Mechanisms of Action of Compounds That Enhance Storage Lipid Accumulation in Daphnia magna.

Accumulation of storage lipids in the crustacean Daphnia magna can be altered by a number of exogenous and endogenous compounds, like 20-hydroxyecdysone (natural ligand of the ecdysone receptor, EcR), methyl farnesoate, pyrirproxyfen (agonists of the methyl farnesoate receptor, MfR), and tributyltin (agonist of the retinoid X acid receptor, RXR). This effect, analogous to the obesogenic disruption in mammals, alters Daphnia's growth and reproductive investment. Here we propose that storage lipid accumulation in droplets is regulated in Daphnia by the interaction between the nuclear receptor heterodimer EcR:RXR and MfR. The model was tested by determining changes in storage lipid accumulation and on gene transcription in animals exposed to different effectors of RXR, EcR, and MfR signaling pathways, either individually or in combination. RXR, EcR, and MfR agonists increased storage lipid accumulation, whereas fenarimol and testosterone (reported inhibitors of ecdysteroid synthesis and an EcR antagonist, respectively) decreased it. Joint effects of mixtures with fenarimol, testosterone, and ecdysone were antagonistic, mixtures of juvenoids showed additive effects following a concentration addition model, and combinations of tributyltin with juvenoids resulted in greater than additive effects. Co-exposures of ecdysone with juvenoids resulted in deregulation of ecdysone- and farnesoid-regulated genes, accordingly with the observed changes in lipid accumulation These results indicate the requirement of ecdysone binding to the EcR:RXR:MfR complex to regulate lipid storage and that an excess of ecdysone disrupts the whole process, probably by triggering negative feedback mechanisms.

Introduction

Recent studies have suggested the involvement of endocrine disrupting chemicals in the obesity epidemia occurring in many modern human societies.[1] Obesity increases the risk of coronary artery diseases, diabetes, and related health detrimental effects, such as hypertension and lipidemia.[1,2] Many widely used chemicals are known or suspected promoters of weight gain at low doses, in an extensive list that includes organotin antifouling agents, among others.[3] It has been proposed that exposure to these so-called obesogens in the uterus may lead to obesity later in life.[4]

Obesogenic effects in vertebrates have often been related to the disruption of the peroxisome proliferator-activated receptor (PPARγ) signaling pathway. This receptor is a master regulator of adipocyte differentiation and lipid metabolism in vertebrates, binding to the promoter of target genes and forming an heterodimer with the retinoid X receptor (RXR).[3] Although PPAR has not been found outside deuterostomes, a recent study showed that the suspected vertebrate obesogen tributyltin (TBT), also disrupts the dynamics of neutral lipids’ storage in the crustacean Daphnia magna.[5] As TBT is the only known ligand of RXR in arthropods,[6] this increases the scope of the search for obesogenic effects to Arthropods and other Protostomata through the interaction with this nuclear receptor, which is present in virtually all Metazoans. In Daphnia, TBT impairs the transfer of triacylglycerols to eggs and hence promotes their accumulation in lipid droplets inside fat cells in postspawning adult females,[5] resulting in a lower fitness for offspring and adults. TBT increased mRNA levels of the RXR gene and of several genes regulated by the ecdysteroid (EcR) and the methyl farnesoate hormone (MfR) receptors.[5] These results suggest a genetic interaction between the regulation of lipid storage by RXR and other endocrine signaling pathways in D. magna.

More recently, Jordão et al.[7] found that in addition to TBT, agonists of EcR (20-hydroxyecdysone), and MfR (methyl farnesoate, pyriproxyfen) increased the accumulation of storage lipids in a concentration-related manner. Conversely fenarimol, which is known to deplete the levels of ecdysone in D. magna,[8] decreased storage lipids. These previous results suggest that the accumulation of storage lipids in D. magna is promoted by agonists of the three transcription factors and that antiecdysteriods inhibited the whole process. There is therefore a need to understand how the different transcription factors interact regulating storage lipid dynamics in Daphnia.

