Polyunsaturated fatty acids are sensitive to heat, light and oxygen. Therefore, care must be taken when preparing fatty acid supplementation plates such that fatty acids are not exposed to excess heat and light. NGM media containing 0.1% Tergitol (NP-40) is autoclaved and partially cooled, after which fatty acid sodium salts are added with constant stirring. The plates are allowed to dry in the dark. Uptake of fatty acids by C. elegans cultured on these plates can then be monitored by gas chromatography.
1. Preparation of Fatty Acid Supplemented Media
2. Inducing Germ Cell Destruction by Supplementation of DGLA
3. Confirming Fatty Acid Uptake by Gas Chromatography
Overall fatty acid composition of C. elegans can be determined by producing fatty acid methyl esters (FAMEs) which are then separated and quantified using gas chromatography4.
Supplementation of the C. elegans diet is limited by the ability of the bacterial food source to uptake and incorporate fatty acid into the bacterial membrane. To determine the ability of E. coli OP50 to assimilate various fatty acids into its membranes, OP50 was plated onto media with no supplement, 0.1 mM and 0.3 mM concentrations of stearic acid (18:0), sodium oleate (18:1n-9), and sodium DGLA (20:3n-6). Plates were dried at room temperature for 2 days in the dark, and incubated at 20 °C for 3 days. Bacterial lawns were collected by gently scraping the lawn into water with a flame-sterilized spatula. Bacteria were pelleted by centrifugation, and treated with 2.5% H2SO4 in methanol to produce fatty acid methyl esters, which were analyzed by GC/MS following the methods listed in the Procedure, step 3. The results demonstrate that unsaturated fatty acids (oleate and DGLA) incorporate into OP50 in higher amounts than the saturated fatty acid stearic acid (Figure 1A).
Additionally, L1 stage N2 larvae were grown on the same batch of supplemented plates and harvested after three days growth at 20 °C. Worms were washed off of the plates and fatty acids in total worm preps were analyzed by GC/MS. The change in supplemented fatty acids is graphed in Figure 1B. These studies demonstrate that supplementation of saturated fatty acids does not change the relative amount of saturated fatty acids in worm tissues, while supplementation of unsaturated fatty acids increased the relative amounts of unsaturated fatty acids in C. elegans lipids. Taken together, the data shown in Figure 1A and Figure 1B demonstrate that the relative accumulation of supplemented fatty acids in C. elegans correlates directly with the relative accumulation of fatty acids in the dietary E. coli.
We have previously shown that dietary DGLA causes sterility in C. elegans10. Figure 2 illustrates the dose response of DGLA induction of sterility in C. elegans. The concentration of DGLA in worm lipids in which 50% of the population will be sterile is approximately 12%. Interestingly, the response to DGLA can be altered by genetic mutations in C. elegans. A recent finding is that the insulin growth factor-dependent stress pathways can suppress the DGLA-induced germ cell destruction8. Supplementing the diet of worms containing deleterious mutations in either the daf-2 insulin/IGF receptor, daf-2(e1370), or the daf-16/FOXO transcription factor, daf-16(mu86), illustrates the usefulness of this method to unravel genetic pathways that influence the physiological effects of dietary fats. Synchronized L1 larvae were pipetted onto DGLA supplemented media. After 3-4 days of growth, worms were scored for sterility, as determined by the absence of eggs in the uterus of adult worms. DGLA supplemented daf-2(e1370)mutants were fertile, with little to no induced germ cell loss compared to wild type (N2) worms at both 0.15 mM and 0.3 mM supplementations (Figure 3).In contrast, DGLA supplemented worms with inactive FOXO (daf-16(mu86)) displayed a higher percentage of sterile worms compared to wild type when fed on plates containing 0.15 mM DGLA (Figure 3).
