BUSCADOR RECOLECTA

1 figure. -- Effect of Ph on the stability of trypsin (TRY), chymotripsin (CHY), leucine aminopeptidase (LAP), lipase (LIP) and pepsin (PP) activities from pyloric caeca and stomach of S. Dumerili adapted to different rearing temperatures (18, 22 and 26º C)., The study of fish digestive biochemistry is essential to understand factors that affect the net efficiency of food transformation and growth, and therefore aquaculture profitability. The aim of the present study was to assess the activity and functional characteristics of key digestive enzymes in juveniles of greater amberjack (Seriola dumerili), as well as the possible modulation of their relative importance by water temperature. For that, a combination of biochemical assays and substrate-SDS-PAGE were used. Under physiological conditions pepsin activity was negligible. Chymotrypsin was the most active enzyme in the digestive tract of the greater amberjack, while lipase was the enzyme with lower activity, though both enzymes in addition to trypsin were responsive to water temperature as revealed by discriminant analysis. Seriola dumerili showed to have pH-sensitive and, except for chymotrypsin, thermally robust proteases. Inhibition assays showed the major importance of serine proteases and revealed inverse trypsin and chymotrypsin responses to environmental temperature, with higher trypsin contribution in 26°C-fish while higher chymotrypsin contribution in 18°C-fish. Zymograms revealed three isotrypsin and three isochymotrypsin enzymes, with no variation in the presence of particular isoforms among rearing temperatures. However, they confirmed the role of chymotrypsin activity in providing digestive plasticity, with one of the isoforms being more active at lower temperatures. Thus, results indicate that variation in the relative contribution of chymotrypsin isoenzymes to a particular environmental temperature occurs due to different physic-chemical features of isoforms as a source of functional flexibility. This study assessed for the first time the effects of rearing temperature on greater amberjack digestive enzymes, increasing the knowledge on its digestive biochemistry, and aiding in the improvement of management practices for this species industrialization., Peer reviewed

1 figure. -- 13% substrate SDS-PAGE showing caseinolysc activity bands in pyloric caeca extracts of individual S. dumerili adapted to different rearing temperatures (18, 22 and 26º C). Get incubation temperature 37º C., The study of fish digestive biochemistry is essential to understand factors that affect the net efficiency of food transformation and growth, and therefore aquaculture profitability. The aim of the present study was to assess the activity and functional characteristics of key digestive enzymes in juveniles of greater amberjack (Seriola dumerili), as well as the possible modulation of their relative importance by water temperature. For that, a combination of biochemical assays and substrate-SDS-PAGE were used. Under physiological conditions pepsin activity was negligible. Chymotrypsin was the most active enzyme in the digestive tract of the greater amberjack, while lipase was the enzyme with lower activity, though both enzymes in addition to trypsin were responsive to water temperature as revealed by discriminant analysis. Seriola dumerili showed to have pH-sensitive and, except for chymotrypsin, thermally robust proteases. Inhibition assays showed the major importance of serine proteases and revealed inverse trypsin and chymotrypsin responses to environmental temperature, with higher trypsin contribution in 26°C-fish while higher chymotrypsin contribution in 18°C-fish. Zymograms revealed three isotrypsin and three isochymotrypsin enzymes, with no variation in the presence of particular isoforms among rearing temperatures. However, they confirmed the role of chymotrypsin activity in providing digestive plasticity, with one of the isoforms being more active at lower temperatures. Thus, results indicate that variation in the relative contribution of chymotrypsin isoenzymes to a particular environmental temperature occurs due to different physic-chemical features of isoforms as a source of functional flexibility. This study assessed for the first time the effects of rearing temperature on greater amberjack digestive enzymes, increasing the knowledge on its digestive biochemistry, and aiding in the improvement of management practices for this species industrialization., Peer reviewed

Composition of experimental WD; sensitivity and inter/intra assay precision of the assays used in this study; primer sequences and qPCR conditions; flow cytometry antibodies characteristics; amino acid composition of LPH; raw data of antioxidant parameters; representative standard curve of the antioxidant assays used in this study; body weight monitored over time; and cell proliferation and cell viability., Peer reviewed

