DOI: 10.5061/dryad.6q573n5zz
title: Olfactory Learning Supports an Adaptive Sugar-Aversion Gustatory Phenotype in the German Cockroach

description: An association of food sources with odors prominently guides foraging
 behavior in animals. To understand the interaction of olfactory memory and
 food preferences, we used glucose-averse (GA) German cockroaches. Multiple
 populations of cockroaches evolved a gustatory polymor-phism where glucose
 is perceived as a deterrent and enables GA cockroaches to avoid eating
 glucose-containing toxic baits. Comparative behavioral analysis using an
 operant conditioning paradigm revealed that learning and memory guide
 foraging decisions. Cockroaches learned to associate specific food odors
 with fructose (phagostimulant, reward) within only 1 hr of condi-tioning
 session, and with caffeine (deterrent, punishment) after only three 1 hr
 conditioning ses-sions. Glucose acted as reward in wild type (WT)
 cockroaches, but GA cockroaches learned to avoid an innately attractive
 odor that was associated with glucose. Olfactory memory was retained for
 at least 3 days after three 1 hr conditioning sessions. Our results reveal
 that specific tastants can serve as potent reward or punishment in
 olfactory associative learning, which reinforces gustatory food
 preferences. Olfactory learning therefore reinforces behavioral resistance
 of GA cockroaches to sugar-containing toxic baits. Cockroaches may also
 generalize their olfactory learning to baits that contain the same or
 similar attractive odors even if they do not contain glucose.
description: 2.1. Insects<br> The wild-type (WT) B. germanica colony
 (Orlando Normal) was collected in Florida in 1947 and has served as a
 standard insecticide-susceptible strain in many studies. The
 glucose-averse (GA) colony (T-164) was collected in Florida in 1989 and
 shown to be aver-sive to glucose; continued artificial selection with a
 glucose-containing toxic bait fixed the homozygous GA allele(s) in this
 population (approximately 150 generations as of 2020) resulting in maximal
 sensitivity to glucose as a deterrent. All cockroaches were main-tained on
 rodent diet (Purina 5001, PMI Nutrition International, St. Louis, MO) and
 dis-tilled water at 27°C, ~40% RH and a 12:12 h L:D cycle (8 pm to 8 am
 photophase, 8 am to 8 pm scotophase). We tested 10–18 days-old males
 during scotophase in this study.  2.2.
 Olfactometers<br> Each linear two-choice olfactometer was composed
 of two connected tubes. The first was an acclimation chamber (acrylic
 tube, 15 cm long, 3.2 cm i.d.) with a swivel metal screen gate. The upwind
 end of the acclimation chamber (near the gate) was connected to a bioassay
 tube (acrylic tube, 54.5 cm long, 3.2 cm i.d.) with a 15-cm-long acrylic
 divider sealed vertically in the upwind end. The divided bioassay tube
 formed a linear two-choice assay tube (Fig. 1A). Each olfactometer was
 connected to a vacuum pump that provided a linear air velocity of 25 cm/s
 through the tube; the tubes were exhausted outside the building.
 Fluorescent lights covered with red photographic safety filters placed 60
 cm be-low and above the olfactometers facilitated observation in the dark.
 In each bioassay, an individual cockroach was introduced into the
 acclimation chamber and allowed to accli-mate to the airflow for 5 min.
 Only quiescent insects were used in the assays.   2.3.
 Lures Containing Odors<br> Each red rubber septum lure (#1780 J07,
 Thomas Scientific, Philadelphia, PA, USA) was placed in a 1.5 ml
 microcentrifuge tube. Undiluted odor compounds or compounds dissolved in
 solvents were applied to each lure (total volume 100 µl) and placed in the
 end of the divided bioassay tube. Following traditional associative
 learning terminology, we termed the training odor associated with a
 tastant during the conditioning session as the conditioned stimulus (CS)
 (Fig. 1B). 2.4. Feeding Tubes Containing
 Tastants <br> An aqueous tastant solution (total liquid volume 1.5
 ml) was loaded into a feeding tube (1.5 ml microcentrifuge tube) and
 plugged with a small cotton ball. It was placed downwind of the lure of
 the divided bioassay tube, so that cockroaches could be exposed to
 specific odors as they fed. However, to prevent cockroaches from
 contacting the lure, a metal screen was placed between the feeding tube
 and lure. In the conditioning session, cockroaches were allowed to freely
 drink each of the two tastant solutions. Based on our previous findings
 [33,34], we used the 90% Effective Concentration (EC90) for each tastant,
 obtained from dose-feeding response curves: 300 mM fructose (EC90 for
 appetitive re-sponses in both strains), 1000 mM glucose (EC90 for
 appetitive responses in WT and aver-sive responses in GA cockroaches) and
 10 mM caffeine (EC90 for aversive responses in both strains). In WT
 cockroaches, fructose and glucose were rewards or appetitive
 uncon-ditioned stimuli (US+) for positive association with odors, and
 caffeine was punishment or aversive unconditioned stimulus (US-) for
 negative association with odors (Fig. 1B). In GA cockroaches, fructose
 acted as a reward, whereas caffeine and glucose were deterrent and served
 as punishment. When cockroaches associated a CS with US+ by conditioning
 (training), they were expected to prefer the CS in preference assays. When
 they associated CS with US-, they were expected to avoid or ignore the
 CS.  2.5. Chemicals<br> All chemicals, except
 vanilla and chocolate extracts, were obtained from Sigma Al-drich (St.
