Electron-activated dissociation (EAD) for the complementary annotation of metabolites and lipids through data-dependent acquisition analysis and feature-based molecular networking, applied to the sentinel amphipod Gammarus fossarum.

The past decades have marked the rise of metabolomics and lipidomics as the -omics sciences which reflect the most phenotypes in living systems. Mass spectrometry-based approaches are acknowledged for both quantification and identification of molecular signatures, the latter relying primarily on fragmentation spectra interpretation. However, the high structural diversity of biological small molecules poses a considerable challenge in compound annotation. Feature-based molecular networking (FBMN) combined with database searches currently sets the gold standard for annotation of large datasets. Nevertheless, FBMN is usually based on collision-induced dissociation (CID) data, which may lead to unsatisfying information. The use of alternative fragmentation methods, such as electron-activated dissociation (EAD), is undergoing a re-evaluation for the annotation of small molecules, as it gives access to additional fragmentation routes. In this study, we apply the performances of data-dependent acquisition mass spectrometry (DDA-MS) under CID and EAD fragmentation along with FBMN construction, to perform extensive compound annotation in the crude extracts of the freshwater sentinel organism Gammarus fossarum. We discuss the analytical aspects of the use of the two fragmentation modes, perform a general comparison of the information delivered, and compare the CID and EAD fragmentation pathways for specific classes of compounds, including previously unstudied species. In addition, we discuss the potential use of FBMN constructed with EAD fragmentation spectra to improve lipid annotation, compared to the classic CID-based networks. Our approach has enabled higher confidence annotations and finer structure characterization of 823 features, including both metabolites and lipids detected in G. fossarum extracts.

Trophic transfer, bioaccumulation, and health risk assessment of heavy metals in Aras River: case study-Amphipoda- zander - human.

Heavy metals (As, Pb, Cd, and Cu) were traced in a model of the aqueous food chain in the Aras River, located in northwest of Iran. The selected model included the zander (Sander lucioperca L.) and crustacean species known as amphipods (Gammarus sp.) which belong to the food chain of this ray-finned fish. A total of 172 samples (70 fish and 102 amphipods) were collected randomly and analyzed for heavy metals using atomic absorption spectrophotometry (AAS). The results showed that the accumulation of heavy metals in both taxa are in the order of As > Pb > Cd > Cu, and concentrations of heavy metals in fish muscle are higher than Gammarus sp. in all stations in different seasons. Specimens of station (1) displayed the highest heavy metal content due to local industrial activities. The recorded concentrations of As, Pb, and Cd exceed the permissible limits. There is a close correlation between the concentrations of heavy metals in the amphipods and zanders. Target hazard quotient (THQ), total target hazard quotient (TTHQ), and carcinogenic risk (CR) were calculated to assess risks to human health. The average of THQ for As (1.43) exceeded the international standards and presenting health risks to the consumers of this fish species. The TTHQ for heavy metals was estimated higher than 1. At all stations, the value of CRCd > 1 × 10-3 indicating the degree of carcinogenicity of this metal in all parts of the Aras River. Therefore, according to our results, efficient control measures and regular biomonitoring should be established in this region.

Determination of metal uptake in single organisms, Corophium volutator, via complementary electrothermal vaporization/inductively coupled plasma mass spectrometry and laser ablation/inductively coupled plasma mass spectrometry.

RATIONALE: (Eco-)toxicological effects are mostly derived empirically and are not correlated with metal uptake. Furthermore, if the metal content is determined, mostly bulk analysis of the whole organism population is conducted; thus, biological variability is completely disregarded, and this may lead to misleading results. To overcome this issue, we compared two different solid sampling techniques for the analysis of single organisms. METHODS: In this study, complementary electrothermal vaporization/inductively coupled plasma mass spectrometry (ETV/ICP-MS) ⇔ laser ablation/inductively coupled plasma mass spectrometry (LA/ICP-MS)-based methods for the analysis of individual organisms were developed and the results obtained were compared with the concentrations obtained after digestion and measured using ICP-MS. For this purpose, a common (eco-)toxicological test organism, the mud shrimp Corophium volutator, was selected. As proof-of-concept application, these organisms were incubated with environmentally relevant metals from galvanic anodes, which are often used for protection against metal corrosion in, for example, offshore wind farms. RESULTS: The bulk analysis revealed that large quantities of the incubated elements were detectable. Using the ETV/ICP-MS method, we could identify a high biovariability within the population of organisms tested. Using the LA/ICP-MS method, it could be determined that the large quantities of the elements detected were due to adsorption of the metals and not due to uptake, which correlates well with the absence of (eco-)toxicological effects. CONCLUSIONS: The results obtained imply the efficiency of complementary methods to explain the absence or presence of (eco-)toxicological effects. In particular, methods that allow for single-organism analysis or provide even a spatial resolution support the interpretation of ecotoxicological findings.