In D. magna, like in other crustacean and arthropods, storage lipid dynamics varied along the molt and reproduction cycle, which is regulated by the ecdysteroid and juvenile hormone receptor signaling pathways.[9] Ecdysone exerts its effects through the interaction with the ecdysteroid receptor (EcR), known to heterodimerize with RXR and to bind to the promoters of ecdysone-regulated genes (i.e., HR3, Neverland).[10−12] TBT, which is an agonist of RXR together with methyl farnesoate and other juvenoids, enhanced the ecdysteroid-dependent activation of the EcR: RXR heterodimer.[12] The previous study provided the first evidence for a ternary receptor complex in Daphnia (MfR, EcR, RXR) that would require ecdysteriods to elicit transcriptional responses to the other ligands.[13] Recent findings indicate that MfR in Daphnia is itself a complex of two nuclear proteins of the bHLH-PAS family of transcription factors: the methoprene-tolerant coactivator proteins (MET), which binds to methyl farnesoate and other juvenoid compounds, and the steroid receptor coactivator (SRC).[14,15] Juvenoids promote expression of hemoglobin genes, such as Hb2,[16] and of male sex determining genes in the latter stages of ovarian oocyte maturation.[17] In this study we propose a conceptual model in which EcR, RXR, and MfR act as a molecular complex to regulate lipid accumulation and other key physiological functions through the modulation of the expression of key genes. This model is based in our current knowledge of the PPARγ mechanistic mode of action and incorporates physiological, life-history, and gene expression data from D. magna responses to effectors of the different receptors, both in single exposures and in combination mixtures.

Experimental Section

Studied Compounds

Studied compounds included the juvenile crustacean’s hormone methyl farnesoate (MF, CAS 10485-70-8) and the molting hormone 20-hydroxyecdysone (20E, CAS 5289-74-7);[11] the juvenoid pesticide pyriproxyfen (PP, CAS 95737-68-1); the RXR agonist tributyltin (TBT, CAS 1461-22-9),[11] the ecdysone synthesis inhibitor fenarimol (FEN, CAS 60168-88-9),[8] and the ecdysone receptor antagonist testosterone (T, 58-22-0).[8] All the compounds were obtained from Sigma-Aldrich (U.S.A/Netherlands) except MF, which was supplied by Echelon Bioscience, Utah, U.S.A.

Experimental Animals

All experiments were performed using the well-characterized single clone F of D. magna maintained indefinitely as pure parthenogenetic cultures.[18] Individual cultures were maintained in 100 mL of ASTM hard synthetic water at high food ration levels (5 × 105 cells/ml of Chlorella vulgaris), as described in Barata and Baird.[18]

Experimental Procedures

Experiments follow previous procedures.[5,7] Briefly experiments were initiated with newborn neonates <4–8 h old obtained from synchronized females cultured individually at high food ration levels. Groups of five neonates were reared in 100 mL of ASTM hard water under high food ration conditions until the end of the third juvenile instar (about 4–8 h before molting for the third time). At this point juveniles were exposed individually in 100 mL or in groups of five in 500 mL of test medium to selected chemicals and used to quantify accumulation of tracylglycerols in lipid droplets using Nile Red and/or gene transcription responses, respectively. Treatments for Nile Red and gene transcription determinations were replicated ten times. Exposures were conducted during the adolescent instar, which is the instar where the first brood of eggs are formed in the ovaries. Females used for lipid droplet and gene transcription analyses were sampled just after their fourth molt and having released their first clutch of eggs into the brood pouch. The test medium was renewed every other day.

Model Framework and Experiments

PPARγ binds on DNA response elements in mammals as a heterodimer with RXR[19] (Figura 1A). This heterodimer acts regulating the differentiation of preadipocytes into adipocytes (3T3:L1 cells) and triacylglycerol accumulation.[18] Agonists of PPARγ and RXR are able to induce lipid accumulation and adipocyte differentiation in mammalian 3T·-L1 cells.[20] This feature is most likely related to the permissive nature of the heterodimer PPARγ:RXR.

