10.5061/DRYAD.6Q573N5ZZ
Wada-Katsumata, Ayako
0000-0002-2895-1102
North Carolina State University
Schal, Coby
North Carolina State University
Olfactory Learning Supports an Adaptive Sugar-Aversion Gustatory Phenotype
in the German Cockroach
Dryad
dataset
2021
National Science Foundation
https://ror.org/021nxhr62
IOS-1557864
United States Department of Housing and Urban Development
https://ror.org/014a5gx79
NCHHU0053-19
2021-07-20T00:00:00Z
2021-07-20T00:00:00Z
en
171331 bytes
4
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
2.1. Insects 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 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 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 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 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 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). 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 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 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 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 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 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).