OPEN ACCESS DOCUMENT

Environ. Sci. Technol. 2016, 50, 13565−13573

DOI: 10.1021/acs.est.6b04768

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Environ. Sci. Technol. 2016, 50, 13565−13573 ( open access)

Mechanisms of action of compounds that enhance storage lipid accumulation in Daphnia magna.

Rita Jordão1,2, Bruno Campos1, Benjamín Piña 1, Romà Tauler1, Amadeu M.V.M. Soares 2 and Carlos Barata1*

1Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), Spanish Research Council (IDAEA, CSIC), Jordi Girona 18,

08034 Barcelona, Spain2 Centre for Environmental and Marine studies (CESAM), Department of Biology, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro , Portugal.

*Address correspondence to Carlos Barata, Institute of Environmental Assessment

and Water Research (IDAEA-CSIC), Jordi Girona 18, 08034 Barcelona, Spain.

Telephone: ± 34-93-4006100. Fax: ± 34-93-2045904. E-mail: cbmqam@cid.csic.es

Funding: This work was funded by the Spanish Ministry of Science and Innovation

project (CTM2014-51985-R) and by the Advance grant of the European Research

Foundation ERC-2012-AdG-320737. Funders had no role in study design, data collection

and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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ABSTRACT

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 de-regulation 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.

Keywords: obesogen, metabolic disruption, nuclear receptor, arthropod, reproduction,

juvenile receptor

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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 post-spawning 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.

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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 of TBT, agonists of EcR (20-

hydroxyecdysone), of RXR (tributyltin), and of 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 anti-

ecdysteriods 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 ligands13.

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 co-activator (SRC) 14, 15. Juvenoids promote expression of hemoglobin

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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 (5x105

cells/ml of Chlorella vulgaris), as described in Barata and Baird 18.

Experimental procedures6

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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 5 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 pre-adipocytes 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.

In this study we explored the hypothesis that the regulation of storage lipid accumulation

inside fat cells in Daphnia is regulated by three nuclear receptors: the heterodimer

EcR:RXR and MfR (Fig 1B) that acts in a conditional manner as it has been described

elsewhere 12. Four functional aspects of the proposed receptor model were tested

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experimentally in three experiments: 1) agonists of RXR (TBT), EcR (20E) and MfR

(MF, PP) should enhance the accumulation of storage lipids in post-spawning females in

vivo as far as there are enough ecdysteriods bound to EcR (Fig 1C). Conversely the

ecdysone synthesis inhibitor FEN, and the ecdysteroid receptor antagonist T8, 21 should

inhibit the accumulation of storage lipids (Fig 1C). 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 modelled and regression parameters were determined from the model of eq 1. 2) If

premise 1 is true, mixtures among juvenoids should enhance storage lipid accumulation in

an additive way predicted by the concentration addition model (Fig 1 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

(Fig 1D, Mix 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 co-repressor impairing the

transcription of genes involved in lipid metabolism and hence preventing accumulation of

storage lipids (Fig 1D, 6-9). Such 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 co-repressor of

transcription whereas unbound RXR does not 23.

Functional properties 2,3 and 4 for the receptor model of Figure 1B 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 1D.

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Experiment 2: 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 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: 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

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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 co-exposures 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

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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’s

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. 1.

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R(ci )=E max

100+(EC 50c i )

p(1 )

where

R(ci) – Percentage fluorescent change (%) at concentration ci relative to controls, which

was fixed to 0

Emax – maximal fluorescence effect in %.

ci – concentration of compound (i)

p – is the Hill index

EC50 -. the concentration of compound that corresponds to 50% of the maximal effect.

RESULTS

Effects on Storage lipids 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 1B,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 (Fig 2A), whereas FEN (Fig 2B) decreased them in a concentration related

manner predicted by the Hill regression model (Table 1). Testosterone (T) decreased

significantly (P<0.05; F 9,70 =2.1) storage lipids at the tested concentration range (Fig 2B),

but it was not possible to fit its responses to the regression of eq 1. Higher concentrations

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than 20 µM of T were not tested since detrimental effects on growth and molt were

observed (data not shown).

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 1B,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 3 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 3) showed no evidence for interaction,

and the combined effects were similar to those predicted by independent action model

(IA, green circles, in Figure 3). 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, Mix 1-3, Figure 3) and

synergic or additive effects at lower concentrations of Mf and TBT, respectively; FEN

and T acted antagonistically for all compounds tested (Mix 6-9 in Figure 4).

Three of the mixtures studied in Figure 3 (Mix 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 5 compares dose-response curves from

the individual constituents of the mixture, the observed joint effects as fitted regression

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curves (including 95% confidence intervals), and the predictions joint IA and CA models.

. Further information of fitted regression curves for mixture responses are in Table 1.

Predicted curves for mixture constituents showed that the MF had the largest

contributions in Mix 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 5) were

similar to those predicted by CA at effects levels higher than 25%. Thus Mix 1 and 4

suggest additive or less-than additive interaction between juvenoids and juvenoids and

ecdysteroids, respectively. Conversely, mixtures 5 (MF:TBT) and 10 (PP:TBT) showed

large-than-additive effects, suggesting a cooperative interaction between juvenoids and

rexoids (Figure 5), particularly at effect levels higher than 50%.