Mercury isotopes identify near-surface marine mercury in deep-sea trench biota.

Mercury isotopic compositions of amphipods and snailfish from deep-sea trenches reveal information on the sources and transformations of mercury in the deep oceans. Evidence for methyl-mercury subjected to photochemical degradation in the photic zone is provided by odd-mass independent isotope values (Δ199Hg) in amphipods from the Kermadec Trench, which average 1.57‰ (±0.14, n = 12, SD), and amphipods from the Mariana Trench, which average 1.49‰ (±0.28, n = 13). These values are close to the average value of 1.48‰ (±0.34, n = 10) for methyl-mercury in fish that feed at ∼500-m depth in the central Pacific Ocean. Evidence for variable contributions of mercury from rainfall is provided by even-mass independent isotope values (Δ200Hg) in amphipods that average 0.03‰ (±0.02, n = 12) for the Kermadec and 0.07‰ (±0.01, n = 13) for the Mariana Trench compared to the rainfall average of 0.13 (±0.05, n = 8) in the central Pacific. Mass-dependent isotope values (δ202Hg) are elevated in amphipods from the Kermadec Trench (0.91 ±0.22‰, n = 12) compared to the Mariana Trench (0.26 ±0.23‰, n = 13), suggesting a higher level of microbial demethylation of the methyl-mercury pool before incorporation into the base of the foodweb. Our study suggests that mercury in the marine foodweb at ∼500 m, which is predominantly anthropogenic, is transported to deep-sea trenches primarily in carrion, and then incorporated into hadal (6,000-11,000-m) food webs. Anthropogenic Hg added to the surface ocean is, therefore, expected to be rapidly transported to the deepest reaches of the oceans.

In situ isobaric lipid mapping by MALDI-ion mobility separation-mass spectrometry imaging.

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high-resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.

Conventional and nano-copper pesticides are equally toxic to the estuarine amphipod Leptocheirus plumulosus.

Modern nano-engineered pesticides have great promise for agriculture due to their extended, low dose release profiles that are intended to increase effectiveness but reduce environmental harm. Whether nanopesticides, including copper (Cu) formulations, cause reduced levels of toxicity to non-target aquatic organisms is unclear but important to assess. Predicting how aquatic species respond to incidental exposure to Cu-based nanopesticides is challenging because of the expected very low concentrations in the environment, and the two forms of exposure that may occur, namely to Cu ions and Cu nanoparticles. We conducted Cu speciation, tissue uptake, and 7-day toxicity laboratory experiments to test how a model estuarine organism, the amphipod Leptocheirus plumulosus, responded to two popular Cu-based nanopesticides, CuPRO and Kocide, and conventional CuCl2. Exposure concentrations ranged from 0 to 2.5 ppm, which were similar to those found in estuarine water located downstream of agricultural fields. Cu dissolution rates were much slower for the nanopesticides than the ionic formula, and Cu body burden in amphipods increased approximately linearly with the nominal exposure concentration. Amphipod survival declined in a normal dose-response manner with no difference among Cu formulations. Growth and movement rates after 7 days revealed no difference among exposure levels when analyzed with conventional statistical methods. By contrast, analysis of respiration rates, inferred from biomass measurements, with a bioenergetic toxicodynamic model indicated potential for population-level effects of exposure to very low-levels of the two nanopesticides, as well as the control contaminant CuCl2. Our results indicate that toxicity assessment of environmental trace pollutant concentrations may go undetected with traditional ecotoxicological tests. We present a process integrating toxicity test results and toxicodynamic modeling that can improve our capacity to detect and predict environmental impacts of very low levels of nanomaterials released into the environment.

In vivo comprehensive multiphase NMR.