14 pages. -- Supplementary description of methods: 1.1. Chemical analysis of pesticides: extraction procedure and LC-MS/MS analysis; 1.2. Acetylcholinesterase (AChE) activity. -- Table S1. MRM conditions used in UPLC-MS/MS determination of pesticides selected. Two transitions (precursor ion →product ion) of each compound were analyzed. -- Table S2. Ranges of nominal and quantified concentrations in μg/L for the two pesticides used and the metabolite analyzed in the colonization assays. -- Table S3. Results of the Chi-square test to check statistically significant differences between the percentage of colonization achieved by the organisms in each chamber at 48h in the pesticide experiments and the expected value (100%). -- Table S4. Results of the within-group Kruskal-Wallis test to check statistically significant differences between chambers (source of variation) for the percentage of colonization of D. magna achieved at 48h in each pesticide experiment, together with the significant results of the pairwise post hoc test. -- Table S5. Results of the between-group Kruskal-Wallis test to check statistically significant differences between colonization experiments and chambers (sources of variation) for the percentage of colonization of D. magna at 48h, together with the significant results of the pairwise post hoc test. -- Table S6. Mean, standard error (SE), maximum and minimum values obtained for the mobility variables analyzed in D. magna after 48 h of exposure to the different study treatments. -- Table S7. Results of PERMANOVA ONE-WAY test to check statistically significant differences between study treatments for the mobility variables evaluated, together with the significant results of the pairwise post hoc test. -- Table S8. Results of Kruskal-Wallis test to check statistically significant differences between study treatments for the average vertical speed (study variable with non-normal distribution according to Shapiro-Wilk test), together with the significant results of the pairwise post hoc test and Dunn´s multiple comparisons test (specific comparisons with the control treatment). -- Table S9. One-way ANOVA test to check statistically significant differences among treatments for the remaining mobility variables evaluated, together with the significant results of the pairwise post hoc test and Dunnett´s multiple comparisons test (specific comparisons with the control treatment). -- Table S10. Mean, standard error (SE), maximum and minimum values obtained for the AChE activity in D. magna after 48 h of exposure to the different study treatments. -- Table S11. One-way ANOVA test to check statistically significant differences among treatments for the acetylcholinesterase (AChE) activity, together with the significant results of the pairwise post hoc test. -- Fig. S1. HeMHAS – Heterogeneous Multi-Habitat Assay System (version #3) used in the colonization experiments. -- Fig. S2. Detail of one of the compartments of the HeMHAS together with two gates that connect it to the adjacent chambers throughout a rotary locking system. -- Fig. S3. Picture of D. magna individually disposed in four wells of culture plates for analysis of horizontal swimming behavior. -- Fig. S4. Percentage of organisms (A) and colonization response (B) achieved in each chamber in the control test at 48h with ±SE of four replicates. -- Fig. S5. PCA analysis of the responses obtained for the mobility variables analysed in D. magna exposed to the different study treatments., Peer reviewed

Table S1 Concentrations (ng/g) of organochlorine compounds in tissue (blood and adipose) of caracals in the Greater Cape Town area, South Africa in terms of wet weight (w.w.) and lipid weight (l.w.). Table S2 Spatial covariates considered for models of organochlorine exposure in caracal in the Greater Cape Town area, South Africa. Fig. S3 Boxplots (diamonds = means) of organochlorine compound concentrations (ng/g, logged) in whole blood and adipose of caracals in the Greater Cape Town area, South Africa. Table S4 Estimates (SE) of top linear mixed effects models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and spatial predictors in caracal in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S5 Estimates (SE) of top linear mixed effects models (based on AICc) of adipose organochlorine levels (ng/g, logged) and spatial predictors in caracal in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S6 Diet proportions in several prey groupings for caracals around Cape Town, South Africa as determined by through integrating diet datasets from scat and prey remains found at GPS clusters. Table S7 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and diet (biomass) for broad taxonomic groups for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S8 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and diet (FO) for broad taxonomic groups for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S9 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and diet (biomass) for prey functional groups for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S10 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and diet (FO) for prey functional groups for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S11 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and diet (biomass) for prey foraging habitat for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S12 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and diet (FO) for prey foraging habitat for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S13 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and diet (biomass) for exotic prey groups for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S14 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and diet (FO) for exotic prey groups for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S15 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and blood cell count for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S16 Estimates (SE) for top models (based on AICc) of whole blood organochlorine levels (ng/g, logged) and serum biochemistry measures for caracals in the Greater Cape Town area, South Africa (*P < 0.1; **P < 0.05; ***P < 0.01). Table S17 Comparison of DDT concentrations (ng/g) ± SE in wild terrestrial mammalian predators from published studies. Where possible, like tissue was compared with like, but where not, serum/plasma was compared with caracal whole blood and liver/kidney was compared with caracal adipose. Table S18 Comparison of PCB concentrations (ng/g) ± SE in wild terrestrial mammalian predators from published studies. Where possible, like tissue was compared with like, but where not, serum/plasma was compared with caracal whole blood and liver/kidney was compared with caracal adipose., Peer reviewed

