Quantification of wheat gluten in LGB Department of Entomology, Kansas State University, Manhattan, KS *USDA, ARS, GMPRC, Manhattan, KS Fengyou Jia, Sonny Ramaswamy, Michael Toews*, James Campbell*

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

LGB in Kansas 4-5 generations annually 4-5 generations annually Minimum Life Cycle: 25 days (1month in 90ºF) Minimum Life Cycle: 25 days (1month in 90ºF) Major food is wheat, alternative food resources? Major food is wheat, alternative food 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 LGB populations vary temporally and spatially LGB populations vary temporally and spatially There are two peaks annually There are two peaks annually First peak appears around June, second peak appears around August First peak appears around June, second peak appears around August LGB population is often very low between the two peak LGB population is often very low between the two peak

Field LGB Population monitored with pheromone traps AprilMayJuneJulyAugSept ?

Field LGB Population AprilMayJuneJulyAugSept Bin LGB Bin empty ? Moved to Sold out ? Image of courtesy:

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

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

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

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

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 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. 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. 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. 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 tissues. 6). Finally, let the LGB samples dry on Kimwipes tissues.

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. 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). 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. 3) Added 20 µl of 40% ethanol and homogenized the 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 at room temperature. 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. 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.

ELISA Gluten Assay Kit (Catalogue No: Y) was provided by Tepnel BioSystems Limited (One Newtech Square, Deeside Industrial Park, Deeside, Flintshire. CH5 2NT, U. K.). Gluten Assay Kit (Catalogue No: 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 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. We basically followed the detailed enzyme immunoassay procedure for LGB gluten assay from step 14.3 to 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.

Individual LGB gluten assay based on a blind experiment Laboratory LGB Control On WheatOn Rice LabelIndex*LabelIndex*CKIndex w10.162R10.058A0.58 w20.283R20.062B0.337 w30.103R30.054C0.184 w40.155R40.125*D0.094 w50.102R50.054E0.068 w60.159R w70.093R w80.057*R w w100.1 NCK**0.052 Notes: * either false positive or false negative

Detectable concentration of gluten of one LGB (extraction) Concentrati on* %12345Average

Gluten detection on starved LGB Index Days Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5Average

Gluten detection in frozen LGB Frozen LGB samples for 30 days On WheatOn Rice Repeat*IndexRepeat**Index w10.353R w20.166R w30.141R30.106* w40.101R w50.184R50.056

Preliminary field LGB examination Populations examined Populations examined KSU Farm LandsKSU Farm Lands 7 populations 7 populations From late April to early July From late April to early July 5 individuals examined for each 5 individuals examined for each Konza PrairieKonza Prairie 10 populations 10 populations From late April to early September From late April to early September 5 individuals examined for each 5 individuals examined for each All negative responses All negative responses

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

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

LGB scutellum scanned 3 LGBs scanned from Each Population (expensive)

Result reports KSU Farm landKonzaOn-RiceOn-Wheat C O K P0.010N/A Al N/A SiN/A0.037N/A N 4.953N/A CLN/A Notes: Average element proportion.

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