Figure 1

Simplified scheme of RXR:PPARγ involvement in mammalian adipocyte differentiation and triacylglycerol accumulation[19] (A) and of the putative involvement of EcR:RXR and SCR:MET receptor complexes signaling pathways in regulating lipid storage accumulation in Daphnia magna fat cells (B). In B, EcR and RXR form a heterodimer, which is activated by ecdysteroids. RXR ligands (i.e., tributyltin-TBT) can also activate the previous receptor complex when ecdysteroids are bound to EcR.[12] The putative MfR receptor in crustaceans is formed by the methoprene-tolerant coactivator protein (MET), which binds to methyl farnesoate (MF) and other juvenoid compounds, and the steroid receptor coactivator (SRC).[14,15] Activation of MET:SRC or RXR:EcR receptor complexes by their respective ligands promote lipid accumulation in a conditional manner since it requires ecdysteroids bound to EcR. When RXR:EcR is activated by ecdysteriods, juvenoids act additively and juvenoids and RXR agonists act cooperatively. RXR retinoid X receptor; PPARγ peroxisome proliferator-activated receptor γ; EcR ecdysteroid receptor. Predicted ligand–receptor interactions for single exposures (experiment 1), which assumes the presence of endogenous ecdysteriods (C) and mixtures (experiments 2 and 3) (D), are shown. Further details are in the Experimental Section.

In this study we explored the hypothesis that the regulation of storage lipid accumulation inside fat cells in Daphnia is regulated by three transcription factors: the heterodimer EcR:RXR and MfR (Figure B) that acts in a conditional manner as it has been described elsewhere.[12] Four functional aspects of the proposed receptor model were tested experimentally in three experiments: (1) agonists of RXR (TBT), EcR (20E), and MfR (MF, PP) should enhance the accumulation of storage lipids in postspawning females in vivo as far as there are enough ecdysteriods bound to EcR (Figure C). Conversely the ecdysone synthesis inhibitor FEN, and the ecdysteroid receptor antagonist T[8,21] should inhibit the accumulation of storage lipids (Figure C). In experiment 1 storage lipid accumulation responses were measured as Nile red fluorescence changes in D. magna individuals exposed to 20E, TBT, MF, PP, FEN, and T. Concentration–response curves were modeled and regression parameters were determined from the model of eq . (2) If premise 1 is true, mixtures among juvenoids should enhance storage lipid accumulation in an additive way predicted by the concentration addition model (Figure D, mix 4). (3) If premise 1 is true, mixtures of tributyltin and juvenoids or of the previous ligands with ecdysone should promote the accumulation of storage lipids in an additive manner predicted by the independent action model or in a cooperative way, more than additively (Figure D, mixes 1–3, 5, 10). Cooperative interactions among ligands of PPARγ and RXR promoting the accumulation of tryacylglicerols have been reported in mammalian adipocytes.[20,22] (4) Empty EcR may act as dominant corepressor impairing the transcription of genes involved in lipid metabolism and hence preventing accumulation of storage lipids (Figure D, 6–9). Such a mechanism is in line with the reported enhancement of the ecdysteroid-dependent activation of the EcR:RXR heterodimer by juvenoids and tributyltin.[12] In Drosophila unbound EcR also acts as a dominant corepressor of transcription whereas unbound RXR does not.[23]

Functional properties 2, 3, and 4 for the receptor model of Figure B were tested using single and binary combinations involving agonist of the three nuclear receptors and antagonists (FEN and T) in experiments 2 and 3 as it is depicted in Figure D.