Peer reviewed

13 pages. -- The file includes: Chemical analysis of pesticides. -- Table S1. Mass spectrometer parameters for 2,4-D and fipronil. -- Quality assurance/Quality control. -- Table S2. Linear range, limit of quantification (LOQ) and determination coefficient (R2) for 2,4-D and fipronil using external calibration method. -- Table S2. Linear range, limit of quantification (LOQ) and determination coefficient (R2) for 2,4-D and fipronil using external calibration method. -- Figure S1. Overview of system (Heterogeneous Multi-Habitat Test System - HeMHAS) used in the present study, featuring the compartment, the piece that connect the compartments and the gyratory door. -- Table S4: Values (mean ± standard deviation) of fipronil and 2,4-D determined at each concentration tested in the avoidance experiments to insecticide and herbicide, respectively, in micrograms per liter (μg/L). -- Table S5: Values (mean ± standard deviation) of fipronil and 2,4-D, determined at each concentration tested in the avoidance experiment to herbicide and insecticide, respectively, in micrograms per liter (μg/L). -- Table S6a: Mixed-design ANOVA for avoidance test with D. rerio exposed to fipronil at 20 °C. -- Table S6b:4Within-subjects effects with Greenhouse-Geisser correction for degrees of freedom. -- Table S6c:5Between-subjects effects. -- Table S7a:6Mixed-design ANOVA for avoidance test with D. rerio exposed to fipronil at 24 °C. -- Table S7b:7Within-subjects effects. -- Table S7c:8Between-subjects effects. -- Table S8a:9Mixed-design ANOVA for avoidance test with D. rerio exposed to fipronil at 28 °C. Table S8b:10Within-subjects effects. -- Table S8c:11Between-subjects effects. -- Table S9a:12Mixed-design ANOVA for avoidance test with D. rerio exposed to 2,4-D at 20 °C. -- Table S9b:13Within-subjects effects. -- Table S9c:14Between-subjects effects. -- Table S10a:15Mixed-design ANOVA for avoidance test with D. rerio exposed to 2,4-D at 24 °C. -- Table S10b:16Within-subjects effects. -- Table S10c:17Between-subjects effects. -- Table S11a:18Mixed-design ANOVA for avoidance test with D. rerio exposed to 2,4-D at 28 °C. -- Table S11b:19Within-subjects effects with Greenhouse-Geisser correction for degrees of freedom. -- Table S11c:20Between-subjects effects. -- Table S12:21Final results (mean ± standard error of the percentages of fish) for the avoidance experiments per zones - pesticide mixtures at different air temperatures (20, 24 and 28 degrees)., Peer reviewed

2 pages. -- Supplementary figure 1: Distribution of D. rerio juveniles along the gradients of Glyphosate, Chlorpyrifos and Chlorothalonil, throughout the three-hour period of exposure to the gradient of each substance individually. -- Supplementary figure 2. Distribution of D. rerio juveniles along the gradients of a mixture of Glyphosate-Chlorpyrifos, and Glyphosate-Chlorothalonil, throughout the three-hour period of exposure in the open gradient of each mixture., Peer reviewed

59 pages. -- Contents: Point S1. Gaussian keywords used in the calculations. -- Figure S1. Experimental spectra of PFPG+g+/G-g- and its isotopologues species. -- Table S1-S7. Transition frequencies of PFPG+g+/G-g- and its isotopologues and PFPTg+/Tg-. -- Figure S2. Experimental spectra of the PFPTg+/Tg- monomer. -- Figure S3. Conformational interconversion barrier of the PFP conformers. -- Table S8-S10. Kraitchamn and STRFIT results of PFPG+g+/G-g. -- Table S11-S13. The semi-experimental equilibrium structural parameters of PFPG+g+/G-g-. -- Table S14. Spectroscopic properties of the predicted binary PFP conformers. -- Table S15-S19. Rotational transition frequencies of the five binary PFP conformers. -- Figure S4. QTAIM and NCI analyses of the five PFP and PrOH conformers. -- Figure S5. QTAIM and NCI analyses of the five binary PFP conformers. -- Completion of reference 24., Peer reviewed

29 pages. -- PDF file includes: Strong-field coherence breaking (SFCB) details; Internal rotation methyl barrier of 4-methyl guaiacol (MG). -- Figure S1: Experimental and simulated variation of the internal rotational barrier of methyl group. -- Figure S2: Summary of bond changes of lignin relative to the average bond length rC-C. -- Figure S3: Optimized structure of guaiacol at B3LYP-D3BJ/def2tzvp level of theory. -- Table S1: Experimental and calculated constants derived from the broadband rotational spectrum of guaiacol. -- Table S2: List of spectroscopic parameters used to fit guaiacol isotopomers. -- Table S3: Experimental and calculated constants derived from the broadband rotational spectrum of syringol. -- Table S4: Molecular parameters of 4-methyl guaiacol. -- Table S5: Molecular parameters for Z- and E-4-vinyl guaiacol. -- Table S6: Line list of transitions fitted for guaiacol (G). -- Table S8: Line list of transitions fitted for 13C(2) guaiacol. -- Table S9: Line list of transitions fitted for 13C(3) guaiacol. -- Table S10: Line list of transitions fitted for 13C(4) guaiacol. -- Table S11: Line list of transitions fitted for 13C(5) guaiacol. -- Table S12: Line list of transitions fitted for 13C(6) guaiacol. -- Table S13: Line list of transitions fitted for 13C(7) guaiacol. -- Table S14: Line list of transitions fitted for syringol (S). -- Table S15: Line list of transitions fitted for methyl guaiacol (MG). -- Table S16: Line list of transitions fitted for vinyl guaiacol (Z-VG). -- Table S17: Line list of transitions fitted for vinyl guaiacol (E-VG)., Peer reviewed