional responses were significantly different following warming at high prey densities under all salinities, except under the ambient 10 ppt. Euryhalinity of invasive non-native species from the Ponto-Caspian region thus could allow sustained ecological impacts across a range of salinity regimes. These results corroborate high invasion success and field impacts of Ponto-Caspian gammarids in brackish through to freshwater ecosystems. Climate warming will likely worsen the potential ecological impact of P. maeoticus. With invasions growing worldwide, quantifications of how combined elements of climate change will alter the impacts of emerging invasive non-native species are needed.

Generation studies on benthic amphipod-Quadrivisio bengalensis, Stebbing (Gammaridae) from the Kochin Estuary, Southwest coast of India.

Quadrivisio bengalensis (Stebbing Records of Indian Museum, 1, 159-161, 1907), a eurythermal (26.5-32.2 °C) and euryhaline (0.10-26.2 psu) tropical species, makes a profound contribution as a fodder organism to the benthic biomass of tropical backwaters. Studies on life span, variations in broods, fecundity, sex ratio, brooding behaviour, brood stock assessment, growth rate, antennal segments as an index of growth, moulting frequency, mortality and starvation resistance of Q. bengalensis were made for the first time under controlled laboratory conditions of 12-h photo period for 252 days on 8 pairs of specimens (male and female) collected from the field and their successive broods. The life span of females was found to be higher (maximum 220 days) than males (maximum 175 days). Number of broods varied between 5 and 15, depending on the "status of the brood" (early or late). The maximum number of juveniles in a single brood was 24 and that by a single female over the entire life span was 211. The incubation time varied between 6 and 9 days and the duration of moults (8-18 days) was found to increase with the age of the animals. Maximum growth is usually attained by the offspring arising from the 5th to 7th broods. The 4th to 7th broods were the optimal broods for the maximum number of females attaining maturity. For broods 3 to 7 of the parental set, probability of extinction (ξ) calculated on applying stochastic branching process to generation studies for the first time showed an increasing trend with number of broods while a decreasing trend for ξ for 8th and 9th broods, with least ξ for broods 5 to 7 of the 5th, 6th and 7th generations, suggesting life span and fecundity rates as functions of the "brood status" (early or late). Whether it is true with higher crustaceans remains to be explored.

Amphipod parasites may bias results of ecotoxicological research.

Amphipods are commonly used test organisms in ecotoxicological studies. Nevertheless, their naturally occurring parasites have mostly been neglected in these investigations, even though several groups of parasites can have a multitude of effects, e.g. on host survival, physiology, or behavior. In the present review, we summarize the knowledge on the effects of Microsporidia and Acanthocephala, 2 common and abundant groups of parasites in amphipods, on the outcome of ecotoxicological studies. Parasites can have significant effects on toxicological endpoints (e.g. mortality, biochemical markers) that are unexpected in some cases (e.g. down-regulation of heat shock protein 70 response in infected individuals). Therefore, parasites can bias the interpretation of results, for example if populations with different parasite profiles are compared, or if toxicological effects are masked by parasite effects. With the present review, we would like to encourage ecotoxicologists to consider parasites as an additional factor if field-collected test organisms are analyzed for biomarkers. Additionally, we suggest intensification of research activities on the effects of parasites in amphipods in connection with other stressors to disentangle parasite and pollution effects and to improve our understanding of parasite effects in this host taxon.

Comparison in the response of three European Gammarid species exposed to the growth regulator insecticide fenoxycarb.

Growth regulator insecticides with juvenoid activity can affect the development and reproduction of non-target organisms such as crustaceans. In this perspective, our previous studies revealed deleterious effects of the juvenoid fenoxycarb at 5 µg L-1 on the embryogenesis and at 50 µg L-1 on the reproductive behavior of the amphipod Gammarus fossarum. In the present study, to determine whether data generated with one amphipod species can be extended to other gammarid species, we tested the effects of a 5 µg L-1 fenoxycarb exposure on three European amphipod species: G. fossarum, Gammarus roeseli, and Echinogammarus longisetosus. We exposed individually 60 freshly fertilized females to fenoxycarb throughout the entire oogenesis/embryogenesis cycle (i.e., 19 days). In newborn individuals from exposed embryos, we measured both pigmentation and lipid reserve impairments while in exposed females, we observed reproductive behavior. At 5 µg L-1 fenoxycarb, reproductive behavior was only altered in G. fossarum. This study demonstrates the variability of the toxic response among the three gammaridae species, underlining the need for acquiring data with a broad phylogenetic representation to better predict toxic effects on freshwater ecosystems.

Wastewater alters feeding rate but not vitellogenin level of Gammarus fossarum (Amphipoda).