Traditionally, due to different hardware requirements, nuclear magnetic resonance (NMR) has developed as two separate fields: one dealing with solids, and one with solutions. Comprehensive multiphase (CMP) NMR combines all electronics and hardware (magic angle spinning [MAS], gradients, high power Radio Frequency (RF) handling, lock, susceptibility matching) into a universal probe that permits a comprehensive study of all phases (i.e., liquid, gel-like, semisolid, and solid), in intact samples. When applied in vivo, it provides unique insight into the wide array of bonds in a living system from the most mobile liquids (blood, fluids) through gels (muscle, tissues) to the most rigid (exoskeleton, shell). In this tutorial, the practical aspects of in vivo CMP NMR are discussed including: handling the organisms, rotor preparation, sample spinning, water suppression, editing experiments, and finishes with a brief look at the potential of other heteronuclei (2 H, 15 N, 19 F, 31 P) for in vivo research. The tutorial is aimed as a general resource for researchers interested in developing and applying MAS-based approaches to living organisms. Although the focus here is CMP NMR, many of the approaches can be adapted (or directly applied) using conventional high-resolution magic angle spinning, and in some cases, even standard solid-state NMR probes.

Mercury source changes and food web shifts alter contamination signatures of predatory fish from Lake Michigan.

To understand the impact reduced mercury (Hg) loading and invasive species have had on methylmercury bioaccumulation in predator fish of Lake Michigan, we reconstructed bioaccumulation trends from a fish archive (1978 to 2012). By measuring fish Hg stable isotope ratios, we related temporal changes in Hg concentrations to varying Hg sources. Additionally, dietary tracers were necessary to identify food web influences. Through combined Hg, C, and N stable isotopic analyses, we were able to differentiate between a shift in Hg sources to fish and periods when energetic transitions (from dreissenid mussels) led to the assimilation of contrasting Hg pools (2000 to present). In the late 1980s, lake trout δ202Hg increased (0.4‰) from regulatory reductions in regional Hg emissions. After 2000, C and N isotopes ratios revealed altered food web pathways, resulting in a benthic energetic shift and changes to Hg bioaccumulation. Continued increases in δ202Hg indicate fish are responding to several United States mercury emission mitigation strategies that were initiated circa 1990 and continued through the 2011 promulgation of the Mercury and Air Toxics Standards rule. Unlike archives of sediments, this fish archive tracks Hg sources susceptible to bioaccumulation in Great Lakes fisheries. Analysis reveals that trends in fish Hg concentrations can be substantially affected by shifts in trophic structure and dietary preferences initiated by invasive species in the Great Lakes. This does not diminish the benefits of declining emissions over this period, as fish Hg concentrations would have been higher without these actions.

First evidence of microplastics ingestion in benthic amphipods from Svalbard.

The present paper provides the first record of ingestion of microplastics in natural context by Gammarus setosus from Svalbard Archipelago. The plastic particles were identified both by Nile Red staining and Micro FT-IR spectroscopy. The species studied ingests microplastic particles in natural conditions if present in its habitat, probably mistaking them as food. The microplastic particles ingested may be available for uptake to predators that consume this Arctic amphipod, producing consequences to the food web.

Evaluation of macroalgae and amphipods as bioindicators of petroleum hydrocarbons input into the marine environment.

The brown alga Sargassum furcatum and three families of amphipods (Ampithoidae, Caprellidae and Hyalidae) associated to that algae were evaluated as bioindicators of petroleum hydrocarbons input into the marine environment of São Sebastião Channel, in southeastern region of Brazil. The n-alkanes pattern were mainly associated with the natural composition of the macroalgae and amphipods, although some indicatives of petroleum hydrocarbons such as unresolved complex mixture and the no predominance of odd over even n-alkanes have been observed in some samples. Total PAHs ranged from 33.4 to 2010 ng g-1 dry weight with the predominance of low molecular weight PAHs, mostly of naphthalene and alkyl-naphthalenes, which also suggested petroleum input. Even in low concentration, Sargassum furcatum and amphipods species studied seems to be good indicators of the introduction of petroleum hydrocarbons.

Will giant polar amphipods be first to fare badly in an oxygen-poor ocean? Testing hypotheses linking oxygen to body size.