 Louis, MO, USA). Vanilla (All Natural Pure Vanilla Extract, McCormick,
 Hunt Valley, MD, USA) and Chocolate (Chocolate Extract, OliveNation, Avon,
 MA, USA) were obtained from local grocery stores.  2.6.
 Conditioning Session<br> The two-choice olfactometers were also used
 for the conditioning session, defined as the training of insects before
 bioassays. One day before any bioassays or conditioning, 20 adult males
 (10–12 days-old) were placed in a plastic cage (14.3 × 10.5 × 9.5 cm;
 T-79, Alt-hor Products, Windsor Locks, CT, USA) containing distilled
 water, rodent diet and egg carton shelter. The cage had two ports in line
 with each other to accept the bioassay tube upwind (air inlet) and a
 downwind exhaust tube (Fig. 1B). These ports were kept closed during the
 1-day-acclimation phase. In the Unconditioned odor preference assay,
 indi-vidual males were obtained directly from this cage. In the
 conditioning session for the other three bioassays, the downwind port was
 connected to a vacuum pump, as described in section 2.2 (Olfactometer).
 The upwind port was connected to the bioassay tube. After removing the
 rodent diet from the arena, feeding tubes and lures were placed upwind in
 the divided portion of the bioassay tube. Cockroaches were allowed to
 freely visit the feeding tubes and thus trained themselves to associate
 tastants and odors for 1 hr from 12 to 1 pm (mid-scotophase), when
 cockroaches actively forage [52]. All insects were attract-ed to odor
 sources and contacted the feeding tubes within 5 min after starting the
 condi-tioning, and all of them returned to the cage (and shelter) within 1
 hr. After the condition-ing session the ports were closed, rodent diet was
 returned to the cage, and males were kept in the cage until the next
 conditioning session or bioassays (Fig. 1B). <br> Traditional
 classical and operant conditioning rely on the assumption that changes in
 conditioned response probability observed during training adequately
 represent neu-ronal plasticity, and commonly, behavioral plasticity is
 quantified by averaging over a population of identically treated animals.
 On the other hand, recent studies using honeybees and American cockroach
 suggest that, even if insects were trained by well controlled methods, the
 average behavioral score of the group does not represent in-dividual
 behavior, which is driven by unique personality traits. The individual
 learning of such species may be influenced by various factors including
 their sensory sensitivity, their ability to learn a task, the speed of
 learning and their asymptotic performance. In this study, however, we did
 not identify individuals, because the group of insects was self-trained.
 Therefore, learning curves of individuals were not evaluated during the
 con-ditioning session, and in Bioassays 2–4 we tested the retained
 olfactory memory as the average behavioral score of groups after
 training. 2.7. Bioassay Procedures <br> Bioassays
 were conducted between noon and 4 pm, in mid-scotophase. Unless stated
 otherwise, a single male was tested only once in a clean olfactometer.
 When the male was quiescent in the acclimation chamber, the gate was
 opened carefully, the lures containing odor stimuli were introduced at the
 upwind end of the olfactometer, and the insect’s re-sponse was noted by
 direct observation. A positive response was scored when the male entered
 the divided bioassay tube within 2 min and remained there for 15 sec.
 After each bioassay, each olfactometer was flushed out with fresh air for
 2 min. The positions of the two lures were randomly switched between the
 left and right sides of the divided section of the bioassay tube. Every
 five bioassays, the olfactometers were washed with distilled water and
 ethanol. The percentage of males responding was calculated by the formula:
 % responding = # of insects making a choice / total # of tested insects.
 The percentage choice for each lure was calculated as: % choice = # of
 insects choosing the lure either on the right side or left side / total #
 of insects making a choice. Chi-square tests (α = 0.05) and Tukey’s Wholly
 Significant Difference (WSD) tests (α = 0.05) were used to compare
 treatments, the two lures, and cockroach strains. 2.8.
