DOI: 10.5061/dryad.jsxksn08r
title: Data from: Salivary digestion extends the range of sugar-aversions in the German cockroach

description: Saliva has diverse functions in feeding behavior of animals. However, the
 impact of salivary digestion of food on insect gustatory information
 processing is poorly documented. Glucose-aversion (GA) in the German
 cockroach, Blattella germanica, is a highly adaptive heritable behavioral
 resistance trait that protects the cockroach from ingesting
 glucose-containing-insecticide-baits. In this study, we confirmed that GA
 cockroaches rejected glucose, but they accepted oligosaccharides.
 However, whereas wild-type cockroaches that accepted glucose also satiated
 on oligosaccharides, GA cockroaches ceased ingesting the oligosaccharides
 within seconds, resulting in significantly lower consumption. We
 hypothesized that saliva might hydrolyze oligosaccharides, releasing
 glucose and terminating feeding. By mixing artificially collected
 cockroach saliva with various oligosaccharides, we demonstrated
 oligosaccharide-aversion in GA cockroaches. Acarbose, an alpha-glucosidase
 inhibitor, prevented the accumulation of glucose and rescued the
 phagostimulatory response and ingestion of oligosaccharides. Our results
 indicate that pre-oral and oral hydrolysis of oligosaccharides by salivary
 alpha-glucosidases released glucose, which was then processed by the
 gustatory system of GA cockroaches as a deterrent and caused the rejection
 of food. We suggest that the genetic mechanism of glucose-aversion support
 an extended aversion phenotype that includes glucose-containing
 oligosaccharides. Salivary digestion protects the cockroach from ingesting
 toxic chemicals and thus could support the rapid evolution of behavioral
 and physiological resistance in cockroach populations.
description: <strong>2. Materials and Methods</strong>
 2-1. Two cockroach strains<br> The wild-type strain
 (Orlando Normal) was collected in Florida in 1947. The glucose-averse
 strain (T164) was collected in Florida in 1989 and was artificially
 selected with glucose-containing toxic bait to fix the glucose-averse (GA)
 trait in this population. The two strains were maintained on rodent diet
 (Purina 5001, PMI Nutrition International, St. Louis, MO) and distilled
 water at 27°C and ~40% RH on a 12:12 h L:D cycle. We tested sexually
 mature virgin females (5-day old) in bioassays because at this stage
 females forage extensively in support of oocyte maturation.
 2-2. Acceptance-rejection assays and consumption assays<br>
 We developed two independent assays to assess feeding.
 <em>Acceptance-rejection assay.</em> This was a
 qualitative yes-no assay that evaluated the instantaneous responses of
 cockroaches to tastants. It involved a small amount of test solution
 placed on the paraglossae—the most proximal mouthpart where sugar
 responsive gustatory sensilla have been observed —and a rapid assessment
 of either its acceptance or rejection. In this assay, the test solution
 stimulated gustatory sensilla on the paraglossae, with only brief
 interaction with saliva. Either one-day starved or non-starved insects
 were restrained in a plastic pipette tip with only the head exposed.
 Before starting the assay, insects were satiated with water, except when
 they were tested with deterrents. The paraglossae, were carefully touched
 under a stereo-microscope with a 0.3 µl drop of test solution dyed with 1
 mM blue food dye (Erioglaucine disodium salt). Acceptance of the taste
 substance was defined as ingestion within 1 sec, and rejection was defined
 as lack of ingestion within 1 sec. During ingestion of the stimulus
 solution the blue dye could be seen through the clypeus and frons, the
 translucent front-middle area of the head capsule. The cockroaches
 ingested &lt;0.01 µl of the test solution before the solution was
 withdrawn to avoid satiation. <em>Consumption
 assay</em>. This assay quantitatively measured the amount of test
 solution ingested by each insect. Non-starved females were individually
 restrained in a pipette tip as in the feeding assay. A test solution
 containing 0.5 mM blue food dye in a microcapillary (10 µl, Kimble Glass)
 was brought in contact with the paraglossae and the volume consumed was
 recorded. We observed each female until she stopped drinking, and we
 considered this a single bout of feeding. Therefore, in this assay the
 test solution stimulated gustatory sensilla on the paraglossae, and more
 of the test solution had longer contact with saliva.  
 2-3. Sugar acceptance and consumption<br> To assess the
 feeding responses of WT and GA females in the acceptance-rejection assays,
 monosaccharides (d-glucose and d-fructose), disaccharides (d-maltose,
 d-trehalose, d-sucrose) and a trisaccharide (d-maltotriose) were tested.
