Quantification of wheat gluten in LGB Department of Entomology, Kansas State University, Manhattan, KS 66506 *USDA, ARS, GMPRC, Manhattan, KS 66502 Fengyou.

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

*USDA, ARS, GMPRC, Manhattan, KS 66502*USDA, ARS, GMPRC, Manhattan, KS 66502

Fengyou Jia, Sonny Ramaswamy, Michael Toews*, James Campbell*

LGB (Lesser Grain Borer)(Rhyzopertha dominica) (Coleoptera: Bostrichidae)

LGB in KansasLGB in Kansas

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?

LGB laboratory populationsLGB laboratory populations

Wheat gluten detection (ELISA)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.).

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.

ELISA ELISA Gluten Assay Kit (Catalogue No: 802002 Y) Gluten Assay Kit (Catalogue No: 802002 Y)

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

Populations examinedPopulations examined• KSU Farm Lands KSU Farm Lands

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

Caution:Caution:• Fact: wheat gluten undetectable Fact: wheat gluten undetectable

following 36 h digestion in LGBfollowing 36 h digestion in LGB• Locations of LGB trapped? Locations of LGB trapped?

Additional tests Additional tests

Objective: variation detection among Objective: variation detection among LGB populations LGB populations

Elemental component proportion (C, Elemental component proportion (C, O, and K) in LGBO, and K) in LGB

LGB scutellum scannedLGB scutellum scanned3 LGBs scanned from Each Population (expensive)

Result reportsResult 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.

Future experimentsFuture experiments

Near-bin-trapping vs. farther-bin-Near-bin-trapping vs. farther-bin-trapping from June to early Sept. ?trapping from June to early Sept. ?

More field LGB tested using wheat More field LGB tested using wheat gluten kitsgluten kits

LGB population variation LGB population variation examination using mass examination using mass spectrometer? spectrometer?

More release-capture experiments More release-capture experiments