Experiment 2

This aimed to determine joint effects of nine binary mixtures and their individual constituents simultaneously of selected compounds with agonists of the EcR (20E), MfR (MF, PP), and RXR (TBT), and with the ecdysone synthesis inhibitor (FEN), and with ecdysone receptor antagonist (T). Mixture combinations included low and high concentration effect responses of the selected compounds and followed a two-way ANOVA design that allowed for testing statistically for the null hypothesis that joint responses were additive and predicted by the independent action model, which means that compounds act dissimilarly disrupting storage lipid accumulation in lipid droplets.[24] Deviations from the null hypothesis was further tested comparing observed joint effects with those predicted by independent action and concentration addition concepts to asses concentration addition additivity, antagonistic or synergic deviations.[25]Mixtures are described in detail in the Supporting Information (SI “Methods”). Property 2 was tetsed with mixtures between MfR agonists (MF, PP) that should act additively and according to the concentration addition model promoting the accumulation of storage lipids; according to property 3 mixtures involving TBT with MF or PP, 20E with TBT, MF or PP should act additively and according to the independent action model, or alternatively, more than additive in a cooperative manner. Following property 4, mixtures of MF and TBT with FEN or T should not promote the accumulation of lipids.

Experiment 3

This test aimed to support results obtained in experiment 2 and to further test joint effects in mixtures involving 20E:MF (mix 1), MF:PP (mix 4), and MF:TBT (mix 5). The experiment was conceived to distinguish between additive effects following independent action, concentration addition predictions, and between antagonistic and synergistic effects.[25] One additional mixture: PP with TBT (mix 10) was included to provide more conclusive evidence that juvenoids act in a similar manner. Binary combinations of test compounds were conducted using a design in which each compound was dosed using a fixed ratio of the total concentration of the mixture (designs are provided in Table S1, SI Methods). For each studied pairing, fixed ratios of its mixture constituents were selected to maximize the observable response range. Designs were based on the regression responses obtained in single exposures of experiment 1. Joint effects of binary mixtures of model compounds were compared with model predictions of effects according to the models of CA and IA, respectively. These experiments served to determine whether EcR, RXR, and MfR ligands target similar or different nuclear receptors, behave antagonistically or synergically.

Finally, to identify positive and negative feedback mechanisms of ecdysteriods and juvenoids at the transcriptional level, changes in mRNA abundance on genes related to ecdysteriod (EcR, HR3, Neverland) and juvenoids (Hb2, MET) were studied under single and mixture exposures in experiment 4.[5,11] Treatments included exposure to low and high concentrations of 20E, MF, and PP alone and coexposures of MF, PP with 0.2 μM of 20E.

Nile Red Assay to Quantify Storage Lipids into Lipid Droplets

Quantification of storage lipids into lipid droplets follow previous methods[5] that are described in SI Methods.

Transcriptomic Analyses

Extraction, purification and quantification methods of mRNA from the studied genes and their primers follow previous procedures[5] that are described in SI Methods.

Chemical Analyses

Physicochemical water quality and test concentrations were monitored in freshly and old test solutions. Further information is in SI Methods.

Data Analyses

ANOVA Analyses

The null hypothesis of independent action for binary combinations in experiment 2 can be tested by determining the significance of the interaction in the two-way ANOVA carried out on log transformed observational data. A significant interaction term (p < 0.05) implies a statistically significant deviation from IA.[24]

Gene transcription responses obtained in experiment 4 in organisms exposed to the tested compounds alone were compared with those of controls using one way ANOVA followed by Dunnett’s post hoc test. Mixture effects were compared with those of single exposures of its constituents using ANOVA followed by Dunnett’s post hoc test. Prior to analyses, data were checked for ANOVA assumptions of normality and variance homoscedasticity. When necessary, log transformed data was tested. Significant values were adjusted to multiple comparisons using the Bonferroni correction.

Curve Fitting and Mixture Analyses

Quantitative prediction of single and combined effects was performed by adapting previously established approaches[26] that have been modified to fit responses having different Emax.[27] The procedure is fully explained in SI Methods. Concentration–response relationships for individual and mixture combinations were estimated using the three parameter Hill regression model of eq .where R(c) is percentage fluorescent change (%) at concentration c relative to controls, which was fixed to 0; Emax is maximal fluorescence effect in percent; c is concentration of compound (i); p is is the Hill index; EC50 is the concentration of compound that corresponds to 50% of the maximal effect.

