DNA Fingerprinting of Saginaw Bay Walleye and Spawning Populations Jennifer Richter, Gracie Lefler, Taylor Shivers, Taylor Anderson, Rachel Laskowski, Ben Cooper, Craig Coopersmith, and Dr. David Stanton Department of , Saginaw Valley State University, 7400 Bay Road, University Center, MI, 48710. 4: DNA Fingerprinting
1: Introduction •
There is a large population of walleye in Saginaw Bay.
• Walleye serve as top predators and are critical to the ecosystem. • Walleye are also economically important both for sport and commercial fishing. • The population is heavily managed and has been extensively stocked.
5: Results
• A technique used by forensic scientists to identify individuals based on highly polymorphic loci • Specific primers are used to amplify these regions by PCR (Polymerase Chain Reaction) • Fragments are sized by capillary electrophoresis in order to determine a multi locus genotype • Results can then be compared between two individuals or among a large database
1. For Svi L6, eight to eleven alleles were observed in each population (average 9.4). 2. Heterozygosities were generally high (average .79) and usually near expected values (average .76). 3. One allele was found in the Rifle River and Saginaw Bay and was not found in any other spawning population.
• We use DNA fingerprinting in order to access genetic diversity, population substructure and spawning site fidelity.
7: Estimating Amounts of Antimicrobial Compounds Present in LRRFF
Alleles and Heterozygosity (Svi L6)
Alleles
A
B1
B2
C
D
E
F
10
10
8
11
10
9
8
2: Materials and Methods • Extract total genomic DNA (DNeasy kit, Qiagen). • Amplify by PCR using dye labeled primers to VNTR regions (IDT). • Perform capillary electrophoresis to determine fragment sizes (CEQ 8000, BC). • Determine genotypes, calculate allele frequencies, heterozygosity, genetic distances (D) and population substructure (FST).
PCR machine DNeasy Kit Micro centrifuge Vortex machine 1.5 mL Eppendorf tubes Micro pipettes CEQ 8000 PCR tubes Polypropylene pestles
Collection Sites
F D C
B
A
Ho
.80
.83
.65
.90
.82
.74 .76
He
.73
.81
.73
.76
.78
.77 .75
4. Most populations were not in Hardy Alleles
E
Range (bp)
Svi 2
188 – 222
13
except the Saginaw Bay summer
Svi 4
106 – 122
8
population.
Svi 6
126 – 168
16
Svi 7
140 – 178
13
(average.06). Bay populations were most
Svi 18
114 – 126
7
similar to the Kawkawlin population.
Svi 33
82 – 106
13
6. Population substructure was low (FST = 0.01).
Svi L6
104 – 140
16
Weinberg equilibrium (P < 0.05),
DNA Fingerprint (Svi L6) 112
Date
A Tittabawassee River
4/4/13
N 48
B Saginaw Bay
7/15/15
72
C Kawkawlin River
4/3/15
48
D Saginaw Bay
1/14/14
33
E Shiawassee River
4/10/14
48
F Rifle River
3/18/16
48
140
B1 B2
.06
.06
C
.02
.05
B2
C
D
Batch 1 (8 oz) Batch 2 (1 kg)
Batch 3 (8 oz)
Isolated and purified salicylic acid (g/50 mL)
0.31
0.12
0.19
Didecyldimethylammonium salts as chloride (mg/50 mL)
8
16
10
• Samples of salicylic acid and didecyldimethylammonium salt isolated from LRRFF were submitted for carbon dating • Based on the amount of 14C present, the compounds were dated to 52000 ± 2900 and 21140 ± 100 years old, respectively • This strongly suggests that both compounds are derived from petroleum based precursors and that neither compound is a product of a recent fermentation of the plant material
9: Summary
Genetic Distances (Svi L6) B1
2
8: Determining Origins of Antimicrobial Compounds
5. Genetic distances were low
A .10
Batch #
1
Table 3: Amount of salicylic acid and didecyldimethylammonium salts obtained from commercial LRRFF from two companies
Locus
Molecular Ecology 12:1689-1702 (2003)
Location
• 50 mL aliquots were drawn from three samples of commercial LRRFF to estimate the amounts of salicylic acid and didecyldimethyl ammonium salt present • Results are summarized in Table 3 Sample
Stizostedion vitreum
• • • • • • • • •
• The solid residue obtained from concentrating the base-washed EtOAc layer was examined by 1H NMR and was consistent with didecyldimethylammonium bromide with a salicylate impurity • The salicylate impurity was removed by dissolving the solid in water and passing it thorough an anion exchange resin AG 1-X8 (Cl- form) • The solid residue obtained from concentrating this mixture gave 1H 13C and NMR spectra that were consistent with didecyldimethylammonium bromide without any trace of salicylate (Figure 5) • Didecyldimethylammonium bromide has been observed to inhibit growth of Gram-positive bacteria6 and this was confirmed on S. aureus with the solid residue and a commercially-available sample
E
• LRRFF and its extracts inhibit the growth of both Gram-negative and Gram-positive bacteria • Salicylic acid and didecyldimethylammonium salt are responsible for this activity, respectively • No antimicrobial peptides were detected in any sample
10: Acknowledgements
.04
D
.04
.09
.07
.03
E
.07
.07
.12
.08
.05
F
.05
.05
.06
.06
.06
.03
• University of Alberta • NSERC • Alberta Innovates Health Solutions • CIHR (postdoctoral fellowship) • Canada Research Chair in • Saginaw Valley State University Bioorganic and Medicinal (travel grant) Chemistry • Dr. Randy Whittal (U of A)