Wastewater treatment plant (WWTP) effluents release complex mixtures of organic and inorganic micropollutants, including endocrine disrupting compounds, into receiving water bodies. These substances may cause adverse effects in aquatic communities as well as in ecosystem functions they provide. The aim of this study was to determine the potential impact of secondary treated wastewater released into a small Swiss stream on leaf litter decomposition based on feeding rates of the amphipod shredder Gammarus fossarum measured in situ. Additionally, endocrine disrupting effects downstream of the WWTP were investigated by measuring vitellogenin (vg) induction in male gammarids exposed in situ, as well as estrogen receptor activation using the Yeast Estrogen Screen (YES) involving passive sampler and grab water sample extracts. Extracts were also analysed for 424 organic micropollutants and selected transformation products. Gammarid feeding rate was significantly reduced 100, 200 and 400 m downstream of the WWTP effluent relative to the upstream site. While YES results showed significantly elevated estrogenicity at downstream sites, vg production in male gammarids was not induced. A laboratory experiment, in which gammarids were exposed to WWTP effluent, supported this observation. These results, hence, suggest that treated wastewater released into aquatic ecosystems impairs the ecosystem function of leaf litter decomposition. Vg levels in male gammarids measured by UPLC-MS/MS did, however, not alter.

Variation in the immune state of Gammarus pulex (Crustacea, Amphipoda) according to temperature: Are extreme temperatures a stress?

Temperature is known to impact host-parasite interactions in various ways. Such effects are often regarded as the consequence of the increased metabolism of parasites with increasing temperature. However, the effect of temperature on hosts' immune system could also be a determinant. Here we assessed the influence of temperature on the immunocompetence of the crustacean amphipod Gammarus pulex. Amphipods play a key ecological role in freshwater ecosystems that can be altered by several parasites. We investigated the consequences of three weeks of acclimatization at four temperatures (from 9 °C to 17 °C) on different immunological parameters. Temperature influenced both hemocyte concentration and active phenoloxidase enzymatic activity, with lower values at intermediate temperatures, while total phenoloxidase activity was not affected. In addition, the ability of gammarids to clear a bacterial infection was at the highest at intermediate temperatures. These results suggest a dysregulation of the immune system of gammarids in response to stress induced by extreme temperature.

Additive effects of temperature and infection with an acanthocephalan parasite on the shredding activity of Gammarus fossarum (Crustacea: Amphipoda): the importance of aggregative behavior.

Climate change can have critical impacts on the ecological role of keystone species, leading to subsequent alterations within ecosystems. The consequences of climate change may be best predicted by understanding its interaction with the cumulative effects of other stressors, although this approach is rarely adopted. However, whether this interaction is additive or interactive can hardly be predicted from studies examining a single factor at a time. In particular, biotic interactions are known to induce modifications in the functional role of many species. Here, we explored the effect of temperature on leaf consumption by a keystone freshwater shredder, the amphipod Gammarus fossarum. This species is found at high densities in the wild and relies on aggregation as an antipredator behavior. In addition, gammarids regularly harbor acanthocephalan parasites that are known to induce multiple effects on their hosts, including modifications on their functional role. We thus assessed the cumulative effect of both intraspecific interactions and parasitism. Consumption tests were conducted on gammarids, either naturally infected with Pomphorhynchus tereticollis or uninfected, feeding alone or in groups. Our results show that increased temperatures induced a significant increase in consumption, but only to a certain extent. Interestingly, consumption at the highest temperature depended on amphipod density: Whereas a decrease was observed for single individuals, no such effect on feeding was observed for individuals in groups. In addition, infection by acanthocephalan parasites per se significantly negatively impacted the shredding role of gammarids. Overall, the combined effects of parasitism and temperature appeared to be additive. Thus, future studies focusing on the impact of climate change on the functional role of keystone species may benefit from a multimodal approach under realistic conditions to derive accurate predictions.

Active biomonitoring for assessing effects of metal polluted sediment resuspension on gammarid amphipods during fluvial traffic.