Results

Effects on Storage Lipid Disruption Measured as Nile Red Fluorescence in Single Exposures

We hypothesized (premise 1) that agonists of the three receptors involved in the model depicted in Figures B and C (MF, PP, 20E, TBT) should enhance the accumulation of storage lipids, whereas antagonist of EcR (FEN, T) should inhibit it. Nile Red fluorescence changes relative to unexposed controls increased upon exposure to MF, PP, 20E, and TBT (Figure A), whereas FEN (Figure B) decreased them in a concentration related manner predicted by the Hill regression model (Table ). Testosterone (T) decreased significantly (P < 0.05; F9,70 = 2.1) storage lipids at the tested concentration range (Figure B), but it was not possible to fit its responses to the regression of eq . Higher concentrations than 20 μM of T were not tested since detrimental effects on growth and molt were observed (data not shown).

Figure 2

Responses and fitted regression curves to observed fluorescence changes of Nile Red relative to unexposed controls of D. magna individuals exposed to the studied chemicals alone. Each symbol is a single observation. For clarity testosterone results are depicted in graph B. Further information on the responses in graph A is found in the work of Jordão et al.[7] The * in graph B indicates significant (P < 0.05) differences from control (0 treatment) following ANOVA and Dunnett’s tests.

Table 1

Nile Red Fluorescence Assay Results of the Six Tested Compoundsa

	Emax ± SE	EC50 ± SE	p ± SE	r2	N
TBT (nM)	118 ± 10	2.4 ± 0.2	3.7 ± 0.6	0.87	90
MF (μM)	83 ± 14	0.34 ± 0.10	1.3 ± 0.3	0.72	88
PP (nM)	49 ± 16	1.2 ± 0.2	0.8 ± 0.1	0.61	70
20E (μM)	49 ± 4	0.27 ± 0.03	2.6 ± 0.7	0.69	78
FEN (μM)	–29 ± 3	0.5 ± 0.05	7.1 ± 6.7	0.79	40
Mixtures
mix 1 (MF/20E)b	24 ± 7	ud	ud	0.28	78
mix 4 (MF/PP)b	41 ± 4	0.19 ± 0.03	7.9 ± 3.6	0.57	80
mix 5 (MF/TBT)b	161 ± 22	0.27 ± 0.01	3.7 ± 0.6	0.67	80
mix 10 (PP/TBT)b	179 ± 6	0.003 ± < 0.001	5.3 ± 1.6	0.77	80

Emax, EC50, and p are the regression parameters of fitting Nile Red fluorescence to eq . All regression models and coefficients were significant (P < 0.05). Negative Emax values mean inhibition of fluorescence relative to unexposed solvent control individuals. N, sample size; SE, standard error.

Emax was computed as the mean fluorescence change at the highest tested mixture concentration; ud, undetermined regression parameters (not significantly (P < 0.05) different than 0).

Joint Effects of Binary Combinations

Up to 10 different binary mixtures were used to study interactive effects (i.e., deviations from the independent action model, IA) between agonists and antagonists of the three receptors depicted in Figure B and D. According to premises 2–4 we should expect that joint effects of agonists of the MfR should act additively as predicted by the concentration addition model (premise 2), that joint effects of agonists of the MfR, EcR, and RXR should be additive and predicted by the independent action model or more than additive (premise 3) and that joint effects of agonists of MfR and RXR with antagonists of EcR should be antagonistic (premise 4). Figures and 4 show results from binary combinations of single and combined exposures analyzed by a two way ANOVA. Only the pairing involving PP with MF (mix 4, Figure ) showed no evidence for interaction, and the combined effects were similar to those predicted by independent action model (IA, green circles, in Figure ). Further information on statistical results are in Table S3 (SI “Results”). Mixture treatments including 20E showed antagonistic effects at high exposure levels of MF, PP, or TBT (upper panel of graphs, mixes 1–3, Figure ) and synergic or additive effects at lower concentrations of MF and TBT, respectively; FEN and T acted antagonistically for all compounds tested (mixes 6–9 in Figure ).

