Chemicals. Tributyltin chloride (TBT; CAS No. 1461-22-9) and Nile red (CAS No. 7385-67-3) were purchased from Sigma-Aldrich, and lipid standards were from Advanti Polar Lipids. All other chemicals were analytical grade and were obtained from Merck.
TBT treatments. TBT was dissolved in acetone; the same amount of acetone (< 0.1 mL/L) was used for a solvent control and in all experimental treatments except in the untreated control (control) to account for any carrier effect. Actual TBT concentrations in test solutions were measured as total tin using a Perkin-Elmer Elan 6000 inductively coupled plasma mass spectrometer (ICP-MS) (Barata et al. 2005 (link)), and were confirmed to be within 10% of nominal concentrations (0.036 and 0.36 μg/L for 0.1 and 1 μg/L doses, respectively).
Experimental animals. All experiments were performed using the well-characterized clone F of D. magna maintained indefinitely as pure parthenogenetic cultures (Barata and Baird 1998 ). Individual cultures were maintained in 100 mL of ASTM hard synthetic water at low and high food-ration levels (Chlorella vulgaris, 1 × 105 and 5 × 105 cells/mL, respectively), as described by Barata and Baird (1998) .
Experimental design. Experiments were initiated with newborn neonates < 4–8 hr old obtained from synchronized females cultured individually at high food-ration levels. Groups of five neonates (F0) were reared in 150 mL of ASTM hard water under high food-ration conditions until the end of the third juvenile instar (about 4–8 hr before molting for the third time). At this point, juveniles were used in three sets of experiments using two TBT treatments, 0.1 μg/L (low; TBT L) and 1 μg/L (high; TBT H). Five to 10 replicates per treatment were used.
The first experiment studied effects of exposure to TBT during the adolescent instar (i.e., 3 days) on the life history of these females (F0) through five consecutive clutches. Their first clutch of neonates, exposed during the egg-provisioning stage (F1) was similarly studied during four consecutive clutches. Following exposure to TBT, F1 females were cultured individually under high food conditions without TBT, and their growth and reproduction performance monitored until the fifth clutch. The tolerance of F1 neonates to starving conditions was studied monitoring the time to death of 10 neonates individually cultured in 50 mL of ASTM hard water alone. The medium was renewed every day. Life-history performance of F1 neonates was studied by culturing them individually in 100 mL of ASTM hard water at high food conditions until the release of the fourth clutch. Measured life-history traits were survival, reproduction, body length of each adult instar (including that of the adolescent instar), age at first reproduction, the size of neonates of each clutch, and the population growth rate (r) estimated from the age-dependent survival and reproduction rates according to the Lotka equation (Barata et al. 2001 ).
The second set of experiments (experiments 1 and 2) aimed to study lipid droplet changes across food and TBT treatments using the Nile red assay. In experiment 1, animals were exposed to three food regimes: starving (no food added), low food (1 × 105 cells/mL C. vulgaris), and high food (5 × 105 cells/mL C. vulgaris). In experiment 2, animals were exposed to two TBT concentrations (TBT L and TBT H) across low and high food levels. Exposures lasted through all the adolescent instars, and females were sampled just after their fourth molt and having released their eggs into the brood pouch (48 hr), as shown in Supplemental Material, Figure S1.
The third set of experiments aimed to determine effects of TBT L and TBT H on the dynamics of lipids, lipid droplets, and mRNA levels of selected genes across an entire adolescent intermolt cycle. Experiments were conducted only at high food levels and included five samplings: 0 hr (just after the third molt), 8 hr, 16 hr, 24 hr, and just after the fourth molt (48 hr). At each sampling, three and five replicates of 5 individuals were collected and processed for total lipid determination and mRNA gene transcription measurement, respectively, and 10 animals were processed for Nile red determination. At the 48-hr sampling period, females were de-brooded by gently flushing water into the brood pouch. Obtained eggs and de-brooded females were then collected and used for lipid and gene transcription analyses. Because of the large number of synchronized animals needed, three different independent but consecutive experiments were performed and used for lipidomic, gene transcription, and Nile red determinations, respectively.