 Bioassay 1: Unconditioned Odor Preference<br> Considering that in
 their natural environment, cockroaches approach certain innate-ly
 preferred odors and avoid innately repellent odors, we screened for
 attractive food odors in various food sources, including plant materials
 and human food for use in Bioassays 2 through 4 (Fig. 1C). Distilled
 water, ethanol and mineral oil were used as solvents. As general odors
 contained in sweet snacks and chocolate drink, we used
 3-methyl-1,2-cyclopentanedione (coffee and caramel flavor, 10-4, 10-3,
 0.01, 0.1, 1, 10 µg in 100 µl mineral oil per lure),
 4,5-dimethyl-2-ethylthiazole (burnt hazelnut odor, 10-8, 10-7, 10-6, 10-5,
 10-4, 10-3 µg in 100 µl mineral oil per lure) and 2,4,5-trimethylthiazole
 (musty, nutty, and brown cocoa and coffee odor, 10-6, 10-5, 10-4, 10-3,
 0.01, 0.1 µg in 100 µl mineral oil per lure). As general plant terpenes,
 we used beta-caryophyllene (sweet woody spicy and peppery odor) and
 farnesene (sweet, woody, herbal and green aroma) at 10-4, 10-3, 0.01, 0.1,
 1, and 10 µg in 100 µl mineral oil in each lure. As aromatic aldehydes, we
 used benzaldehyde (almond odor, 10-4, 10-3, 0.01, 0.1, 1, 10 µg in 100 µl
 mineral oil per lure) and vanillin (vanilla odor,
 4-hydroxy-3-methoxybenzaldehyde, 10-4, 10-3, 0.01, 0.1, 1, 10 µg in 100 µl
 ethanol per lure). As a blend of sweet odors, commercial vanilla extract
 (All Natural Pure Vanilla Extract, McCormick, Hunt Valley, MD, USA) and
 chocolate extract (OliveNa-tion, Avon, MA, USA) were dissolved in
 distilled water at 0.01, 0.1, 1 equivalents of the original product to
 find the optimal concentrations for our bioassays. Each odor was ap-plied
 to a single red rubber septum lure and placed in one side of the divided
 bioassay tube. A solvent-only lure was placed in the other side of the
 divided tube. We tested 20–30 insects at each concentration of each odor
 source. Additionally, a two-choice test using vanilla and chocolate was
 conducted with the undiluted original products to assess the unconditioned
 (innate) odor preferences for these two attractive odors (Table S1 and
 S2). 2.9. Bioassay 2: Conditioned Odor Preference After
 Conditioning with a Single Odor<br> To test whether the insects
 associated odor with either rewarding or punishing tastants, cockroaches
 self-trained (operantly conditioned) with a combination of a single odor
 and a single tastant in the conditioning session, then olfactometer
 bioassays were carried out (Fig. 1C). During conditioning, one side of the
 divided bioassay tube contained a single combination of ‘odor (lure) +
 tastant (feeding tube)’. Six types of combinations were prepared: ‘Vanilla
 (CS) + Frucotse, Glucose or Caffeine (US+ or US-)’ and ‘Chocolate (CS) +
 Fructose, Glucose or Caffeine (US+ or US-)’. The other side of the tube
 contained a lure and the feeding tube contained distilled water. To test
 the impact of the training, we tested two types of conditioning sessions.
 The first was a single conditioning session of 1 hr, after which the
 insects were tested for their odor preference in the two-choice preference
 assay using both vanilla and chocolate odors, namely, the CS (either
 vanilla or chocolate) and US (either vanilla or chocolate) approximately
 24 hrs later (Fig. 1C). The second training paradigm was three successive
 1 hr conditioning sessions (1 hr at 12–1 pm each day for three days),
 followed by odor preference assays approximately 24 hrs later. In a
 comparison of odor preference among the treatments, we used the results of
 the innate odor preferences from Bioassay 1 as control. If trained
 cockroaches chose the CS more than untrained cockroaches do, we considered
 that they associated the CS with the US+. If trained cockroaches preferred
 the CS less than the untrained cockroaches do, we consid-ered that they
 associated CS with the US-. We tested 30–40 males in each
 treatment. 2.10. Bioassay 3: Conditioned Odor
 Preference After Conditioning with Two Odors <br> To test whether
 insects associated two combinations of tastants and odors, males were
 trained with both vanilla and chocolate odors using two types of tastants.
 The di-vided tubes contained different combinations of ‘lure + tastant’:
 ‘Either Vanilla or Choco-late + either Fructose or Caffeine’, ‘Either
 Vanilla or Chocolate + either Fructose or Glucose’ and ‘Either Vanilla or
 Chocolate + either Glucose or Caffeine’. Males received either one or
 three successive 1 hr conditioning sessions. In this paradigm, both
 vanilla and chocolate used in the two-choice olfactometer bioassays acted
 as CS associated with either US+ or US- (Fig. 1C). Data analysis was by
 the same methods described in section 2.9 (Bioassay 2). 
 2.11. Bioassay 4: Retention of Olfactory Memory<br> To test
 the retention of olfactory memory, we exposed insects to three successive
 1 hr conditioning sessions (1 hr at 12–1 pm each day for three days). The
 combinations of ‘lure + tastant’ were ‘Vanilla + Fructose and Chocolate +
 Caffeine’, ‘Vanilla + Fructose and Chocolate + Glucose’ and ‘Vanilla +
 Glucose and Chocolate + Caffeine’. Conditioned pref-erence bioassays with
 vanilla and chocolate were conducted 2, 3 and 5 days later (Fig. 1C). Data
 analysis was by the same methods described in section 2.9 (Bioassay
 2).