The storage lipid metabolism of two caridean shrimp species (Crangon crangon and Pandalus montagui) was studied through a combination of enzyme assays, total lipid determination and transcriptome analyses. The initital sampling was carried out in June, July and August 2021 by the research vessel R/V Uthörn. Freshly caught shrimps from the North Sea were measured, weighted and dissected and brought back to the laboratory facilites of the Alfread-Wegener-Institute, Bremerhaven, Germany. Individual midgut glands were weighted to determine the wet mass and freeze-dried. Dry mass was termined and lipids were extracted after Hagen (2000, see in Postel et al. 2000). The total lipid content of individual midgut glands of Crangon crangon and Pandalus montagui was determined gravimetrically. The synthesis of the storage lipid triacylglycerol (TAG) was measured in pooled microsomal fraction of midgut gland tissue of both shrimp species through the activity of the enzyme diacylglycerol acyltransferase (DGAT) at 37 °C in a water bath (McFie & Stone 2011). Here a fluorescent activated fatty acid (NBD-palmitoyl-CoA, 810229 Avanti Polar Lipids) was used. Lipids in the reaction mix were extracted and lipid classes separated on a thin layer chromatography plate. DGAT activity was measured through arbitrary fluorescent units (AFU/min/mg protein) of the correscponding TAG product. Annotated transcriptomes of both species (C. crangon Bioproject: PRJNA479562, NCBI; P. montagui Bioproject PRJNA798226, NCBI) were screened for enzymes involved in the lipid metabolism. Transcripts identified as relevant enzymes using BLAST were translated into amino acid sequences.
The hydrolytic degradation of biodegradable and conventional plastics by the gastric fluid of the edible crab Cancer pagurus were assayed in-vitro with pH Stat titration. Suspensions of different microplastics were incubated with gastric fluid at 15 °C in artificial seawater. Rates of hydrolysis, as determined by counter-titration with a diluted base (NaOH), was recorded for two hours. The gastric fluid was separated by anion exchange chromatography into 65 fractions. Again, three pools of fractions were tested for their hydrolytic potential with a plastic based on polylactic acid at 30°C. Enzyme assays with fluorogenic substrates were performed with each of the 65 fractions of the gastric fluid. Methylumbelliferone derivatives of fatty acid esters (MUF-butyrate, MUF-heptanoate and MUF-oleate) were used as substrates in buffer at different pH ranging from 5 to 9 and at a temperature of 25°C. The increase in fluorescence was measured with a microplate reader. Analogously, the effect of a detergent (sodium dodecyl sulfate) on the enzymatic activity was measured. All measurements were conducted under controlled laboratory conditions.