Reproduction Stage Differentiates the Time-Course Regulation of Metabolites in Daphnia magna

Abstract

Daphnia magna is a keystone indicator zooplankton used in environmental quality assessments. Comparative metabolomics, which contrasts small biomolecular regulations under different conditions, has emerged as a sensitive and informative investigation tool for the assessment of environmental stressors on D. magna. Baseline metabolomic variation is likely impacted by the asexual reproduction cycle of D. magna; however, the relationship between metabolite concentration regulation and reproduction cycle has not been investigated. This study investigated the time-course regulation of 51 metabolites during different reproduction stages to determine how the reproduction cycle controlled the metabolite profile of D. magna. Two-way analysis of variance (ANOVA) results reveal that most metabolites show significantly differentiated concentrations by individual or a combination of reproduction stages and sampling time. On the basis of the partial least-squares-discriminant analysis (PLS-DA) and ANOVA-simultaneous component analysis (ANOVA-SCA), stages 2 and 3 of reproduction show similarity in metabolite abundance regulation compared to stage 1. Metabolites were classified as being either dependent or independent of reproduction in the pairwise concentration correlation based on Pearson correlation coefficients. This study observed that the D. magna reproduction stage is an important consideration and potential variable and should be considered carefully when conducting metabolomic experiments using D. magna.

Targeted Metabolomic Assessment of the Sub-Lethal Toxicity of Halogenated Acetic Acids (HAAs) to Daphnia magna

“…Daphnia metabolites were extracted based on the method of Jeong and Simpson [53]. This method was specifically developed to isolate polar metabolites from individual daphnids and has high recovery rates for the suite of metabolites analyzed [53].…”

“…Daphnia metabolites were extracted based on the method of Jeong and Simpson [53]. This method was specifically developed to isolate polar metabolites from individual daphnids and has high recovery rates for the suite of metabolites analyzed [53]. Briefly, the Daphnia were individually placed into 2.5 mL Eppendorf tubes (Eppendorf, Mississauga, ON, Canada) and 160 µL of 10:9 methanol (Fisher Scientific, Toronto, ON, Canada; 99.9% purity): water (v/v) was added to each tube ( Figure 6).…”

“…The concentration of metabolites in the Daphnia extracts was calculated using generated calibration curves from external standards and then subject to further analysis of the data [53,88]. Absolute metabolite concentrations were imported into MetaboAnalystR (Xia Lab, Montreal, QC, Canada), where the data were filtered to remove outliers based on the interquartile range [58,89,90].…”

“…The normalized concentration data points were then scaled using the autoscaling function included in the MetaboAnalystR package [89,93]. The methods previously described have been used across metabolomics experiments with varying contaminants and target organisms [51,53,57,94].…”

“…Here liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to decode the metabolic responses of D. magna to three commonly observed HAAs (DCAA, TCAA and DBAA) in acute sub-lethal exposure studies. This method targets amino acids and derivatives, neurotransmitters, nucleosides/nucleotides, saccharide derivatives, vitamins, polyamines and carboxylic acids and has been used to assess sub-lethal toxicity in previous studies [51][52][53][54]. Previous studies reported that exposure to HAAs leads to disruptions in energy metabolism [23,24,39,41], and, as such, it is hypothesized that D. magna will also exhibit energy disruption with contaminant exposure.…”