The resuspension of polluted sediments by boat traffic could release substantial amounts of metals to the water column, affecting at the same time their bioavailability. In order to characterize the impact of sediment resuspensions on biota, caged amphipods have been deployed on three different channelized watercourses in Northern France. Firstly, the biological responses of transplanted freshwater gammarid amphipods, Gammarus fossarum, described by trace metal accumulation, feeding and reproduction activities were quite similar for the three water courses despite the differences of metal contamination and navigability. Secondly, the concentrations of metals accumulated in gammarids never exceeded the contamination thresholds previously defined for Co, Cu, Cr and Zn. Values were in the same order of magnitude whatever the studied site despite: (i) large differences noticed in the sediment quality and (ii) some concentrations in the overlying waters exceeding the Environmental Quality Standards (EQS) defined by the Water Framework Directive. Conversely, Pb was highly bioaccumulated with values systematically exceeding the threshold value whatever the site. Therefore, the impact of navigation cannot be proved and the difference between the 3 monitoring periods is rather attributed to environmental variability, probably linked to the seasonality. Moreover, this study also confirms that organisms sampled from a local population in the vicinity of the three studied watercourses could be used as test organisms, leading to similar results than the ones obtained with reference gammarids initially used for developing all the biological responses. This would simplify and then promote the development of studies based on gammarid amphipod, G. fossarum, as bioindicators.

A biodynamic model predicting waterborne lead bioaccumulation in Gammarus pulex: Influence of water chemistry and in situ validation.

Metals bioaccumulated in aquatic organisms are considered to be a good indicator of bioavailable metal contamination levels in freshwaters. However, bioaccumulation depends on the metal, the species, and the water chemistry that influences metal bioavailability. In the laboratory, a kinetic model was used to describe waterborne Pb bioaccumulated in Gammarus pulex. Uptake and elimination rate constants were successfully determined and the effect of Ca(2+) on Pb uptake was integrated into the model. Thereafter, accumulated Pb concentrations in organisms were predicted with the model and compared with those measured in native populations from the Seine watershed (France). The predictions had a good agreement with the bioaccumulation levels observed in native gammarids and particularly when the effect of calcium was considered. To conclude, kinetic parameters experimentally derived for Pb in G. pulex are applicable in environmental conditions. Moreover, the consideration of the water's chemistry is crucial for a reliable interpretation of bioaccumulation.

Invertebrates as indicators for chemical stress in sewage-influenced stream systems: toxic and endocrine effects in gammarids and reactions at the community level in two tributaries of Lake Constance, Schussen and Argen.

The present study investigates the impact of releases from waste water treatment plants and storm water overflow basins on gammarids and other macrozoobenthos. The study relates to a recent upgrading of a waste water treatment plant (Langwiese) at the Schussen river, an important tributary to Lake Constance. Samples were taken at different sites at the Schussen river upstream and downstream of a storm water overflow basin and the waste water treatment plant Langwiese and, in parallel, at the Argen river, a less polluted reference stream. We assessed the influence of water quality on the distribution of macrozoobenthos and on the health of gammarid populations by a variety of ecotoxicological methods including biomarkers prior to the expansion of the waste water treatment plant. Through histopathological studies, the impact of parasites on host tissue health was evaluated. Analyses of heat shock protein (hsp70) levels allowed us to draw conclusions about the proteotoxicity-related stress status of the organisms. Furthermore, gammarid populations from all sites were investigated in respect to sex ratio, parasitism rate, and fecundity. Macrozoobenthos community integrity was determined by means of the saprobic index and the abundance as well as by the number of taxa. In gammarids, the sex ratio was significantly shifted towards females, fecundity was significantly decreased, and the hsp70 level was significantly increased downstream of the waste water treatment plant Langwiese, compared to the upstream sampling site. Similarly, these effects could be detected downstream of three small storm water overflow basins. In the macrozoobenthos communities, the abundance of taxa, the number of taxa, the number of ephemeroptera, plecoptera, and trichoptera taxa (EPT-taxa), and the number of sensitive taxa decreased downstream of the storm water overflow basin Mariatal as well as downstream of the waste water treatment plant Langwiese. Our study showed, that waste water treatment plants and storm water overflow basins affected macroinvertebrate communities and the health of gammarids.

Combined effect of UV-irradiation and TiO2-nanoparticles on the predator-prey interaction of gammarids and mayfly nymphs.

Although nanoparticle production and application increases continuously, their implications in species interactions, especially in combination with other environmental stressors, are rarely assessed. Therefore, the present study investigated the influence of 2 mg/L titanium dioxide nanoparticles (nTiO2; <100 nm) on the interaction between the prey Ephemerella ignita (Ephemeroptera) and the predator Gammarus fossarum (Amphipoda) over 96 h considering UV-irradiation at field relevant levels (approximately 11.4 W/m(2)) as an additional environmental factor (n = 16). At the same time, gammarid's consumption of an alternative food source, i.e. leaf discs, was assessed. All endpoints covered were not affected by nTiO2 alone, while the combination of nTiO2 and UV caused a reduction in gammarid's predation (68%), leaf consumption (60%) and body weight (22%). These effects were most likely triggered by the UV-induced formation of reactive oxygen species by nTiO2. The present study, hence, highlights the importance to cover UV-irradiation during the risk assessment of nanoparticles.