It has been suggested that giant Antarctic marine invertebrates will be particularly vulnerable to declining O2 levels as our ocean warms in line with current climate change predictions. Our study provides some support for this oxygen limitation hypothesis, with larger body sizes being generally more sensitive to O2 reductions than smaller body sizes. However, it also suggests that the overall picture is a little more complex. We tested predictions from three different, but overlapping, O2-related hypotheses accounting for gigantism, using four Antarctic amphipod species encompassing a wide range of body sizes. We found a significant effect of body size, but also of species, in their respiratory responses to acutely declining O2 tensions. The more active lifestyle of intermediate-sized Prostebbingia brevicornis was supported by a better respiratory performance than predicted by the oxygen limitation hypothesis alone, but consistent with the symmorphosis hypothesis. We suggest that giant polar amphipods are likely to be some of the first to fare badly in an O2-poor ocean. However, the products of past evolutionary innovation, such as respiratory pigments that enhance O2-transport and novel gas exchange structures, may in some species offset any respiratory disadvantages of either large or small body size. This article is part of the theme issue 'Physiological diversity, biodiversity patterns and global climate change: testing key hypotheses involving temperature and oxygen'.

Species-Specific (Hyalella azteca and Lymnea stagnalis) Dietary Accumulation of Gold Nano-particles Associated with Periphyton.

Ecological effects of gold nano-particles (AuNP) are examined due to growing use in consumer and industrial materials. This study investigated uptake and movement of AuNPs through an aquatic food chain. Simple (single-species) and diverse (multi-species) periphyton communities were exposed to AuNP (0, 100, 500 µg L-1 treatments). AuNP quickly aggregated and precipitated from the water column, suggesting it is an insignificant route of AuNP exposure even at elevated concentrations. Gold was measured in 100 and 500 µg L-1 periphyton treatments. Gold accumulation was similar between periphyton treatments, suggesting physical processes were important for AuNP basal accumulation. Hyalella azteca and Lymnea stagnalis whole body tissue analysis indicated gold accumulation may be attributed to different feeding mechanisms, general versus selective grazing, respectively. Results suggest trophic transfer of AuNP is organism specific and aggregation properties of AuNP are important when considering fate of nano-particles in the environment and movement through aquatic food webs.

Microplastics in Talitrus saltator (Crustacea, Amphipoda): new evidence of ingestion from natural contexts.

Using Fourier transform infrared spectroscopy (FT-IR) measurements and comparing the spectrum peaks (range 4000-600 cm-1) with reference spectra database and instrument libraries, we observed new evidence of the ingestion of microplastic particles analyzing the digestive tracts of Talitrus saltator. Specimens, sampled in central Italy, probably ingested the particles with natural detritus. Since worldwide many species of invertebrates and vertebrates (e.g., birds) feed on Amphipoda along coastal ecosystems, we hypothesized that microplastic in these crustaceans can be accumulated along the food chain.

Fenoxycarb exposure disrupted the reproductive success of the amphipod Gammarus fossarum with limited effects on the lipid profile.

Insect growth regulator insecticides mimic the action of hormones on the growth and development of insect pests. However, they can affect the development of non-target arthropods. In the present study, we tested the effects of the growth regulator insecticide fenoxycarb on several endpoints in the freshwater crustacean Gammarus fossarum (Amphipoda). Females carrying embryos in their open brood pouch were exposed to 50 µg L-1 fenoxycarb throughout the entire oogenesis (i.e. 21 days). After exposure, newborn individuals from exposed embryos were removed from the maternal open brood pouch for lipidomic analysis, while males were added to assess the reproductive success. After fertilization, the lipid profile, energy reserve content (lipids, proteins and glycogen), and activity of phenoloxidase - an enzyme involved in the immune response - were measured in females. No significant effect of fenoxycarb exposure was observed on the lipid profile of both newborn individuals and females, while reproductive success was severely impaired in exposed females. Particularly, precopulatory behavior was significantly reduced and fertilized eggs were unviable. This study highlighted the deleterious effects of the insect growth regulator fenoxycarb on gammarid reproduction, which could have severe repercussions on population dynamics.

Metal accumulation in sediments and amphipods downstream of combined sewer overflows.

