2003042 SOW (RFQ).docx

NOAA/NATIONAL MARINE FISHERIES SERVICE

SOUTHWEST FISHERIES SCIENCE CENTER

Fisheries Ecology Division

STATEMENT OF WORK

Klamath River Thiamine Deficiency Complex Egg Analysis

I. Background/Overview:

Thiamine Deficiency Complex (TDC) is a nutritional deficiency of thiamine (vitamin B1) recently linked with high mortalities of early life stage spring- and fall-run Chinook salmon in Central Valley (CV) hatcheries and coho salmon in the Klamath Basin at Iron Gate Hatchery. Clinical signs of TDC are anorexia and sporadic spinning prior to death (Lee et al. 2009; Ketola et al. 2000). Offspring of female salmon with reduced levels of thiamine are prone to TDC. The current hypothesis: a narrowing of the diversity of salmon marine diet and/or a more southerly ocean distribution where prey are either deficient in thiamine levels or high in thiaminase (Northern anchovies). Thiaminase is an enzyme that destroys or inactivates thiamine, and has been identified as the primary explanation for low levels of thiamine and the subsequent onset of TDC in Great Lakes and Baltic Sea salmonids.

There is an urgent need to document the scope of TDC by directly assessing egg-thiamine concentrations in California’s salmonids. To date, TDC in hatchery fish has been inferred from unusual fry behavior, higher than normal fry mortality rates, and rapid positive fry responses to thiamine bath treatments. However, the extent of both lethal and sub-lethal TDC is unknown. Likewise, although evidence supports thiaminase consumption as a causative factor for TDC in fishes, research is still needed to identify and describe ecological and environmental factors linked to ocean processes that may influence the manifestation of TDC. Investigating under what conditions thiamine deficiency occurs for salmon in natural systems will support development of predictive and preventative management strategies.

We propose to document the scope of TDC in Pacific salmonids in the Klamath Basin. The proposed activity is to measure the thiamine concentration in broodyear 2020 Chinook salmon, coho salmon, and steelhead eggs from Klamath Basin hatcheries and from Chinook salmon collected from lower Klamath River fisheries. Once collected, egg samples will be shipped for analysis of egg thiamine concentrations (each sample: 10 g per female). Funding has been acquired to support sampling and analysis of CV Chinook salmon. This is a comparison study to the funded effort for Central Valley Chinook salmon to provide insight to the prevalence of TDC in the Klamath Basin and provide within basin (Central Valley and Klamath Basin) and regional context of TDC to inform conservation and management actions of Pacific salmonids.

II. Scope of Work Free-flowing eggs collected at two Klamath Basin hatcheries (Chinook salmon, coho salmon, and steelhead) and eggs collected from Chinook salmon in the lower Klamath River will provide a synoptic view of the scope of TDC in populations of anadromous fish in the Klamath Basin.

Egg samples from Chinook salmon, coho salmon, and steelhead (10g of eggs per individual fish) will be collected by NMSF scientists and collaborators. Samples will be shipped on dry ice to contractor where they will be stored at -80℃ prior to biochemical analysis. Total number of individual samples will be 300 (30 individuals from six hatchery populations and four lower river groups of Chinook salmon).

III. Tasks

· Task 1: Total thiamine in 80% of our salmon/steelhead eggs will be measured using the rapid solid-phase extraction fluorometric method (RSPE; Zajicek et al. 2005a), because this method is more cost-effective than high-performance liquid chromatography. For 20% of the egg samples, free thiamine, thiamine monophosphate, and thiamine pyrophosphate will be quantified using high-performance liquid chromatography per established techniques (Brown et al. 1998, Futia et al. 2017). Total thiamine will be determined as the sum of the three vitamers. Doing both methods will allow for comparisons among Klamath samples, and between Klamath and Central Valley salmon egg samples that are being analyzed in another project. Regardless of the method, thiamine will be extracted following Brown et al. (1998) using 1 g of egg homogenized in a 2% trichloroacetic acid solution. The resulting homogenates will be placed into a boiling water bath for 10 min at 96-100oC to free the thiamine and its phosphorylated esters from denaturated binding proteins and to inactivate thiamine-altering enzymes (e.g., thiaminases, kinases, phosphatases). Then, the homogenates will be centrifuged at 14,000 g for 15 min to eliminate potentially interfering high-molecular weight materials. The supernatants (TCA extract) will be collected and frozen at -80oC until thiamine assay. Two method blanks will be included after every 20 samples for quality insurance.

