Quantification%20of%20wheat%20gluten%20in%20LGB - PowerPoint PPT Presentation

Title: Quantification%20of%20wheat%20gluten%20in%20LGB


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Quantification of wheat gluten in LGB
Fengyou Jia, Sonny Ramaswamy, Michael Toews,
James Campbell

• Department of Entomology, Kansas State
  University, Manhattan, KS 66506
• USDA, ARS, GMPRC, Manhattan, KS 66502

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LGB (Lesser Grain Borer)
(Rhyzopertha dominica)
(Coleoptera Bostrichidae)
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LGB in Kansas

• 4-5 generations annually
• Minimum Life Cycle 25 days (1month in 90ºF)
• Major food is wheat, alternative food resources?
• Overwinter in wooded areas?
• Female can lay up to 500 eggs in lifetime

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Pheromone-baited Traps

• LGB populations vary temporally and spatially
• There are two peaks annually
• First peak appears around June, second peak
  appears around August
• LGB population is often very low between the two
  peak

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Field LGB Population monitored with pheromone
traps
?
April
May
June
July
Aug
Sept
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Field LGB Population
Sold out
Bin
Bin
Bin
Bin
Bin
Bin
empty
Moved to
LGB
?
LGB
?
April
May
June
July
Aug
Sept
Image of courtesy http//www.freefoto.com/preview
.jsp?id07-50-12kWheatHarvesting
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Hypothesis?

• Winter wheat harvested in the late May and early
  June in Kansas
• Then wheat is moved to bins where the food is
  abundant for LGB
• LGBs attracted to farm bins following wheat
  harvesting?
• Later on farmers empty bins (sell out) and store
  other crops (e. g., corn and other crops)
• Thereafter LGBs back to field?

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Hypothesis examination

• Assumption if LGBs stay in wheat bins during
  this period and they must consume wheat kernels
  which contain gluten (protein).
• If the gluten is detected in LGBs,
• ? The LGBs must consume wheat
• ? inferring LGBs from wheat bins?

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LGB laboratory populations

• Wheat gluten detection (ELISA)
• Gluten Assay Kit (Catalogue No 802002 Y) from
  Tepnel BioSystems Limited (One Newtech Square,
  Deeside Industrial Park, Deeside, Flintshire. CH5
  2NT, U. K.).

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Methods

• Field population traps/collections (By Dr.
  Michael Toews)
• LGB washing procedure
• Wheat gluten extraction from LGB
• ELISA

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Washing

• 1). Randomly picked up live LGBs from laboratory
  colonies that feed on wheat or rice kernels, or
  randomly selected frozen LGB samples collected
  from fields
• 2). Place the LGB samples into a beaker.
• 3). Added 100 ml of 0.1 Triton X 100 to rinse
  the LGB by shaking the beaker for one minute, and
  then used tap water to rinse the samples 3-4
  times until the water became clean.
• 4). Repeated step 3 washing procedure twice.
• 5). Rinsed the LGB samples twice using ddH2O.
• 6). Finally, let the LGB samples dry on Kimwipes
  tissues.

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Wheat gluten extraction

• 1) Added one individual LGB sample into a 1.5 ml
  micro-centrifuge-tube with 30 µl of 40 ethanol
  and homogenized the sample by hand using pestle.
• 2) Then, homogenized the sample for one minute
  using Pellet Pestle Motor (Kontes Company).
• 3) Added 20 µl of 40 ethanol and homogenized the
  sample for another one minute.
• 4) Added 200 µl of 40 ethanol.
• 5) Centrifuged the tube at 2000 rpm for 15
  minutes at room temperature.
• 6) Transferred 200 µl of supernatant to a new 1.5
  micro-centrifuge-tube and added 300 µl of Working
  Diluent Solution (from Gluten Assay Kit) for
  Gluten Assay.

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ELISA

• Gluten Assay Kit (Catalogue No 802002 Y) was
  provided by Tepnel BioSystems Limited (One
  Newtech Square, Deeside Industrial Park, Deeside,
  Flintshire. CH5 2NT, U. K.).
• We basically followed the detailed enzyme
  immunoassay procedure for LGB gluten assay from
  step 14.3 to 14.13 of Gluten Assay Kit. Only Step
  14.4 and Step 14.7 were modified slightly by
  placing the microwell plate on a plate shaker at
  200 rpm and incubated at 37ºC instead of room
  temperature.

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Individual LGB gluten assay based on a blind
experiment
Laboratory LGB Laboratory LGB Laboratory LGB Laboratory LGB Control Control
On Wheat On Wheat On Rice On Rice Control Control
Label Index Label Index CK Index
w1 0.162 R1 0.058 A 0.58
w2 0.283 R2 0.062 B 0.337
w3 0.103 R3 0.054 C 0.184
w4 0.155 R4 0.125 D 0.094
w5 0.102 R5 0.054 E 0.068
w6 0.159 R6 0.053    
w7 0.093 R7 0.053    
w8 0.057 R8 0.057    
w9 0.147        
w10 0.1     NCK 0.052
Notes either false positive or false negative
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Detectable concentration of glutenof one LGB
(extraction)
Concentration 1 2 3 4 5 Average
50 0.271 0.162 0.234 0.134 0.368 0.2338
25 0.383 0.201 0.281 0.166 0.424 0.291
12.5 0.437 0.177 0.254 0.142 0.362 0.2744
6.26 0.354 0.159 0.168 0.263 0.255 0.2398
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Gluten detection on starved LGB
Index Index Index Index Index Index
Days Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Average
1.5 0.075 0.051 0.055 0.045 0.049 0.055
3 0.065 0.043 0.045 0.042 0.066 0.0522
6 0.046 0.047 0.046 0.049 0.075 0.0526
9 0.045 0.048 0.049 0.05 0.054 0.0492
12 0.055 0.049 0.067 0.048 0.049 0.0536
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Gluten detection in frozen LGB
Frozen LGB samples for 30 days Frozen LGB samples for 30 days Frozen LGB samples for 30 days Frozen LGB samples for 30 days
On Wheat On Wheat On Rice On Rice
Repeat Index Repeat Index
w1 0.353 R1 0.051
w2 0.166 R2 0.066
w3 0.141 R3 0.106
w4 0.101 R4 0.073
w5 0.184 R5 0.056
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Preliminary field LGB examination

• Populations examined
• KSU Farm Lands
• 7 populations
• From late April to early July
• 5 individuals examined for each
• Konza Prairie
• 10 populations
• From late April to early September
• 5 individuals examined for each
• All negative responses

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Preliminary conclusion

• More tests are needed to prove the hypothesis
• Caution
• Fact wheat gluten undetectable following 36 h
  digestion in LGB
• Locations of LGB trapped?

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Additional tests

• Objective variation detection among LGB
  populations
• Elemental component proportion (C, O, and K) in
  LGB

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LGB scutellum scanned
3 LGBs scanned from Each Population (expensive)
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Result reports
  KSU Farm land Konza On-Rice On-Wheat
C 72.937 74.360 70.047 73.987
O 27.033 25.550 24.973 25.953
K 0.007 0.037 0.027 0.050
P 0.010 N/A N/A N/A
Al 0.013 0.037 N/A N/A
Si N/A 0.037 N/A N/A
N N/A N/A 4.953 N/A
CL N/A N/A N/A 0.007
Notes Average element proportion.
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Future experiments

• Near-bin-trapping vs. farther-bin-trapping from
  June to early Sept. ?
• More field LGB tested using wheat gluten kits
• LGB population variation examination using mass
  spectrometer?
• More release-capture experiments