The aim of this study was to investigate the concentrations of the metals Ag, Cd, Cu, Mn, Mo, Pb, Pt and Zn in sediments and amphipods along a 100m transect downstream of three different combined sewer overflows (CSOs). Moreover, the study was implemented to better understand the biological availability of metals downstream of CSOs as well as to identify a potential risk of CSOs for benthic or pelagic biota. Samples were taken at downstream sampling sites, which were 1, 5, 10, 20, 50 and 100m away from the outlets of the CSOs. An upstream-located sampling site each was used as reference for the respective transect. Additionally, sediments in two retention zones, located between the CSOs and the receiving creeks were analyzed. All downstream located creek sediments showed a similar metal pattern. Metal concentrations were found to be highest within the first 20m of the creek sediments. Elevated metal concentrations were also detected in the sediments of the retention zones. Metal accumulation in the amphipods was different at all three locations, but highest levels were always found downstream of the sediment accumulation hot spots. This might indicate that the metals were remobilized and became available for the amphipods further downstream of the CSOs. Although the general contamination pattern with respect to each reference site was similar, the degree of contamination was different at each location as demonstrated by anthropogenic enrichment factors. The discharge frequency as well as anthropogenic influences from upper reaches appear to be the most important factors for high metal concentrations in the sediments. Accordingly, in one case high background concentrations in combination with the CSO lead to an exceedance of the predicted effect level of Zn for aquatic organisms. Therefore, sediment contamination should be included in risk assessment when constructing CSOs to protect aquatic life.

Metal release from contaminated leaf litter and leachate toxicity for the freshwater crustacean Gammarus fossarum.

Industrialization has left large surfaces of contaminated soils, which may act as a source of pollution for contiguous ecosystems, either terrestrial or aquatic. When polluted sites are recolonized by plants, dispersion of leaf litter might represent a non-negligible source of contaminants, especially metals. To evaluate the risks associated to contaminated leaf litter dispersion in aquatic ecosystems, we first measured the dynamics of metal loss from leaf litter during a 48-h experimental leaching. We used aspen (Populus tremula L.), a common tree species on these polluted sites, and collected leaf litter on three polluted sites (settling pond of a former steel mill) and three control sites situated in the same geographic area. Then, toxicity tests were carried out on individuals of a key detritivore species widely used in ecotoxicology tests, Gammarus fossarum (Crustacea, Amphipoda), with uncontaminated and contaminated leaf litter leachates, using a battery of biomarkers selected for their sensitivity to metallic stress. Leaf litters collected on polluted sites exhibited not only significantly higher cadmium and zinc concentrations but also lower lignin contents. All leaf litters released high amounts of chemical elements during the leaching process, especially potassium and magnesium, and, in a lesser extent, phosphorus, calcium, and trace metals (copper, cadmium, and zinc but not lead). Toxicity tests revealed that the most important toxic effects measured on G. fossarum were due to leaf litter leachates by themselves, whatever the origin of litter (from polluted or control sites), confirming the toxicity of such substances, probably due to their high content in phenolic compounds. Small additional toxic effects of leachates from contaminated leaf litters were only evidenced on gammarid lipid peroxidation, indicating that contaminated leaf litter leachates might be slightly more toxic than uncontaminated ones, but in a very reduced manner. Further studies will be required to verify if these patterns are generalizable to other species and to investigate the effects of contaminated leaf litter ingestion by consumers on aquatic food webs. Nevertheless, our results do not permit to exclude potential chronic effects of an exposure to contaminated leaf litter leachates in aquatic ecosystems.

Treated wastewater effluent as a source of pyrethroids and fipronil at todos santos bay, Mexico: Its impact on sediments and organisms.

Pyrethroids are insecticides widely used to control pests and disease vectors in residential areas and agricultural lands. Pyrethroids are emerging pollutants, and their use is a growing concern because of their toxicity potential to aquatic organisms. Todos Santos Bay and the Punta Banda estuary, 2 coastal bodies located to the south of the Southern California Bight, were studied to establish a baseline of the current conditions of pollution by pyrethroids and fipronil. Eight pyrethroids, along with fipronil and its 2 metabolites, were determined in effluents from wastewater-treatment plants (n = 3), surface sediments (n = 32), and 3 locations with mussels (Mytilus californianus, n = 9). Bifenthrin, permethrin, and cypermethrin were the most common pyrethroids found in the study areas and were widespread in sediments, mussels, and wastewater-treated effluents. Fipronil and its metabolites were detected in mussels and wastewater-treated effluents only. Total pyrethroid concentrations in sediments ranged from 0.04 to 1.95 ng/g dry weight in the Punta Banda estuary (n = 13) and from 0.07 to 6.62 ng/g dry weight in Todos Santos Bay (n = 19). Moreover, total pyrethroids in mussels ranged from 1.19 to 6.15 ng/g wet weight. Based on the toxic unit data calculated for pyrethroids and fipronil for Eohaustorius estuarius and Hyalella azteca, little to no impact is expected to the benthic population structure. Environ Toxicol Chem 2017;36:3057-3064. © 2017 SETAC.

