Lipotropes Protect against Pathogen-Aggravated Stress and Mortality in Low Dose Pesticide-Exposed Fish

<div><p>The decline of freshwater fish biodiversity corroborates the trends of unsustainable pesticide usage and increase of disease incidence in the last few decades. Little is known about the role of nonlethal exposure to pesticide, which is not uncommon, and concurrent infection of opportunistic pathogens in species decline. Moreover, preventative measures based on current knowledge of stress biology and an emerging role for epigenetic (especially methylation) dysregulation in toxicity in fish are lacking. We herein report the protective role of lipotropes/methyl donors (like choline, betaine and lecithin) in eliciting primary (endocrine), secondary (cellular and hemato-immunological and histoarchitectural changes) and tertiary (whole animal) stress responses including mortality (50%) in pesticide-exposed (nonlethal dose) and pathogen-challenged fish. The relative survival with betaine and lecithin was 10 and 20 percent higher. This proof of cause-and-effect relation and physiological basis under simulated controlled conditions indicate that sustained stress even due to nonlethal exposure to single pollutant enhances pathogenic infectivity in already nutritionally-stressed fish, which may be a driver for freshwater aquatic species decline in nature. Dietary lipotropes can be used as one of the tools in resurrecting the aquatic species decline.</p></div

Secondary stress response to low dose endosulfan exposure in fish unfed or fed with lipotropes for 37 days: Cellular responses.

<p>Secondary cellular stress responses included activities/levels of: antioxidant enzymes superoxide dismutase (SOD) and catalase; phase II metabolism enzymes glutathione-s-transferase (GST) and SAM-dependent methyl transferase (MT); heat shock protein (HSP70); and caspase. While MT was measured in serum, all other attributes were quantified in the liver and gills. Abbreviations for exposure/diet treatments of fish are the same as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093499#pone-0093499-g001" target="_blank">Figure 1:</a> Ctr, control; EE, endosulfan-exposed; Cho, choline; Bet, betaine and Lec, lecithin. The values reported in bar charts represent the mean±SE. Bars bearing different letters (a, b, c) indicate significant differences between treatment means for the level/activity of a marker in respective tissue or serum. Probability (P) values: SOD liver (P = 0.002), SOD gill (P = 0.005), catalase gill (P = 0.003), catalase liver (P = 0.005), GST gill (P = 0.02), GST liver (P = 0.03), serum MT (P<i> = </i>0.001), HSP70 and caspase (P = 0.001). Number of observations (n): n = 6 for SOD, GST, catalase, HSP70 and caspase, and n = 7 for MT.</p

Particle size distributions of chitosan-LHRH (A) and chitosan-gold-LHRH nanoparticles (B) and their respective TEM Images (a, b).

<p>Particle size distributions of chitosan-LHRH (A) and chitosan-gold-LHRH nanoparticles (B) and their respective TEM Images (a, b).</p

Secondary stress response to low dose endosulfan exposure in fish unfed or fed with lipotropes for 37 days: Pre- and post-challenge immunological responses.

<p>Pre-challenge samples were collected after 37 days of experiment. Fish were challenged with a pathogen, <i>A. hydrophila</i>, injected intraperitoneally and samples were collected at the end of 7 days post-challenge in surviving fish, or just before sacrificing severely morbid fish. Abbreviations for exposure/diet treatments of fish are the same as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093499#pone-0093499-g001" target="_blank">Figure 1:</a> Ctr, control; EE, endosulfan exposed; Cho, choline; Bet, betaine; and Lec, lecithin. The values reported in bar charts represent the mean±SE. Bars bearing different letters (a, b, c) indicate significant differences between treatment means for respective attributes during a separate comparison of pre- or post-challenge data. Probability (P) values during pre-challenge: A: G ratio (P = 0.53), NBT (P = 0.013), lysozyme (P = 0.46). P values during post-challenge: A: G (P = 0.018), NBT (P = 0.002), lysozyme (P = 0.02) and agglutination score (P = 0.002). Number of observations (n): n = 6 for A: G, lysozyme and agglutination score, and n = 3 for NBT. Comparisons by t-test indicated significantly reduced (P<0.05) lysozyme post-challenge compared to pre-challenge within these respective groups: Ctr/Ctr, EE/Ctr and EE/Cho.</p

Schematic diagram showing the functional structure of chitosan encapsulated gold nanoparticles and subsequent conjugation of LHRH on chitosan encapsulated gold nanoparticles.

