Quantification of Quantification of wheat gluten in LGB wheat gluten in LGB Department of Entomology, Kansas State Department of Entomology, Kansas State University, Manhattan, KS 66506 University, Manhattan, KS 66506 *USDA, ARS, GMPRC, Manhattan, KS 66502 *USDA, ARS, GMPRC, Manhattan, KS 66502 Fengyou Jia, Sonny Ramaswamy, Michael Toews*, James Campbell*
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Quantification of wheat gluten in LGB Department of Entomology, Kansas State University, Manhattan, KS 66506 *USDA, ARS, GMPRC, Manhattan, KS 66502 Fengyou.
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Quantification of wheat Quantification of wheat gluten in LGBgluten in LGB
Department of Entomology, Kansas State University, Department of Entomology, Kansas State University, Manhattan, KS 66506Manhattan, KS 66506
4-5 generations annually4-5 generations annually Minimum Life CycleMinimum Life Cycle:: 25 days (1month in 25 days (1month in
90ºF) 90ºF) Major food is wheat, alternative food Major food is wheat, alternative food
resources?resources? Overwinter in wooded areas?Overwinter in wooded areas? Female can lay up to 500 eggs in lifetime Female can lay up to 500 eggs in lifetime
Pheromone-baited Traps Pheromone-baited Traps
LGB populations vary temporally and LGB populations vary temporally and spatially spatially
There are two peaks annually There are two peaks annually First peak appears around June, First peak appears around June,
second peak appears around Augustsecond peak appears around August LGB population is often very low LGB population is often very low
between the two peakbetween the two peak
Field LGB Population
monitored with pheromone traps
April May June July Aug Sept
?
Field LGB Population
April May June July Aug Sept
Bin Bin Bin
LGB
LGB
Bin Bin Binempty
?
Moved to
Sold out
?
Image of courtesy: http://www.freefoto.com/preview.jsp?id=07-50-12&k=Wheat+Harvesting
Hypothesis?Hypothesis?
Winter wheat harvested in the late May Winter wheat harvested in the late May and early June in Kansasand early June in Kansas
Then wheat is moved to bins where the Then wheat is moved to bins where the food is abundant for LGBfood is abundant for LGB
LGBs attracted to farm bins following LGBs attracted to farm bins following wheat harvesting?wheat harvesting?
Later on farmers empty bins (sell out) and Later on farmers empty bins (sell out) and store other crops (e. g., corn and other store other crops (e. g., corn and other crops)crops)
Thereafter LGBs back to field?Thereafter LGBs back to field?
Hypothesis examinationHypothesis examination
Assumption: if LGBs stay in wheat Assumption: if LGBs stay in wheat bins during this period and they must bins during this period and they must consume wheat kernels which consume wheat kernels which contain contain glutengluten (protein). (protein).
If the gluten is detected in LGBs,If the gluten is detected in LGBs, The LGBs must consume wheatThe LGBs must consume wheat inferring LGBs from wheat bins?inferring LGBs from wheat bins?
Gluten Assay Kit (Catalogue No: 802002 Y) from Tepnel BioSystems Limited (One Newtech Square, Deeside Industrial Park, Deeside, Flintshire. CH5 2NT, U. K.).
MethodsMethods
Field population traps/collections Field population traps/collections (By (By Dr. Michael Toews)Dr. Michael Toews)
LGB washing procedureLGB washing procedure
Wheat gluten extraction from LGBWheat gluten extraction from LGB
ELISAELISA
WashingWashing 1). Randomly picked up live LGBs from laboratory 1). Randomly picked up live LGBs from laboratory
colonies that feed on wheat or rice kernels, or colonies that feed on wheat or rice kernels, or randomly selected frozen LGB samples collected randomly selected frozen LGB samples collected from fields from fields
2). Place the LGB samples into a beaker.2). Place the LGB samples into a beaker. 3). Added 100 ml of 0.1% 3). Added 100 ml of 0.1% TritonTriton X 100 to rinse X 100 to rinse
the LGB by shaking the beaker for one minute, the LGB by shaking the beaker for one minute, and then used tap water to rinse the samples 3-4 and then used tap water to rinse the samples 3-4 times until the water became clean. times until the water became clean.
