Obesogens beyond Vertebrates: Lipid Perturbation by Tributyltin in the Crustacean Daphnia magna

Abstract

Background: The analysis of obesogenic effects in invertebrates is limited by our poor knowledge of the regulatory pathways of lipid metabolism. Recent data from the crustacean Daphnia magna points to three signaling hormonal pathways related to the molting and reproductive cycles [retinoic X receptor (RXR), juvenile hormone (JH), and ecdysone] as putative targets for exogenous obesogens.

Objective: The present study addresses the disruptive effects of the model obesogen tributyltin (TBT) on the lipid homeostasis in Daphnia during the molting and reproductive cycle, its genetic control, and health consequences of its disruption.

Methods: D. magna individuals were exposed to low and high levels of TBT. Reproductive effects were assessed by Life History analysis methods. Quantitative and qualitative changes in lipid droplets during molting and the reproductive cycle were studied using Nile red staining. Lipid composition and dynamics were analyzed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer. Relative abundances of mRNA from different genes related to RXR, ecdysone, and JH signaling pathways were studied by qRT-PCR.

Results and conclusions: TBT disrupted the dynamics of neutral lipids, impairing the transfer of triacylglycerols to eggs and hence promoting their accumulation in adult individuals. TBT's disruptive effects translated into a lower fitness for offspring and adults. Co-regulation of gene transcripts suggests that TBT activates the ecdysone, JH, and RXR receptor signaling pathways, presumably through the already proposed interaction with RXR. These findings indicate the presence of obesogenic effects in a nonvertebrate species.

Figure 1

Quantitative assessment of lipid droplets in Daphnia magna individuals. (A) Lateral partial view under fluorescent microscopy of adolescent females just after molting and releasing the first brood of eggs across different food ration regimes (starving, low food, and high food) and treatments [control and TBT H (1 μg/L)]; top left, bright field microscopy image of a female, with the studied area indicated by a rectangle. Lipid droplets stained with Nile red are in green. (B) Nile red fluorescence [mean ± SE fluorescence units (FU); n = 5–10] in 48-hr females across starving, low, and high food rations, and (C) across TBT L and TBT H at low and high food rations. (D) Nile red fluorescence (mean ± SE; n = 5–10) measured at different time points within the adolescent instar and just after molting across TBT L and TBT H at high food rations. In B and C, different letters indicate significant (p < 0.05) differences among food levels or across food levels and TBT treatments, respectively, following ANOVA and Tukey’s post hoc tests. Further details are in the text.

Figure 2

Lipidomic profiles of major lipid classes (mean ± SE; n = 3) in control, TBT L, and TBT H treatment groups during the adolescent instar in females at 0, 8, 16, and 24 hr, in de-brooded females just after the fourth molt (48 hr), and in eggs. Abbreviations: TG, triacylglycerols; DG, diacylglycerols; CE, cholesterylesters; PC, phosphocholines; LPC, lysophosphatidylcholine; SM, sphingolipids; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol.

Figure 3

Transcription patterns (mean ± SE; n = 5) shown by the number of mRNA copies of the eight studied genes (HR3, EcRB, Neverland, HR38, MET, SRC, Hb2, and RXR), relative to G3PDH, across the adolescent instar in females exposed to TBT L (gray triangles), TBT H (black triangles), or the carrier solvent (open circles).