Figure 3

Single and joint effects on Nile Red fluorescence changes relative to controls (mean ± SE, N = 10) of five binary mixture combinations and their mixture constituents of 20E, MF, PP, and TBT. White and filled bars represent single and binary mixture treatments, respectively. In each graph different letters mean significant (P < 0.05) differences among treatments following two-way ANOVA and Tukeỳs post hoc tests. – or + indicates absence or presence of a compound. Predicted joint effects following CA–concept of concentration addition and IA–concept of independent action are shown as red and green circles, respectively.

Figure 4

Single and joint effects on Nile Red fluorescence changes relative to controls (mean ± SE, N = 10) of four binary mixture combinations and their mixture constituents of FEN and T with MF and TBT. White and filled bars represent single and binary mixture treatments, respectively. In each graph different letters mean significant (P < 0.05) differences among treatments following two way ANOVA and Tukeỳs post hoc tests. – or + indicates absence or presence of a compound. Predicted joint effects following IA–concept of independent action are shown as green circles.

Three of the mixtures studied in Figure (mixes 1, 4, 5) plus that of TBT with PP (mix 10) were further tested using fixed ratio designs that facilitated testing for additivity and adequacy to predicted IA or CA responses. Figure compares dose–response curves from the individual constituents of the mixture, the observed joint effects as fitted regression curves (including 95% confidence intervals), and the predictions joint IA and CA models. Further information on fitted regression curves for mixture responses are in Table . Predicted curves for mixture constituents showed that the MF had the largest contributions in mixes 1 and 4. Mixture 1 (MF:20E) showed combined effects below those predicted by the IA and CA, whereas those of mix 4 (MF with PP, Figure ) were similar to those predicted by CA at effects levels higher than 25%. Thus, mixtures 4 and 1 suggest additive or less-than-additive interaction between juvenoids and juvenoids and ecdysteroids, respectively. Conversely, mixtures 5 (MF:TBT) and 10 (PP:TBT) showed larger-than-additive effects, suggesting a cooperative interaction between juvenoids and rexoids (Figure ), particularly at effect levels higher than 50%.

Figure 5

Concentration-effect-curves of fluorescence changes including 95% confidence intervals of D. magna individuals exposed to the studied four mixtures. Data from ten replicates per concentration are depicted. Predicted joint effects following CA–concept of concentration addition and IA–concept of independent action are also plotted. Estimated mixture constituents effects using eq and data from Table are shown. Labels 1, 2, 3, and 4 refer to mixture constituents MF, 20E, PP, and TBT, respectively.

Gene Transcription

Exogenous administration of ecdysone resulted in up and down-regulation of EcR and Hb2, respectively (Figure ).

Figure 6

Quantitative RT-PCR of mRNA of genes belonging to the ecdysteriod EcR, (HR3, Neverlan) and methyl farnesoate (Hg2) receptor signaling pathways (mean ± SE, N = 10). Single and mixture combination concentrations included 20E (0.2 μM), low (MF L, 0.2 μM) and high (MF H, 1 μM), and PP (5 nM). Different letters indicate significant (P < 0.05) treatment differences, following one way ANOVA and Tukey’s test. For clarity the y-axis for Hb2 is in log scale.

In addition, exposure to juvenoids (MF or PP) resulted in a strong increase of mRNA levels of the Hb2 gene, and, to a lesser extent, of those from genes EcR and HR3 (only PP).

In contrast, mRNA levels of the oxygenase gene neverland showed a significant decrease in the presence of 20E or MF. Ecdysone when coexposed with MF did not affect the transcription of EcR relative to single exposures, increased that of neverland and decreased that of Hb2 only at low concentrations (MF L). Ecdysone and PP mixtures decreased the transcripts of HR3 and neverland genes relative to single exposures of PP.

