Exchange of polar lipids from adults to neonates in Daphnia magna: Perturbations in sphingomyelin allocation by dietary lipids and environmental toxicants

Abstract

Because xenosensing nuclear receptors are also lipid sensors that regulate lipid allocation, we hypothesized that toxicant-induced modulation of HR96 activity would alter lipid profiles and the balance between adult survival and neonate production following exposure in Daphnia magna. Adult daphnids were exposed to unsaturated fatty acid- and toxicant- activators or inhibitors of HR96 and later starved to test whether chemical exposure altered allocation toward survival or reproduction. The HR96 activators, linoleic acid and atrazine, decreased reproduction as expected with concomitant changes in the expression of HR96 regulated genes such as magro. The HR96 inhibitors, docosahexaenoic acid (DHA) and triclosan, increased reproduction or neonate starvation survival, respectively. However, pre-exposure to triclosan increased in neonate survival at the expense of reproductive maturation. Lipidomic analysis revealed that sphingomyelins (SM) are predominantly found in neonates and therefore we propose are important in development. DHA and triclosan increased neonatal SM, consistent with HR96's regulation of Niemann-Pick genes. While DHA altered expression of magro, Niemann-Pick 1b, mannosidase, and other HR96-regulated genes as expected, triclosan primarily perturbed sphingomyelinase and mannosidase expression indicating different but potentially overlapping mechanisms for perturbing SM. Overall, SM appears to be a key lipid in Daphnia maturation and further support was provided by carmofur, which inhibits sphingomyelin/ceramide metabolism and in turn severely represses Daphnia maturation and initial brood production. In conclusion, toxicants can perturb lipid allocation and in turn impair development and reproduction.

Fig 1. Timeline for chemical exposures, starvation, and polar lipid determinations in D. magna as described in materials and methods.

Fig 2

Adult starvation survival (A), reproduction (B) and neonate starvation survival (C) in daphnids exposed to DHA, LA, PA, atrazine or triclosan. Data are presented as number of daphnids alive per day (A and C) or number of neonates per adult daphnid (B). Survival of daphnids was analyzed using Fishers 2x2 test (n = 12). An asteriak represents differences from DHA, LA, PA and atrazine groups (p<0.05). Reproduction (B) was analyzed using one-way ANOVA with a general linear model followed by Fisher’s LSD (n = 12) (p<0.05), with higher reproduction observed in group A than UT daphnids, and lower reproduction in groups D and F compared to UT and group A treated daphnids (n = 12).

Fig 3. Relative levels of different polar lipid classes in adult and neonatal daphnids.

(A) Relative concentrations of different classes of polar lipids in (A) untreated adult and neonatal daphnids, (B) treated adult daphnids, and (C) treated neonatal daphnids. Phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidic acid (PA), sphingomyelin (SM). Data are analyzed by two-way ANOVA followed by Fisher’s LSD as the post-hoc test (n = 5). ‘An a’ is different from UT, ‘b’ from DHA, ‘c’ from LA, ‘d’ from atrazine (Atr), ‘e’ from triclosan (Tric). No asterisk indicates a p<0.01 and an asterisk indicates a p<0.001. All significant data is p < 0.0001 in (A).

Fig 4. Changes in sphingomyelin (SM) composition among different exposure groups in adult and neonatal daphnids.

Changes in concentrations (nmol) of individual SM species following chemical exposure (UT, DHA, LA, atrazine and triclosan) in adult and neonatal daphnids were analyzed by two-way ANOVA followed by Fisher’s LSD (n = 5) (p<0.05). An ‘a’ refers to UT different from DHA, ‘b’ refers to UT different from LA, ‘c’ refers to UT different from Atr, ‘d’ refers to UT different from Tric, ‘e’ refers to DHA different from LA, ‘f’ refers to DHA different from Atr, ‘g’ refers to DHA different from Tric, ‘h’ refers to Tric different from LA, and ‘i’ refers to Tric different from Atr.

Fig 5. Hierarchal clustering and principle component analysis in adult Daphnia magna.

(A) Hierarchal Clustering (HCL) was used to cluster and visualize significant changes in the percentage signal of individual lipids in the adult daphnids using MultiExperiment Viewer (MeV). One–way ANOVA (p<0.01) with MEV was used to identify significantly altered lipid species. (B) Principle component analysis (PCA) demonstrates associations between chemical exposures and lipid profiles in adult D. magna. Variability among the polar lipids was observed following chemical treatment.

Fig 6. Hierarchal clustering and principle component analysis in neonatal Daphnia magna.

(A) Hierarchal Clustering (HCL) was used to cluster and visualize significant changes in the percentage signal of individual lipids in the adult daphnids using MultiExperiment Viewer (MeV). One–way ANOVA (p<0.01) was used to identify significantly altered lipid species. (B): Principle component analysis (PCA) demonstrates associations between chemical exposures and lipid profiles in neonatal D. magna. Variability among the polar lipids was observed following chemical treatment.

Fig 7. Sphingomyelin metabolism pathway.

Genes regulated by HR96 play important roles in breakdown of sphingomyelin to ceramide and sphingosine.

Fig 8. Altered expression of the HR96-regulated genes magro, mannosidase, NPC1b, sphingomyelinase, in addition to ceramidase in 8 day-old D. magna exposed to fatty acids or toxicants for 24 hours.

Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD with *p ≤0.05 considered significant. Data are provided as mean ± SEM (n = 5).

Fig 9. Altered expression of the HR96-regulated genes magro, mannosidase, NPC1b, sphingomyelinase, in addition to ceramidase in 8 day-old D. magna exposed to fatty acids or toxicants for 96 hours.

Statistical significance was determined by one way ANOVA followed by Fisher’s LSD test with *p ≤0.05 considered significant. Data are provided as mean ± SEM (n = 5).

Fig 10. Altered fecundity of adult daphnids exposed to the ceramidase inhibitor, carmofur.

Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD with *p<0.001 considered significant (n = 10).