Determination of Adenylate Nucleotides in Amphipod Gammarus fossarum by Ion-Pair Reverse Phase Liquid Chromatography: Possibilities of Positive Pressure Micro-Solid Phase Extraction

Adenine nucleotides—adenosine monophosphate, diphosphate, and triphosphate—are of utmost importance to all living organisms, where they play a critical role in the energy metabolism and are tied to allosteric regulation in various regulatory enzymes. Adenylate energy charge represents the precise relationship between the concentrations of adenosine monophosphate, diphosphate, and triphosphate and indicates the amount of metabolic energy available to an organism. The experimental conditions of adenylate extraction in freshwater amphipod Gammarus fossarum are reported here for the first time and are crucial for the qualitative and quantitative determination of adenylate nucleotides using efficient and sensitive ion-pair reverse phase LC. It was shown that amphipod calcified exoskeleton impeded the neutralization of homogenate. The highest adenylate yield was obtained by homogenization in perchloric acid and subsequent addition of potassium hydroxide and phosphate buffer to achieve a pH around 11. This method enables separation and accurate detection of adenylates. Our study provides new insight into the complexity of adenylate extraction and quantification that is crucial for the application of adenylate energy charge as a confident physiological measure of environmental stress and as a health index of G. fossarum.

Shotgun lipidomics and mass spectrometry imaging unveil diversity and dynamics in lipid composition in Gammarus fossarum

SummarySentinel species are playing an indispensable role in monitoring environmental pollution in aquatic ecosystems. Many pollutants found in water prove to be endocrine disrupting chemicals that could cause disruptions in lipid homeostasis in aquatic species. A comprehensive profiling of the lipidome of these species is thus an essential step towards understanding the mechanism of toxicity induced by pollutants. We here extensively examined both the composition and spatial distribution of lipids in freshwater crustacean Gammarus fossarum. The baseline lipidome of gammarids of different gender and reproductive stage was established by high throughput shotgun lipidomics. Spatial lipid mapping by high resolution mass spectrometry imaging led to the discovery of sulfate-based lipids in hepatopancreas and their accumulation in mature oocytes. We uncovered in G. fossarum a diverse and dynamic lipid composition that deepens our understanding of the biochemical changes during development and which could serve as a reference for future ecotoxicological studies.

Effects of parasites upon non-host predator avoidance behaviour in native and invasive gammarids

The acanthocephalan parasite, Polymorphus minutus, manipulates its intermediate hosts' (gammarids) behaviour, presumably to facilitate its transmission to the definitive hosts. A fundamental question is whether this capability has evolved to target gammarids in general, or specifically sympatric gammarids. We assessed the responses to chemical cues from a non-host predator (the three-spined sticklebacks Gasterosteus aculeatus) in infected and non-infected gammarids; two native (Gammarus pulex and Gammarus fossarum), and one invasive (Echinogammarus berilloni) species, all sampled in the Paderborn Plateau (Germany). The level of predator avoidance was assessed by subjecting gammarids to choice experiments with the presence or absence of predator chemical cues. We did not detect any behavioural differences between uninfected and infected G. pulex and E. berilloni, but an elevated degree of predator avoidance in infected G. fossarum. Avoiding non-host predators may ultimately increase the probability of P. minutus' of predation by the definitive host. Our results suggested that P. minutus' ability to alter the host's behaviour may have evolved to specifically target sympatric gammarid host species. Uninfected gammarids did not appear to avoid the non-host predator chemical cues. Overall the results also opened the possibility that parasites may play a critical role in the success or failure of invasive species.