Gene transcription

Exogenous administration of ecdysone resulted in up and down-regulation of EcR and Hb2, respectively (Figure 6). In addition, exposure to juvenoids (MF or PP) resulted in a strong increase of mRNA levels of the Hb2 gene, and, in a less extend, 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 co-exposed 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.

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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 2, Table 1). 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 1B, C,

and in line with Wang and LeBlanc 12. These results, thus, support the first premise of the

model proposed in Figure 1B 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 3,5 ) 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 1B,D. Joint effects of binary mixtures of TBT with MF or PP (Mix

5, 10, Figures 3, 5) indicated that the RXR:EcR and the MfR signaling pathways acted

more than additively enhancing storage lipid accumulation in lipid droplets in post-

spawning females, thus supporting premise 3 (Figure 1B, 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 co-exposures with low levels of the

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ecdysone synthesis inhibitor FEN or the partial ecdysone receptor antagonist T (Mix 6-9,

Figure 4). 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 1B, 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 signalling of multiple pathways that control many processes

of development, growth and reproduction in arthropods11. Ecdysteriod signalling 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 pathway11, 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 signalling 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 co-repressor impairing the transcription of genes involved in

lipid metabolism are also in line with the ecdysteriod- dependant activation of the

Daphnia heterodimer EcR: RXR by juvenoids and TBT 12.

The antagonistic effects of 20E when co-administered 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

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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

binding33. 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

present33. The first mechanism, combined with the reported ecdysteriod dependent nature

of the EcR:RXR complex12, 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 Fig1B. Exogenous

administration of ecdysone alone activates the three receptor model complex proposed in

Fig 1B and enhances lipid accumulation since there is no competition for SRC. In co-

exposures 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 binding33 also explain the s hormetic

response observed in Figure 3 in Mix 1 and 3 that shows additive/ synergic effects when

co-exposing 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

MF14 so competition between MfR and EcR for SRC binding is likely already high even

at low concentrations of PP in Mix 2 (Fig 3). This suggests that the paradoxical,

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inhibitory effect of 20E in the allocation of lipids when co-administered 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 1B).

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 co-activator33. 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.

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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 receptors34, which are

known to form heterodimeric transactivation complexes with RXR37. 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 evidences

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 process14 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/co-activators (i.e. HR78, HR96, ERR) on lipid metabolism.

ASSOCIATED CONTENT

Supporting Information

Details of experimental procedures and further explanation of results: This material is

available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

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Corresponding Author

Address: Jordi Girona 18, 08034 Barcelona, Spain. Email: cbmqam@cid.csic.es .

Telephone: (34)93-400-6100

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This study was supported by the Spanish MEC CTM2014-51985-R and Advance grant of

the European Research Foundation ERC-2012-AdG-320737. The Portuguese Foundation

for Science and Technology (FCT), supported the doctoral fellowship of Rita Jordão

(SFRH/BD/79453/2011).

ABBREVIATIONS

CA Concentration additionIA Independent actionMF Methyl farnesoate20E 20-hydroxyecdysonePP PyriproxyfenTBT TributyltinFEN FeniramolT Testosterone

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Figure 1. Simplified scheme of RXR:PPAR involvement in mammalian adipocyte differentiation and triacylglycerol accumulation 19 (A) and of the putative involvement of EcR : RXR and SCR: MET receptor complexes signalling pathways in regulating lipid storage accumulation in Daphnia magna fat cells (B). In scheme B, EcR and RXR forms an 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 co-activator (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; PPARperoxisome proliferator-activated receptor EcR – ecdysteroid receptor. Predicted ligand- receptor interactions for single exposures (experiment 1), which assumes the presence of endogenous ecdysteriods (C ) and mixtures (experiments 2,3) (D) are shown. Further details are in experimental section.

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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 Jordao et al.7 * in graph B indicates significant (P<0.05) differences from control (0 treatment) following ANOVA and Dunnett’s tests.

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vvvv Figure 3.

26Figure 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 means significant (P<0.05) differences among treatments following two way ANOVA and Tukey`s 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.

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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 TE with MF and TBT. White and filled bars represent single and binary mixture treatments, respectively. In each graph different letters means significant (P<0.05) differences among treatments following two way ANOVA and Tukey`s 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.

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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 1 and data from Table 1 are shown. Labels 1,2,3,4 refer to mixture constituents MF, 20E, PP and TBT, respectively.

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Figure 6. Quantitative RT-PCR of mRNA of genes belonging to the ecdysteriod EcR, HR3, Neverlan) and methyl farnesoate (Hg2) receptor signalling 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 y axis for Hb2 are in log scale.

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Table 1. Nile red fluorescence assay results of the six tested compounds. Emax, EC50 and

p are the regression parameters of fitting Nile red fluorescence to eq. 1 . 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 ± 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)* 24 ± 7 ud ud 0.28 78

Mix 4(MF/PP)* 41 ± 4 0.19 ± 0.03 7.9 ± 3.6 0.57 80

Mix 5 (MF/TBT) * 161± 22 0.27 ± 0.01 3.7 ± 0.6 0.67 80

Mix 10 (PP/TBT)* 179 ± 6 0.003± <0.001 5.3 ± 1.6 0.77 80

*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).

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