 All chemicals were prepared at 0.01, 1, 10, 100 and 1000 mM with 1 mM blue
 food dye. Two groups of females were prepared: Group A was prepared using
 non-starved females to assess their acceptance responses of phagostimulant
 sugars. Each insect was satiated with water before the test, to ensure
 that their acceptance of phagostimulants was not due to thirst; these
 females would reject the negative control (water with blue dye) and
 various deterrents. For each sugar 21 WT and 31 GA females were tested.
 Group B was prepared to assess their rejection responses of various
 tastants. Females were starved for one day without food and water to
 produce hungry and thirsty females that were motivated to accept water and
 phagostimulants, but would reject deterrents at high concentration. Data
 were obtained from 20 GA females for glucose. The effective concentration
 (EC50) of each sugar was obtained from dose-response curves. For Group A,
 dose-response curves and EC50 values were obtained for WT and GA females
 in response to six and five sugars, respectively. Glucose was tested as a
 deterrent in GA females in Group B. In the consumption assay, the amount
 consumed (µl) of six types of sugar solutions with 1 mM blue food dye was
 measured at 0, 10, 100 and 1000 mM. Each female received a single
 concentration of a single test solution. For assays using plain water
 consumption, 26 WT and 13 GA females were used. For sugar consumption,
 21–25 females were tested at each concentration of each test
 solution. 2-4. Effects of saliva on sugar degradation
 and feeding<br> To evaluate the effects of saliva mixed with sugar
 solutions on the feeding response in the acceptance-rejection assay,
 saliva from 5 day-old WT and GA females was collected. Individual females
 were briefly anesthetized with carbon dioxide under a microscope and
 gently squeezed at the side of the thorax to avoid contamination with
 regurgitates from the gut. Saliva droplets were collected at the
 mouthparts into a microcapillary (10 µl, Kimble Glass). Collected fresh
 saliva was immediately used in experiments. Test solutions were prepared
 by mixing 3 ml of 200 mM maltose, maltotriose, trehalose, sucrose or 600
 mM glucose or fructose with 3 ml of either HPLC-grade water or saliva of
 WT or GA females. The four tri- and disaccharides were presented to
 females at a final concentration of 100 mM, and the two monosaccharides
 (glucose and fructose) were presented at 300 mM in a total volume of 6 ml.
 These concentrations were chosen based on the EC70 in the
 acceptance-rejection assay. Saliva and sugars were incubated for 5 min at
 25oC. We tested 18–22 females in each strain. To evaluate the effect of
 saliva alone on the feeding response, either non-starved females or
 females that were starved for one day without food and water were tested
 with water only and the mixture of saliva and water at a 1:1 ratio (n =
 20). All test solutions contained blue food dye. 2-5.
 Involvement of salivary glucosidases in sugar degradation<br> To
 evaluate whether salivary enzymes are involved in the hydrolysis of
 disaccharides and trisaccharides, the contribution of salivary
 alpha-glucosidases was tested in the feeding acceptance-rejection assay
 using the glucosidase inhibitor acarbose (CAS 56180-94-0, Sigma Aldrich).
 We first confirmed that the range of 0–125 mM acarbose in HPLC-grade water
 did not disrupt feeding acceptance or rejection of cockroaches. Test
 solutions were prepared as follows: 2 µl of either HPLC-grade water or
 saliva of GA females was mixed with 1 µl of either 250 µM of acarbose or
 HPLC-grade water, then the mixture was added to 1 µl of 400 mM of six
 different sugar solutions. The total volume was 4 µl with the final
 concentration of sugar being 100 mM. As before, saliva and sugars were
 incubated for 5 min at 25°C and all test solutions contained blue food
 dye. Only 5 day-old GA females (n = 20–25) were tested in each
 assay.   2-6. Salivary protein and alpha-glucosidase
 activity<br> Salivary protein was measured by the Bradford method
 (Coomassie protein assay kit, Thermo Scientific, 23200, Rockford, IL, USA)
 to compare differences between sexes and strains. We conducted 6–8
 replications with both sexes of the two strains. To estimate
 alpha-glucosidase activity of saliva, a colorimetric assay using
 p-nitrophenol (alpha-glucosidase activity colorimetric assay kit,
 Biovision, K690-100, Milpitas, CA, USA) was carried out. Either 1 µl of
 HPLC-grade water or 250 mM acarbose was added to 1 µl of saliva and
 incubated for 5 min at 25 oC. We conducted 5 replications with both sexes
 of the two strains.