4). Repeated step 3 washing procedure twice.4). Repeated step 3 washing procedure twice. 5). Rinsed the LGB samples twice using ddH2O.5). Rinsed the LGB samples twice using ddH2O. 6). Finally, let the LGB samples dry on Kimwipes 6). Finally, let the LGB samples dry on Kimwipes
tissues. tissues.
Wheat gluten extractionWheat gluten extraction 1) Added one individual LGB sample into a 1.5 ml 1) Added one individual LGB sample into a 1.5 ml
micro-centrifuge-tube with 30 µl of 40% ethanol and micro-centrifuge-tube with 30 µl of 40% ethanol and homogenized the sample by hand using pestle.homogenized the sample by hand using pestle.
2) Then, homogenized the sample for one minute 2) Then, homogenized the sample for one minute using Pellet Pestle Motor (Kontes Company).using Pellet Pestle Motor (Kontes Company).
3) Added 20 µl of 40% ethanol and homogenized the 3) Added 20 µl of 40% ethanol and homogenized the sample for another one minute.sample for another one minute.
4) Added 200 µl of 40% ethanol.4) Added 200 µl of 40% ethanol. 5) Centrifuged the tube at 2000 rpm for 15 minutes 5) Centrifuged the tube at 2000 rpm for 15 minutes
at room temperature.at room temperature. 6) Transferred 200 µl of supernatant to a new 1.5 6) Transferred 200 µl of supernatant to a new 1.5
micro-centrifuge-tube and added 300 µl of Working micro-centrifuge-tube and added 300 µl of Working Diluent Solution (from Gluten Assay Kit) for Gluten Diluent Solution (from Gluten Assay Kit) for Gluten Assay. Assay.
was provided by Tepnel BioSystems Limited was provided by Tepnel BioSystems Limited (One Newtech Square, Deeside Industrial (One Newtech Square, Deeside Industrial Park, Deeside, Flintshire. CH5 2NT, U. K.).Park, Deeside, Flintshire. CH5 2NT, U. K.).
We basically followed the detailed enzyme We basically followed the detailed enzyme immunoassay procedure for LGB gluten immunoassay procedure for LGB gluten assay from step 14.3 to 14.13 of Gluten assay from step 14.3 to 14.13 of Gluten Assay Kit. Only Step 14.4 and Step 14.7 Assay Kit. Only Step 14.4 and Step 14.7 were modified slightly by placing the were modified slightly by placing the microwell plate on a plate shaker at 200 microwell plate on a plate shaker at 200 rpm and incubated at rpm and incubated at 37ºC37ºC instead of room instead of room temperature. temperature.
Individual LGB gluten assay based on a blind experiment
Laboratory LGB
ControlOn Wheat On Rice
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
Detectable concentration of glutenDetectable concentration of glutenof one LGB (extraction)of 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
Gluten detection on starved LGBGluten detection on starved LGB
Index
DaysRepeat
1Repeat
2Repeat
3Repeat
4Repeat
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
Gluten detection in frozen LGBGluten detection in frozen LGB
Frozen LGB samples for 30 days
On Wheat 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
Preliminary field LGB examinationPreliminary field LGB examination
7 populations7 populations From late April to early JulyFrom late April to early July 5 individuals examined for each5 individuals examined for each
• Konza PrairieKonza Prairie 10 populations10 populations From late April to early SeptemberFrom late April to early September 5 individuals examined for each5 individuals examined for each
All negative responses All negative responses
Preliminary conclusionPreliminary conclusion
More tests are needed to prove the More tests are needed to prove the hypothesishypothesis