Nile red determination. The Nile red stock solution was prepared in acetone and stored protected from light following Tingaud-Sequeira et al. (2011) (link). Just before use, the working solution was prepared by diluting the stock solution to 1.5 μM in ASTM. Live individuals were then exposed to Nile red working solution in the dark for 1 hr at 20°C. After incubation, animals were placed in 100 mL ASTM for 1 min to allow clearance of Nile red residuals. Following clearance, animals were placed individually in 1.5-mL centrifuge tubes, the remaining water was removed, and samples were sonicated in 300 μL of isopropanol. The homogenized extract was then centrifuged at 10,000 × g. We used 200 μL of supernatant to measure Nile red fluorescence using an excitation/emission wavelength of 530/590 nm and a microplate fluorescence reader (Synergy 2, BioTek). Each treatment had one animal per sample (10 replicates in total). For each quantification and treatment, 10 blanks (animals not exposed to Nile red) were used to account for background levels of fluorescence. After exposure to Nile red, images were taken in the area surrounding the midgut for visualization of lipid droplets. Fluorescence and bright file images were obtained using a Nikon SMZ1500 microscope and a Nikon Intensilight C-HGFI with a GFP filter (EX 472/30, EM 520/35; Nikon).
Lipidomic analyses. Lipidomic analyses were performed as described by Gorrochategui et al. (2014) (link), with minor modifications. Each replicate consisted of a pool of five animals that were homogenized in 500 μL phosphate-buffered saline (PBS), pH 7.4, with 2,6-di-tert-butyl-4-methylphenol (BHT; 0.01%) as an antioxidant. Lipid extraction was performed using a modification of Folch’s method (Folch et al. 1957 (link)). Briefly, 100 μL of the homogenized sample was mixed with 500 μL of chloroform and 250 μL of methanol. Internal standards (200 pmol) (described in Supplemental Material, Table S1) were also added. Samples were heated at 48°C overnight and dried under N2 the next day. Lipid extracts were solubilized in 150 μL methanol. The liquid chromatograph–mass spectrometer consisted of a Waters Aquity UPLC system connected to a Waters LCT Premier Orthogonal Accelerated Time of Flight Mass Spectrometer (Waters) operated in positive and negative electrospray ionization (ESI) mode. Full-scan spectra from 50 to 1,500 Da were obtained. Mass accuracy and reproducibility were maintained by using an independent reference spray (LockSpray; Waters). A 100-mm × 2.1-mm i.d., 1.7-μm C8 Acquity UPLC BEH (Waters) analytical column was used. Further chromatographic details of mobile phases were described by Gorrochategui et al. (2014) (link).
Quantification was carried out using the ion chromatogram obtained for each compound using 50-mDa windows. The linear dynamic range was determined by injection of standard mixtures. Positive identification of compounds was based on the accurate mass measurement, with an error < 5 mg/L, and its LC retention time compared with that of a standard (± 2%).
A total of 116 lipids were identified and quantified by UPLC-TOF ESI-positive mode that were distributed as follows: five classes of glycerophospholipids [phosphocholine (PC) with 20 lipids, lysophosphatidylcholine (LPC) with 6 lipids, phosphatidylethanolamine (PE) with 9 lipids, phosphatidylserine (PS) with 7 lipids, and phosphatidylinositol (PI) with 3 lipids]; diacylglycerols (DG) with 20 lipids; triacylglycerols (TG) with 39 lipids; cholesterylesters (CE) with 4 lipids; and sphingolipids (SM) with 8 lipids. Glycerophospholipids, diacylglycerol, triacylglycerol, and cholesterylesters were annotated as :. Sphingolipids were annotated as :.
Transcriptomic analyses. Methods of extraction, purification, and quantification of mRNA from the studied genes and their primers follow previous procedures (Campos et al. 2013 (link)). Eight genes were selected for representation of different pathways/gene families: EcRB, HR3, HR38, Neverland, Hb2, RXR, MET, and SRC. The gene glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was used as an internal control. For each of the genes, primers were designed using Primer Quest (IDT Technologies) and are listed in Supplemental Material, Table S2. Aliquots of 10 ng were used to quantify specific transcripts in a LightCycler® 480 real-time PCR system (Roche) using LightCycler 480 SYBR Green I Master® (Roche). Relative abundance values of all genes were calculated from the second derivative of their respective amplification curve (Cp; crossing point) values calculated by technical triplicates. Cp values of target genes were compared with the corresponding reference genes.
Data analyses. The effect of food rations and/or treatment or sampling period or juvenile stage on Nile red fluorescence, lipidomic profiles, mRNA abundance, and life history and physiological responses were analyzed by two-way and/or one-way analysis of variance (ANOVA). Post hoc Dunnett’s or Tukey’s tests were performed to compare exposure treatments with solvent controls. Prior to analyses, all data except survival responses were log transformed to achieve normality and variance homoscedasticity. If not indicated otherwise, significance levels were set at p < 0.05. Survival responses were assessed by Wilcoxon-Gehan tests. Tests were performed with IBM-SPSS statistics software, version 19. Lipidomic data were further analyzed using cluster and K-means analyses in R (R Core Team 2014 ) to identify clusters of lipid families similarly affected by TBT.