Figure 1. Uptake and incorporation of supplemented fatty acids by E. coli OP50 and C. elegans. A. E. coli OP50 was grown on plates containing 0.1 mM or 0.3 mM stearic acid, sodium oleate, or sodium DGLA as well as un-supplemented plates. After five days of growth on plates at 20 °C, E. coli were harvested and fatty acid methyl esters were generated for analysis by GC/MS. Because OP50 does not produce oleic acid or DGLA, and produces only trace amounts of stearic acid, the percentage of each supplemented fatty acid in the E. coli lipids reveals the ability of OP50 to incorporate the supplemented fatty acid. Error bars are SD. B. Change in C. elegans fatty acids in young adults grown for three days, starting at L1 stage, on E. coli plates containing 0.1 mM or 0.3 mM stearic acid, sodium oleate, or DGLA. The values for change in stearic acid and DGLA were obtained by subtracting the relative amount of 18:0 or 20:3 in worms grown on supplemented plates from those of worms grown on unsupplmented plates. To monitor uptake of oleic acid, the sum of oleic acid plus downstream C20 PUFAs (20:3, 20:4n-6, 20:4n-3, and 20:5) were calculated in supplemented and unsupplemented plates, because incorporated oleic acid is further desaturated and elongated. Error bars are SD. Click here to view larger image.
Figure 2. Increasing concentrations of DGLA in worm lipids correlate with increasing sterility in C. elegans. Wild type (N2) worms were treated with various concentrations of DGLA. The % DGLA in total worm lipids and the % of the population that is sterile is plotted for is plotted for 17 data points from five independent feeding experiments using dietary DGLA concentrations ranging from 0-0.3 mM DGLA. Click here to view larger image.
Figure 3. Physiological effects of supplementing C. elegans with DGLA. Starved L1 larval wild type, daf-2(e1370), or daf-16(mu86) were plated onto un-supplemented, 0.15 mM or 0.3 mM DGLA supplemented media and grown to the adult stage. At least 150 individual worms were then scored for sterility. Thedaf-2(e1370) mutants were almost completely fertile, even at 0.3 mM DGLA, while thedaf-16(mu86) mutants display an increased number of sterile worms compared to wild type at 0.15 mM DGLA. Error bars are SEM. Click here to view larger image.
Bacto-Agar | Difco | 214010 | |
Tryptone | Difco | 211705 | |
NaCl | J.T. Baker | 3624-05 | |
Tergitol | Sigma | NP40S-500mL | |
Cholesterol | Sigma | C8667-25G | (5 mg/mL in ethanol) |
MgSO4 | J.T. Baker | 2504-01 | |
CaCl2 | J.T. Baker | 1311-01 | |
K2HPO4 | J.T. Baker | 3254-05 | |
KH2PO4 | J.T. Baker | 3246-05 | |
Sodium dihomogamma linolenate | NuCHEK | S-1143 | |
Warm sterile Millipore water | |||
Sterile water for collecting worms | |||
Nuclease-free Water for DGLA stock solution | Ambion | AM9932 | |
Ampicillin | Fisher Scientific | BP1760-25 | 100 mg/ml in water (for RNAi plates) |
Isopropyl-beta-D-thiogalactopyranoside (IPTG) | Gold Biotechnology | 12481C100 | 1 M in water (for RNAi plates) |
HSO4 | J.T. Baker | 9681-03 | |
Methanol | Fisher Scientific | A452-4 | |
Hexane | Fisher Scientific | H302-4 | |
diamindinophenylindole (DAPI) | Sigma | D9542 | |
VectaShield | Vector Laboratories | H-1000 | |
Glass Flask | Corning | 4980-2L | |
Autoclaveable Glass bottles with stirbars | Fisherbrand | FB-800 | |
Autoclaveable Glass Graduated Cylinder | Fisherbrand | 08-557 | |
Stir Plate | VWR | 97042-642 | |
Waterbath at 55+ °C | Precision Scientific Inc. | 66551 | |
Screwcap Brown Glass Vial | Sun SRI | 200 494 | |
Argon gas tank | |||
Automated Pipette aid | Pipette-Aid | P-90297 | |
Sterile Serological Pipettes (25 ml) | Corning | 4489 | |
Bunsen Burner | VWR | 89038-534 | |
Dissection microscope | Leica | TLB3000 | |
Silanized glass tube | Thermo Scientific | STT-13100-S | for FAMEs derivitization |
PTFE Screw caps | Kimble-Chase | 1493015D | |
Clinical tabletop centrifuge | IEC | ||
GC Crimp Vial | SUN SRi | 200 000 | |
GC Vial Insert | SUN SRi | 200 232 | |
GC Vial cap | SUN SRi | 200 100 | |
Gas Chromatograph | Agilent | 7890A | |
Mass Spectrometry Detector | Agilent | 5975C | |
Column for gas chromatography | Suppelco | SP 2380 | 30 m x 0.25 mm fused silica capillary column |
Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.
Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.
Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.