11: References 1. Li et al., J, Agric. Food Chem. 2015, 63, 3053-3058 and references within 2. Kim et al. J. Food Prot. 2008, 71, 325-332 2. Lee et al., US Patent 2010/0129305 A1, May 27. 2010 3. Duthie and Wood. Food Funct. 2011, 2, 515-520 4. Gershon and Parmegiani, Appl. Microbiol., 1962, 10, 348-353 5. Ioannou et al. Antimicrob. Agents Chemother. 2007, 51, 296-306
DNA Fingerprinting of Saginaw Bay Walleye and Spawning Populations Jing Li, Jennifer L. Chaytor, Brandon Findlay, Lynn M. McMullen, David C. Smith, and John C. Vederas Department of Chemistry, Saginaw Valley State University, 7400 Bay Road, University Center, MI, 48710. Email: jchaytor@svsu.edu Department of Chemistry and Department of Agricultural, Food, and Nutritional Science, University of Alberta, 11227 Saskatchewan Dr., Edmonton, AB, T6G 2G2, Canada. Griffith Laboratories Limited, 757 Pharmacy Avenue, Toronto, Ontario, Canada, M1L 3J8 1: Introduction • Kimchi is a Korean food dish typically made from fermented Chinese cabbage and/or other vegetables • Lactic acid bacteria (specifically Leuconostoc kimchii) are responsible for the distinctive taste but also impart a prolonged shelf life and prevent spoilage2 • Many lactic acid bacteria produce antimicrobial peptides called bacteriocins which are sometimes used in food preservation • A mixture of L. kimchii fermentation products has been patented for use as a preservative for cosmetics, with reported activity against Gram-positive and Gram-negative bacteria and fungi3 • It is possible that this reported activity is due to antimicrobial peptides produced by the fermentation process or the plant itself
• A needle-like white solid precipitated from the 60% and 80% isopropanol fractions overnight
6: Identification of Compound Active Against L. lactis • Unlike commercial salicylic acid, the solid isolated from LRRFF was active against both Gram-positive and Gram-negative organisms (Figure 3, Table 1)
5: Identification of White Solid • The solid was isolated and characterized by Fourier Transform Infrared (FTIR), Nuclear Magnetic Resonance (NMR), elemental analysis, and electron impact (EI) mass spectrometry • A mass of 138 g/mol was identified which suggested a molecular formula of C7H6O3 • All characterization data was consistent with that of salicylic acid with a minor impurity observed by 1H NMR (Figure 1)
7: Estimating Amounts of Antimicrobial Compounds Present in LRRFF
A)
• 50 mL aliquots were drawn from three samples of commercial LRRFF to estimate the amounts of salicylic acid and didecyldimethyl ammonium salt present • Results are summarized in Table 3
2: Objectives • Confirm the antimicrobial activity of Leuconostoc/radish root ferment filtrates (LRRFF, a mixture of Daikon radish [Raphanus sativus] fermented with Leuconostoc kimchii) • Identify the compounds responsible for any observed antimicrobial activity
Figure 3: Spot-on-lawn assay of LRRFF pre- and post-EtOAc extraction and the EtOAc extract following base wash. The samples were tested against A) E.coli DH5a and B) L. lactis subspecies cremonis HP
B)
3: Materials and Methods • Spot-on-lawn assays were performed using Escherichia coli DH5a, E. coli ATCC 25922, Lactococcus lactis subspecies cremonis HP, and Staphylococcus aureus ATCC 6538 as indicator strains. Antimicrobial activity was defined as a clear zone in the bacterial lawn at the location of sample addition. • LRRFF components were separated using a hydrophobic interaction resin (Amberlite XAD-16) and organic extraction with ethyl acetate
Entry
Sample
E. coli DH5a
E. coli 25922
L. lactis subsp. cremonis HP
Leuconostoc/radish root ferment filtrate Following Protease treatment Following heating to 100 °C for 60 min
+
++
++
n/a
++
++
n/a
++
++
4
Flowthrough
+
n/a
++
5
Wash (0% IPA)
n/a
1 2 3
6 7
20% IPA 40% IPA
n/a n/a
• The white solid was analyzed with high-resolution ESI-MS and showed only one signal in the positive mode (m/z 326.3776). This corresponds to a molecular formula of C22H48N+, suggesting a saturated alkyl amine • Extraction of LRRFF with ethyl acetate followed by base wash with sodium hydroxide and subsequent acidification with hydrochloric acid gave salicylic acid without the alkyl ammonium component, and this sample behaved identically to commercial salicylic acid in HPLC (Figure 4) and in spot-onlawn assays
C)
• Salicylic acid (Figure 2) is produced by lactic acid bacteria and plants4 • It has been observed to inhibit growth of certain Gram-negative bacteria but does not usually display anti-Gram-positive activity5 • This was confirmed with commercial salicylic acid (Table 2)
L. lactis subsp. cremonis HP
Entry
Sample (dissolved in MeOH:H2O/1:1)
Table 1: Results of spot-on-lawn assays of LRRFF against indicator organisms. Entries 4-9 represent various stages of Amberlite XAD16 fractionation. ++ indicates the sample was active, + indicates weak activity, - indicatives the sample was inactive, and n/a indicates the sample was not tested against that organism.