Discussion

Our tested hypothesis was that storage lipids accumulating across a molting/reproduction cycle were regulated by three hormonal receptor signaling pathways: RXR, EcR, and MfR and that the whole system was functional when EcR was activated by ecdysteriods. Agonists of the previous mentioned receptors (MF, PP for MfR; 20E for EcR and TBT for RXR) enhanced the accumulation of storage lipids in a concentration related manner (Figure , Table ). Free ecdysteriods are always present during normal growth, although their levels are relatively low before they peak just before molt.[8] This probably implicates a low, but constant, occupation of the EcR:RXR complex, which is therefore susceptible to be further activated by TBT and MF, according to the model proposed in Figure B and C, and in line with the work of Wang and LeBlanc.[12] These results, thus, support the first premise of the model proposed in Figure B that EcR:RXR and MfR act in a conditional manner when EcR is activated by ecdysteriods. Conversely, FEN, which is known to deplete the levels of ecdysone in D. magna,[8] and therefore to decrease the occupancy of the EcR ligand binding site, greatly diminished lipid droplet formation, in full support of premise 4. Testosterone, which it has been described to behave in vivo as a functional antagonist of the EcR receptor in Daphnia,[8,21] inhibited storage lipid accumulation only at high concentrations, thus acting as a partial anti-ecdysteroid and partly supporting premise 4.

Joint effects of juvenoids (MF and PP, see mix 4 in Figures and 5) enhanced the accumulation of storage lipids in an additive way predicted by the concentration addition model, which predicts additivity of similar acting chemicals, therefore, supporting premise 2 in Figure B and D. Joint effects of binary mixtures of TBT with MF or PP (mixes 5 and 10; Figures and 5) indicated that the RXR:EcR and the MfR signaling pathways acted more than additively enhancing storage lipid accumulation in lipid droplets in postspawning females, thus supporting premise 3 (Figure B and D). This means that these two receptors may act cooperatively in a similar way as PPAR and RXR do in in mammalian adipocytes.[20,22] Conversely enhancing lipid droplet accumulation by agonists of MfR (MF) and RXR (TBT) was neutralized or reversed in coexposures with low levels of the ecdysone synthesis inhibitor FEN or the partial ecdysone receptor antagonist T (mixes 6–9, Figure ). These results also support our hypothesis and indicated that observed enhanced levels of lipid droplets by agonists of RXR and MfR are ecdysteroid-dependent (premise 4, Figure B and D). Single and joint effects observed for T at low concentrations, however, indicated that this compound rather than acting as a partial antagonist, disrupted the interactions of EcR with MfR and RXR, and hence prevented the accumulation of storage lipids. Ecdysteroids initiate signaling of multiple pathways that control many processes of development, growth and reproduction in arthropods.[11] Ecdysteriod signaling in Daphnia involves the formation of heterodimeric complexes with RXR and the induction of downstream transcription factors that either positively or negatively regulate aspects of the pathway.[11,12,28−30] Therefore, it is possible that T may interact with other steroid responsive nuclear receptors/transcription factors, negatively regulating the EcR:RXR: MfR signaling pathway on storage lipids.

Storage lipids in Daphnia accumulated during the intermolt cycle and then are allocated to ecdysis and egg provisioning at the end of the cycle.[5,9,31] This means that the whole process is likely to be precisely regulated by ecdysteriods. Our premise that the empty EcR may act as dominant corepressor impairing the transcription of genes involved in lipid metabolism are also in line with the ecdysteriod- dependent activation of the Daphnia heterodimer EcR:RXR by juvenoids and TBT.[12]