<p>Schematic diagram showing the functional structure of chitosan encapsulated gold nanoparticles and subsequent conjugation of LHRH on chitosan encapsulated gold nanoparticles.</p

Secondary stress response to low dose endosulfan-exposure in <i>L. rohita</i> fish fingerlings unfed or fed with lipotropes for 37 days: Pre-challange^┼ and post-challenge^┼┼ hematological profile.

┼<p>Pre-challange blood samples were taken after 37 days of experiment. Subsequently, fish were challenged with the infectious bacteria, <i>A. hydrophila</i>, injected intraperitoneally and ^┼┼post-challange samples were taken at the end of 7 days in surviving fish or just before sacrificing severely morbid fish.</p><p>Units: RBC count (x 10⁶ cells/mm³), WBC count (x 10³ cells/mm³) and Hemoglobin (Hb) g/dL.</p><p>**Indicates significant difference from pre-challenge values (P<0.01) with in a group by student's t-test.</p>a, b, c, d<p>Means bearing different superscript letters in a row differ significantly against the P value indicated in the last column. Data expressed as Mean ± SE (n = 6).</p

Secondary stress response to low dose endosulfan exposure in fish unfed or fed with lipotropes for 37 days: Histoarchitectural response.

<p>Histoarchitecture of the liver revealed a protective role for lipotropes in fish exposed to a nonlethal dose of endosulfan and intraperitoneally injected with the infectious bacteria, <i>A. hydrophila</i>. Pre-challenge samples were collected after 37 days of experiment. Post-challenge samples were collected at the end of 7 days in surviving fish or just before sacrificing severely morbid fish. Section from control fish (A), <i>A. hydrophila</i> injected fish (B), fish exposed to nonlethal dose of endosulfan (C) and also injected with <i>A. hydrophila</i> but either fed with no supplements (D), or fed with choline (E), betaine (F) or lecithin (G). Blue arrowhead: sinusoids, Red arrowhead: hepatocyte with nucleus, Black arrowhead: vacuole in hepatocyte, White arrow: central vein, White arrowhead: ghost cell without nucleus (due to karyolysis), Yellow arrowhead: focal inflammatory infiltrate. Histological changes are described in detail in the text.</p

Reproductive output of the <i>Cyprinus carpio</i> injected with different preparations of salmon luteinising hormone-releasing hormone (LHRH).

<p>Percent relative eggs and fertilised eggs are expressed as relative to 100% in bare LHRH preparation. Fishes were injected with bare LHRH, chitosan LHRH nanoparticles (ChLHRHNPS) and chitosan-gold LHRH (ChAuLHRHNPs).</p

Histology of ovary from <i>Cyprinus carpio</i> injected with different preparations of salmon luteinising hormone- releasing hormone (LHRH).

<p>A]. from control fishes: 1.Ovigerous lamellae with immature oocyte; 2. mature oocyte; 3. maturing oocyte; 4. atretic oocyte. B] from fish injected with bare LHRH. 1. mature/ripe oocyte; 2. Partially spent oocyte; 3. cortical alveoli stage oocyte; 4. spent follicle; 5. immature oocytes in nest C] from fish injected with chitosan LHRH nanoparticles. 1. Oocytes nested in lamellae 2. spent follicle 3. spent oocytes 4. ovigerous lamellae with immature oocytes D] from fish injected with chitosan gold LHRH nanoparticles 1. mature oocytes 2. spent oocytes 3. immature oocytes 4. discharged follicle.</p

Secondary stress response to low dose endosulfan-exposure in <i>L. rohita</i> fish fingerlings unfed or fed with lipotropes for 37 days: Pre-challange^┼ and post-challenge^┼┼ serum protein profile.

┼<p>Pre-challange blood samples were taken after 37 days of experiment. Subsequently, fish were challenged with the infectious bacteria, <i>A. hydrophila</i>, injected intraperitoneally and ^┼┼post-challange samples were taken at the end of 7 days in surviving fish or just before sacrificing severely morbid fish.</p><p>TP indicates Total Protein. Serum proteins expressed as g/dL.</p><p>*Indicates significant difference from pre-challenge value (P<0.01) with in a group by student's t-test.</p>a, b, c<p>Means bearing different superscript letters in a row differ significantly against P value indicated in the last column. Data expressed as Mean ± SE (n = 6).</p