Multiplexed assay for protein quantitation in the invertebrate Gammarus fossarum by liquid chromatography coupled to tandem mass spectrometry.

A highly multiplexed liquid chromatography mass spectrometry-selected reaction monitoring (SRM)-based assay for determination of 40 potential protein biomarkers from Gammarus fossarum, an ecotoxicological relevant species, was described. The assay relies on 71 stable isotope-labeled reported peptide standards for the quantitation of proteins of interest in relation to essential physiological functions such as reproductive cycle, defense mechanism, and enzymes involved in homeostasis process and in energy. A direct linear relationship between the spiked peptide concentration and the area under the peak was clearly demonstrated in biological extracts. Precision and accuracy were determined to be between 1.1 and 21% and between 79 and 120%, respectively, depending on the selected protein in a few samples after optimization of digestion conditions. The validity of the assay was documented for several biomarkers linked with reproduction and the molting process was performed with the assessment of protein levels throughout contrasted physiological process (sex, reproductive status). This assay is easy to use, robust, sensitive, and has high-throughput capabilities. The proposed strategy may be extended to any non-model organisms relevant in environmental science. Graphical abstract ᅟ.

Dietary administration of Pontogammarus maeoticus extract affects immune responses, stress resistance, feed intake and growth performance of caspian roach (Rutilus caspicus) fingerlings.

Dietary administration of immunostimulants showed promising results for elevation of immune responses and disease resistance. The aim of the present study was to investigate the effects of dietary Pontogammarus (Pontogammarus maeoticus) extract on innate immune response, stress resistance, feed intake and growth performance of the Caspian roach fingerling. Two levels of P. maeoticus extract dilution with distilled water 1:25 [T1] and 1:50 [T2] were prepared. Experimental diets were prepared by top-spraying the basal diet with equal amount (2%) of diluted extracts. One hundred and eighty Caspian roach fingerlings (4.30 ± 0.10 g) were supplied, stocked in nine 100-L tanks (three treatment repeated in triplicates) and fed on experimental diets for 8 weeks. At the end of the trial, serum innate immune parameters (Total Ig, ACH50, and lysozyme activity), resistance against salinity stress, feed intake and growth parameters were measured. The results revealed remarkable increase of innate immune parameters and resistance against salinity stress in roach fed P. maeoticus extracts (P < 0.05). Also, growth performance and food intake were notably improved in P. maeoticus extracts fed fish (P < 0.05). These results revealed beneficial effects of P. maeoticus extract on innate immune response, resistance, feed intake as well as growth performance of the Caspian roach.

High-throughput proteome dynamics for discovery of key proteins in sentinel species: Unsuspected vitellogenins diversity in the crustacean Gammarus fossarum.

UNLABELLED: In environmental science, omics-based approaches are widely used for the identification of gene products related to stress response. However, when dealing with non-model species, functional prediction of genes is challenging. Indeed, functional predictions are often obtained by sequence similarity searches and functional data from phylogenetically distant organisms, which can lead to inaccurate predictions due to quite different evolutionary scenarios. In oviparous females, vitellogenin production is vital for embryonic development, ensuring population viability. Its abnormal presence in fish male organisms is commonly employed as a biomarker of exposure to xenoestrogens, named endocrine disruptors. Here, in the freshwater amphipod Gammarus fossarum, we identified vitellogenin proteins by means of a proteome temporal dynamics analysis during oogenesis and embryogenesis. This exhaustive approach allows several functional molecular hypotheses in the oogenesis process to be drawn. Moreover, we revealed an unsuspected diversity of molecular players involved in yolk formation as eight proteins originating from different families of the large lipid transfer protein superfamily were identified as "true vitellogenins". BIOLOGICAL SIGNIFICANCE: In non-model species, next generation sequencing technologies development enables quickly deciphering gene and protein sequences but accuracy of associated functional prediction remains to be established. Here, in the crustacean Gammarus fossarum, a key sentinel species in freshwater biomonitoring, we identified key molecular players involved in the female reproduction by studying the proteome dynamics of ovaries and embryos. An unsuspected diversity of vitellogenin proteins was evidenced. These proteins being vital for offspring development, their high diversity may be advantageous for the organism's reproduction. Phylogenetic analysis showed that some forms are true vitellogenin orthologs while others are included in the apolipoprotein family, a paralogous group from the vitellogenin family. Among crustaceans, Gammarus fossarum is the first documented case where diverse protein families are involved in the yolk formation process.