The antagonistic effects of 20E when coadministered with MfR and RXR agonists indicate negative feedback mechanisms on storage lipid accumulation. Information reporting interactive effects of ecdysteriods on RXR and MfR signaling pathways or on the physiological processes regulated by them are limited and somewhat contradictory in Daphnia. Mu and LeBlanc[32] reported that ecdysteroids did not interfere with the juvenoid activity of PP (the production of male offspring). Conversely Wang et al.[29] found that exogenous administration of 20E increased the impairment of ecdysis by TBT. Antagonistic interactions between juvenoids and ecdysteroids during insect development have been postulated as resulting from competition between EcR and Met for SCR binding.[33] Alternatively, the same authors proposed that SRC:Met and SRC:EcR:RXR complexes may recruit different proteins depending on whether or not both hormones are present.[33] The first mechanism, combined with the reported ecdysteriod dependent nature of the EcR:RXR complex,[12] could explain the observed Daphnia responses across single and mixture exposures. When the levels of ecdysteriods bind to EcR are too low in single exposures with FEN or T or mixtures involving these compounds, the heterodimer receptor EcR:RXR is unstable, and thus it is unable to be activated by other agonists (i.e., TBT) or interact with other receptors (i.e., MfR) as proposed in Figure1B. Exogenous administration of ecdysone alone activates the three receptor model complex proposed in Figure B and enhances lipid accumulation since there is no competition for SRC. In coexposures of ecdysone with high levels of either MfR or RXR agonists (MF, PP, and TBT), competition to recruit SRC is high and consequently there is less activation of the ternary receptor complex.

Competition between EcR and Met for SCR binding[33] also explain the hormetic response observed in Figure in mixtures 1 and 3 show additive/synergic effects when coexposing 20E with low concentrations of MF or TBT, and antagonistic effects at higher concentrations of the same ligands. Pyrirproxyfen has higher affinity for MfR than MF[14] so competition between MfR and EcR for SRC binding is likely already high even at low concentrations of PP in mixture 2 (Figure ). This suggests that the paradoxical, inhibitory effect of 20E in the allocation of lipids when coadministered with other receptor agonists may also apply to different aspects of ecdysteroid/juvenoid signaling pathways.

Analysis of transcription of different genes demonstrated that negative feedback mechanisms between ecdysone and juvenoids dominated over positive ones. Exogenous administration of ecdysone and juvenoids up-regulated 1.2-fold the transcription of neverland gene relative to single exposures but down-regulated 1.6- and 29-fold those of HR3 and Hb2, respectively.

In summary results of single and mixture combinations of agonists of three nuclear receptors and those with the ecdysone synthesis inhibitor fenarimol and the ecdysteriod antagonist testosterone support most of the proposed premises of the three-receptor conceptual model for regulating lipid storage accumulation in D. magna (Figure B). Binary joint effects involving ecdysone, however, were mostly antagonistic, which support the hypothesis that SRC is involved in MfR and EcR:RXR receptor complexes and there is competition for binding to this coactivator.[33] Gene transcription responses were affected mostly antagonistically in mixtures involving ecdysone and juvenoids, thus supporting the argument that cross-talk between ecdysteriods and juvenoids could affect negatively the accumulation of storage lipids at the transcriptional level, which is also in line with the competition SRC binding hypothesis. Note, however, that alternative mechanisms of interaction between MfR and EcR:RXR such as activation/competition at similar promoters, induction of the same genes, or competition for similar resources other than SCR that may cause these interactions are also possible and should be further study.

Daphnia genome contains several other nuclear receptors already known in other species to regulate lipid metabolism (i.e., HNF4, HR96, ERR, HR78[34]). Some of these receptors (HR78) are also regulated during the molt cycle in crustaceans,[35] while others (i.e., HR96) are regulated by fatty acids and some pollutants in Daphnia.[36] The Daphnia HR96 receptor is orthologous to CAR/PXR/VDR mammalian receptors,[34] which are known to form heterodimeric transactivation complexes with RXR.[37] This means that observed effects of TBT on storage lipid accumulation could be also mediated by HR96:RXR receptor complexes. Recent studies, however, were unable to relate HR96 activity with specific changes on polar lipids.[38] There is also experimental evidence indicating that MF binds to the RXR in later larval stages of Drosophila.[39] The evidence for the requirement of ecdysteriods for MF and PP to activate the RXR:EcR receptor complex[12] and for the role of MfR in the process[14] come essentially from reporter assays and/or in vitro methods; we think that our observations represent an in vivo validation of these results. While the proposed mechanistic ternary-receptor model may explain how lipid metabolism is regulated in Daphnia, further research is needed to study more deeply the involvement of SRC on the proposed three receptor complex and of other nuclear receptors/coactivators (i.e., HR78, HR96, ERR) on lipid metabolism.