TBT treatments. TBT was dissolved in acetone; the same amount of acetone (< 0.1 mL/L) was used for a solvent control and in all experimental treatments except in the untreated control (control) to account for any carrier effect. Actual TBT concentrations in test solutions were measured as total tin using a Perkin-Elmer Elan 6000 inductively coupled plasma mass spectrometer (ICP-MS) (Barata et al. 2005 (link)), and were confirmed to be within 10% of nominal concentrations (0.036 and 0.36 μg/L for 0.1 and 1 μg/L doses, respectively).
Experimental animals. All experiments were performed using the well-characterized clone F of D. magna maintained indefinitely as pure parthenogenetic cultures (Barata and Baird 1998 ). Individual cultures were maintained in 100 mL of ASTM hard synthetic water at low and high food-ration levels (Chlorella vulgaris, 1 × 105 and 5 × 105 cells/mL, respectively), as described by Barata and Baird (1998) .
Experimental design. Experiments were initiated with newborn neonates < 4–8 hr old obtained from synchronized females cultured individually at high food-ration levels. Groups of five neonates (F0) were reared in 150 mL of ASTM hard water under high food-ration conditions until the end of the third juvenile instar (about 4–8 hr before molting for the third time). At this point, juveniles were used in three sets of experiments using two TBT treatments, 0.1 μg/L (low; TBT L) and 1 μg/L (high; TBT H). Five to 10 replicates per treatment were used.
The first experiment studied effects of exposure to TBT during the adolescent instar (i.e., 3 days) on the life history of these females (F0) through five consecutive clutches. Their first clutch of neonates, exposed during the egg-provisioning stage (F1) was similarly studied during four consecutive clutches. Following exposure to TBT, F1 females were cultured individually under high food conditions without TBT, and their growth and reproduction performance monitored until the fifth clutch. The tolerance of F1 neonates to starving conditions was studied monitoring the time to death of 10 neonates individually cultured in 50 mL of ASTM hard water alone. The medium was renewed every day. Life-history performance of F1 neonates was studied by culturing them individually in 100 mL of ASTM hard water at high food conditions until the release of the fourth clutch. Measured life-history traits were survival, reproduction, body length of each adult instar (including that of the adolescent instar), age at first reproduction, the size of neonates of each clutch, and the population growth rate (r) estimated from the age-dependent survival and reproduction rates according to the Lotka equation (Barata et al. 2001 ).
The second set of experiments (experiments 1 and 2) aimed to study lipid droplet changes across food and TBT treatments using the Nile red assay. In experiment 1, animals were exposed to three food regimes: starving (no food added), low food (1 × 105 cells/mL C. vulgaris), and high food (5 × 105 cells/mL C. vulgaris). In experiment 2, animals were exposed to two TBT concentrations (TBT L and TBT H) across low and high food levels. Exposures lasted through all the adolescent instars, and females were sampled just after their fourth molt and having released their eggs into the brood pouch (48 hr), as shown in Supplemental Material, Figure S1.
The third set of experiments aimed to determine effects of TBT L and TBT H on the dynamics of lipids, lipid droplets, and mRNA levels of selected genes across an entire adolescent intermolt cycle. Experiments were conducted only at high food levels and included five samplings: 0 hr (just after the third molt), 8 hr, 16 hr, 24 hr, and just after the fourth molt (48 hr). At each sampling, three and five replicates of 5 individuals were collected and processed for total lipid determination and mRNA gene transcription measurement, respectively, and 10 animals were processed for Nile red determination. At the 48-hr sampling period, females were de-brooded by gently flushing water into the brood pouch. Obtained eggs and de-brooded females were then collected and used for lipid and gene transcription analyses. Because of the large number of synchronized animals needed, three different independent but consecutive experiments were performed and used for lipidomic, gene transcription, and Nile red determinations, respectively.