1
MeOH:H2O/1:1
2
Commercial salicylic acid
+
3
Solid obtained from fractionation by 60%, 80% IPA on XAD resin
+
++
• Commercial LRRFF showed activity against E. coli (Gramnegative) and L. lactis (Gram-positive) (Table 1) • Activity was maintained following treatment with Protease Type XIV and following heat treatment, which are conditions that typically degrade peptides • Following fractionation with the hydrophobic interaction resin, activity was only observed in the 60% and 80% isopropanol fractions
4
Solid obtained by EtOAc extract
+
++
n/a
++
9
80% IPA with 0.1% TFA
+
n/a
++
Batch 1 (8 oz) Batch 2 (1 kg)
Batch 3 (8 oz)
Isolated and purified salicylic acid (g/50 mL)
0.31
0.12
0.19
Didecyldimethylammonium salts as chloride (mg/50 mL)
8
16
10
• Samples of salicylic acid and didecyldimethylammonium salt isolated from LRRFF were submitted for carbon dating • Based on the amount of 14C present, the compounds were dated to 52000 ± 2900 and 21140 ± 100 years old, respectively • This strongly suggests that both compounds are derived from petroleum based precursors and that neither compound is a product of a recent fermentation of the plant material
Figure 2: Structure of salicylic acid
E. coli DH5a
+
2
9: Summary
60% IPA
Batch #
1
8: Determining Origins of Antimicrobial Compounds
8
Sample
Table 3: Amount of salicylic acid and didecyldimethylammonium salts obtained from commercial LRRFF from two companies
Figure 1: 1H NMR spectra of (A) commercial salicylic acid, (B) the white solid, and (C) a white solid obtained from extraction of LRRFF with ethyl acetate
4: Results of Spot-on-Lawn Assays
• The solid residue obtained from concentrating the base-washed EtOAc layer was examined by 1H NMR and was consistent with didecyldimethylammonium bromide with a salicylate impurity • The salicylate impurity was removed by dissolving the solid in water and passing it thorough an anion exchange resin AG 1-X8 (Cl- form) • The solid residue obtained from concentrating this mixture gave 1H 13C and NMR spectra that were consistent with didecyldimethylammonium bromide without any trace of salicylate (Figure 5) • Didecyldimethylammonium bromide has been observed to inhibit growth of Gram-positive bacteria6 and this was confirmed on S. aureus with the solid residue and a commercially-available sample
5
Residue obtained after the base wash of EtOAc extract
Figure 4: HPLC trace of commercial salicylic acid (top) and the residue obtained from sodium hydroxide-washed EtOAC extract (bottom)
10: Acknowledgements • University of Alberta • NSERC • Alberta Innovates Health Solutions • CIHR (postdoctoral fellowship) • Canada Research Chair in • Saginaw Valley State University Bioorganic and Medicinal (travel grant) Chemistry • Dr. Randy Whittal (U of A)
11: References
++
Table 2: Results of spot-on-lawn assays of commercial salicylic acid and the isolated solid and residue from LRRFF against indicator organisms. ++ indicates the sample was active, + indicates weak activity, - indicates the sample was inactive.
• LRRFF and its extracts inhibit the growth of both Gram-negative and Gram-positive bacteria • Salicylic acid and didecyldimethylammonium salt are responsible for this activity, respectively • No antimicrobial peptides were detected in any sample
Figure 5: 1H NMR spectrum of didecyldimethylammonium chloride with the chemical structure of the cation shown
1. Li et al., J, Agric. Food Chem. 2015, 63, 3053-3058 and references within 2. Kim et al. J. Food Prot. 2008, 71, 325-332 2. Lee et al., US Patent 2010/0129305 A1, May 27. 2010 3. Duthie and Wood. Food Funct. 2011, 2, 515-520 4. Gershon and Parmegiani, Appl. Microbiol., 1962, 10, 348-353 5. Ioannou et al. Antimicrob. Agents Chemother. 2007, 51, 296-306