HPLC method: Three thiamine vitamers (free thiamine, thiamine monophosphate, and thiamine pyrophosphate will be measured for each sample in duplicate. Following extraction, thiamine concentrations were quantified using HPLC (Agilent Technologies 1200 series, Agilent Technologies, Inc., Santa Clara, California, U.S.A.) (Brown et al., 1998; Futia et al., 2017). The HPLC system includes a delivery pump, an automatic sample injector, a Hamilton PRP-HI column (150x4.1 mm; 5-mm mesh size) with an attached guard column (25 x 2.3 mm; 23 to 30 mm mesh size), and a fluorometic detector (375-nm excitation wavelength and 433-nm emission wavelength for thiochrome detection). The column thermostat will be set to 30oC. Flow rate will be 1.0 ml/min and the total run time will be 25 min. The mobile phase will be 25mM potassium phosphate buffer (pH 8.4) and N,N,dimethylformamide. For 0 to 1 min, the mobile phase will be 25 nM potassium phosphate buffer. From 1 to 5 min, the mobile phase will be 0.5% N,N,dimethylformamide and 25 mM potassium phosphate buffer. From 5 to 9 min, the mobile phase will be 10% N,N,dimethylformamide and 25 mM potassium phosphate buffer. From 9 to 11 min, the mobile phase will be 30% N,N,dimethylformamide and 25 mM potassium phosphate buffer. From 11 to 11.1 min, the mobile phase will be 35% N,N,dimethylformamide and 25 mM potassium phosphate buffer. Afterwards, the mobile phase will return to the initial conditions, equilibrating the column for the next sample. A six-point standard curve with known concentrations of thiamine (0, 1, 2.5, 5, 10, and 30 nmol/g) will be generated at the start of each group of samples run. Concentrations of each vitamer will be then calculated based on the standard curve and expressed as nmol/g. These concentrations will be combined to determine total thiamine (TTH).

RSPE method: Strata SPE syringe-columns (1 ml) packed with C18-E (end capped) stationary phase (100 mg/column), an average particle diameter 57 μm (Phenomenex, Torrance, California) will be used for the extractions. Separate SPE columns will be used for each sample. Each SPE column fractionation will involve column activation, quantitative loading of the extract, and elution with concomitant collection of the fractions associated with thiamine. Column activation will be performed by sequential 500 μl washes of pure methanol, pH2.0 PO4 buffer containing 200 methanol per liter (20% methanol), and two 500 μl washes of pH2.0 PO4 buffer. After column activation, sample extract will be loaded (890 μl) onto the column in two equal portions. The column will be then washed with 250 μl of pH2.0 PO4 buffer. Both the solvent displaced during loading of the 890 ml of sample and the 250 μl of rinse will be collected as SPE fraction 1 (F1 = phosphorylated thiamine). After collection of F1, the SPE column will be eluted twice with 600 μl of 10% methanol in pH 2.0 PO4 buffer. The combined 1.2 ml eluate will be collected and designated as SPE fraction 2 (F2 = free thiamine). Potassium ferricyanide will be added to SPE F1 and F2 to oxidize the thiamine compounds to their corresponding thiochromes. This will be done under alkaline pH conditions. This oxidation method for thiamine and its phosphorylated esters required that the aliquots of aqueous 0.1% K3Fe(CN)6 and 5N NaOH solutions will be added simultaneously, directly to the fraction volume and not to the walls of the tube. After adding the oxidation reagents, each tube will be immediately vortex-mixed. Aliquots of the oxidized F1 and F2 will be then analyzed for thiochrome fluorescence using a FLx800 multi-detection microplate reader (BioTek Instruments, Inc., Vermont). The fluorescence of each sample will be read with a 360 nm excitation/460 nm emission filter pair to detect thiochrome fluorescence.

Brown, S.B., D.C. Honeyfield, L. Vandenbyllaardt. 1998. Thiamine analysis in fish tissues. Pages 73-81 in G. McDonald, J. Fitzsimons and D.C. Honeyfield, editors. Early Life Stage Mortality Syndrome in Fishes of the Great Lakes and the Baltic Sea. American Fisheries Society, Symposium 21, Bethesda, Maryland.

Futia, M.H., S. Hallenbeck, A.D. Noyes, D.C. Honeyfield, G.E. Eckerlin, and J. Rinchard. 2017. Thiamine deficiency and the effectiveness of thiamine treatments through broodstock injections and egg immersion on Lake Ontario steelhead trout. J. Great Lakes Res. 43:352-358.

Zajicek, J.L., D.E. Tillitt, S.B. Brown, L.R. Brown, D.C. Honeyfield, and J.D. Fitzsimons. 2005. A rapid solid-phase extraction fluorometric method for thiamine and riboflavin in salmonid eggs. J. Aquat. Anim. Health 17:95-105.

· Task 2: Contractor to provide biochemical analysis report for each sample.

IV. Period of Performance

V. Place of Performance Contractor’s facility.

VI. Deliverable Schedule All tasks must be completed/delivered within the period of performance.

VII. Government-Furnished Property (GFP) None.

VIII. Security Requirements None.

IX. Required Knowledge and Experience: Demonstrated experience with required procedures and expertise as specifically listed above in salmonids. Demonstration of experience and expertise should include publication in peer reviewed journals.

X. Delivery Address

NOAA/NMFS (Attn: Tommy Williams) 110 McAllister Way Santa Cruz, CA 95060