Nile red determination. The Nile red stock solution was prepared in acetone and stored protected from light following Tingaud-Sequeira et al. (2011) (link). Just before use, the working solution was prepared by diluting the stock solution to 1.5 μM in ASTM. Live individuals were then exposed to Nile red working solution in the dark for 1 hr at 20°C. After incubation, animals were placed in 100 mL ASTM for 1 min to allow clearance of Nile red residuals. Following clearance, animals were placed individually in 1.5-mL centrifuge tubes, the remaining water was removed, and samples were sonicated in 300 μL of isopropanol. The homogenized extract was then centrifuged at 10,000 × g. We used 200 μL of supernatant to measure Nile red fluorescence using an excitation/emission wavelength of 530/590 nm and a microplate fluorescence reader (Synergy 2, BioTek). Each treatment had one animal per sample (10 replicates in total). For each quantification and treatment, 10 blanks (animals not exposed to Nile red) were used to account for background levels of fluorescence. After exposure to Nile red, images were taken in the area surrounding the midgut for visualization of lipid droplets. Fluorescence and bright file images were obtained using a Nikon SMZ1500 microscope and a Nikon Intensilight C-HGFI with a GFP filter (EX 472/30, EM 520/35; Nikon).
Lipidomic analyses. Lipidomic analyses were performed as described by Gorrochategui et al. (2014) (link), with minor modifications. Each replicate consisted of a pool of five animals that were homogenized in 500 μL phosphate-buffered saline (PBS), pH 7.4, with 2,6-di-tert-butyl-4-methylphenol (BHT; 0.01%) as an antioxidant. Lipid extraction was performed using a modification of Folch’s method (Folch et al. 1957 (link)). Briefly, 100 μL of the homogenized sample was mixed with 500 μL of chloroform and 250 μL of methanol. Internal standards (200 pmol) (described in Supplemental Material, Table S1) were also added. Samples were heated at 48°C overnight and dried under N2 the next day. Lipid extracts were solubilized in 150 μL methanol. The liquid chromatograph–mass spectrometer consisted of a Waters Aquity UPLC system connected to a Waters LCT Premier Orthogonal Accelerated Time of Flight Mass Spectrometer (Waters) operated in positive and negative electrospray ionization (ESI) mode. Full-scan spectra from 50 to 1,500 Da were obtained. Mass accuracy and reproducibility were maintained by using an independent reference spray (LockSpray; Waters). A 100-mm × 2.1-mm i.d., 1.7-μm C8 Acquity UPLC BEH (Waters) analytical column was used. Further chromatographic details of mobile phases were described by Gorrochategui et al. (2014) (link).
Quantification was carried out using the ion chromatogram obtained for each compound using 50-mDa windows. The linear dynamic range was determined by injection of standard mixtures. Positive identification of compounds was based on the accurate mass measurement, with an error < 5 mg/L, and its LC retention time compared with that of a standard (± 2%).
A total of 116 lipids were identified and quantified by UPLC-TOF ESI-positive mode that were distributed as follows: five classes of glycerophospholipids [phosphocholine (PC) with 20 lipids, lysophosphatidylcholine (LPC) with 6 lipids, phosphatidylethanolamine (PE) with 9 lipids, phosphatidylserine (PS) with 7 lipids, and phosphatidylinositol (PI) with 3 lipids]; diacylglycerols (DG) with 20 lipids; triacylglycerols (TG) with 39 lipids; cholesterylesters (CE) with 4 lipids; and sphingolipids (SM) with 8 lipids. Glycerophospholipids, diacylglycerol, triacylglycerol, and cholesterylesters were annotated as
Transcriptomic analyses. Methods of extraction, purification, and quantification of mRNA from the studied genes and their primers follow previous procedures (Campos et al. 2013 (link)). Eight genes were selected for representation of different pathways/gene families: EcRB, HR3, HR38, Neverland, Hb2, RXR, MET, and SRC. The gene glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was used as an internal control. For each of the genes, primers were designed using Primer Quest (IDT Technologies) and are listed in Supplemental Material, Table S2. Aliquots of 10 ng were used to quantify specific transcripts in a LightCycler® 480 real-time PCR system (Roche) using LightCycler 480 SYBR Green I Master® (Roche). Relative abundance values of all genes were calculated from the second derivative of their respective amplification curve (Cp; crossing point) values calculated by technical triplicates. Cp values of target genes were compared with the corresponding reference genes.
Data analyses. The effect of food rations and/or treatment or sampling period or juvenile stage on Nile red fluorescence, lipidomic profiles, mRNA abundance, and life history and physiological responses were analyzed by two-way and/or one-way analysis of variance (ANOVA). Post hoc Dunnett’s or Tukey’s tests were performed to compare exposure treatments with solvent controls. Prior to analyses, all data except survival responses were log transformed to achieve normality and variance homoscedasticity. If not indicated otherwise, significance levels were set at p < 0.05. Survival responses were assessed by Wilcoxon-Gehan tests. Tests were performed with IBM-SPSS statistics software, version 19. Lipidomic data were further analyzed using cluster and K-means analyses in R (R Core Team 2014 ) to identify clusters of lipid families similarly affected by TBT.
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