DOI: 10.5061/dryad.xpnvx0kk6
title: Data from: Gustatory polymorphism mediates a new adaptive courtship strategy

description: Human-imposed selection can lead to adaptive changes in sensory traits.
 However, rapid evolution of the sensory system can interfere with other
 behaviors, and animals must overcome such sensory conflicts. In response
 to insecticide baits that contain glucose, German cockroaches evolved
 glucose-aversion (GA), which confers behavioral resistance against baits.
 During courtship, the male offers the female a nuptial gift that contains
 maltose, which expediates copulation. However, the female’s saliva rapidly
 hydrolyzes maltose into glucose, which causes GA females to dismount the
 courting male, which reduces mating success of GA females. Comparative
 analysis revealed two adaptive traits in GA males. They produce
 maltotriose, which is more resilient to salivary glucosidases, and they
 initiate copulation faster than wild-type (WT) males, before GA females
 interrupt their nuptial feeding and dismount the male. Recombinant lines
 of the two strains showed that the two emergent traits of GA males were
 not genetically associated with the GA trait. Results suggest that the two
 courtship traits emerged in response to the altered sexual behavior of GA
 females and independently of the male’s GA trait. Although rapid adaptive
 evolution generates sexual mismatches that lower fitness, compensatory
 behavioral evolution can correct these sensory discrepancies.
description: (a) Cockroach
 strains All cockroaches were maintained on rodent diet (Purina 5001, PMI
 Nutrition International, St. Louis, MO) and distilled water at 27°C, ~40%
 RH and a 12:12 h L:D cycle. The WT colony (Orlando Normal) was collected in
 Florida in 1947 and has served as a standard insecticide-susceptible
 strain. The GA colony (T-164) was also collected in Florida in 1989 and
 shown to be aversive to glucose; continued artificial selection with
 glucose-containing toxic bait fixed the homozygous GA trait in this
 population (approximately 150 generations as of
 2020). As described in detail in our previous study
 [29] and in the electronic supplementary material, two recombinant colonies were initiated in
 2013 by crossing 10 pairs of WT♂ × GA♀ and 10 pairs of GA♂ × WT♀ to
 homogenize the genetic backgrounds of the two strains. At the F8
 generation (free bulk mating without selection), 400 cockroaches were
 separated into glucose-accepting and glucose-rejecting groups. These
 colonies were bred for three more generations, and 200 cockroaches from
 each group were assayed in the F11 generation and backcrossed to confirm
 homozygous glucose-accepting (aa) and glucose-averse (AA) lines. Similar
 results were obtained in both directions of the cross, confirming previous
 findings of no sex linkage of the GA trait [25]. Glucose-aversion
 homozygosity of these two lines were confirmed by <strong>Backcross
 assay</strong> to confirm as WT_aa (homozygotes, glucose-accepting)
 and GA_AA (homozygotes, glucose-averse). Then we cultured these two lines
 for three more generations (F14) and used them for this
 study. To determine the effective concentration
 (EC50 values) for glucose acceptance and rejection in
 females, F14 WT_aa and F14 GA_aa were tested by the
 <strong>Acceptance-rejection assay</strong> (see below).
 (b)
 Mating bioassay A male and a female were placed in a Petri dish
 with fresh water and a piece of rodent food and video-recorded for 24 hrs.
 The males were 10–12-days-old, and females were 5–7-days-old. Four
 treatment pairs were tested: WT♂ and WT♀ (n = 52); WT♂ and GA♀ (n = 67);
 GA♂ and WT♀ (n = 52); GA♂ and GA♀ (n = 80), as described elsewhere
 [29].
 On the other hand, to
 evaluate the association of male courtship traits and the GA trait, before
 mating assays with GA♀, 7-day-old males from the F14 recombinant lines
 (WT_aa and GA_AA) were subjected to a <strong>Two-choice feeding
 assay</strong>. Ten
 males (WT_aa♂,
 non-starved; GA_AA♂, 1-day starved without water) were placed in a Petri dish (90 mm × 15 mm
 height). Each Petri dish contained two agar discs: one disc contained 1%
 agar and 1 mmol l−1 red food dye (Allura Red AC), and the second disc
 contained 1% agar, 0.5  mmol l−1 blue food dye (Erioglaucine disodium
 salt) and 1000  mmol l−1 glucose. The assay duration was 2 h during the
 dark phase of the insects’ L:D cycle. After each assay, the color of the
 abdomen of each cockroach was visually inspected under a microscope to
 assess their
 glucose-appetitive or glucose-aversive behavioral traits. Individuals were
 paired with 0-day-old GA♀ (n = 25 WT_aa♂, n = 24 GA_AA♂). They mated when
 the females became 5–7 days old. The mating behavior of the focal insect was
 video-recorded for several days until they mated. All mating sequences
 were recorded using an infra-red-sensitive camera (Polestar II EQ610,
 EverFocus Electronics) coupled to a data acquisition board and analyzed by
 searchable and frame-by-frame capable software (NV3000, AverMedia
 Information) at 27°C, ~40% RH and a 12:12 h L:D cycle. Tested pairs were
 classified into two groups: mated (successful mating) and not-mated
 (failed mating) within the 24 hr video recording. Three distinct
 behavioral events, described previously [29], included Contact, Wing
 raising, Nuptial feeding and Copulation, and we analyzed four parameters:
 Latency of wing raising display in male (sec), nuptial feeding duration in
 female (sec), copulation latency in male (sec) and copulation duration
 (sec). A successful mating sequence was defined as the courtship events
 from Contact to Copulation in the mated group.  (c) Acceptance-rejection
 assay This is a rapid qualitative assay that assessed
 the instantaneous initial responses (binary: yes-no) of cockroaches to
 tastants, as previously described [26]. Briefly, acceptance means that the
 cockroach started drinking. Rejection means that the cockroach never
 initiated drinking. To conduct dose–response studies with phagostimulants
 and deterrents, adults were deprived of food for 24 h but supplied with
 water. When testing deterrents, the cockroaches were deprived of both food
 and water for 24 h to increase their thirst. The mouthparts were carefully touched with a
 drop of stimulus solution colored with 1 mmol l-1 blue food dye
 (erioglaucine) in a sequence from the lowest to the highest concentration.
 The percentage of positive responders was defined as the Number of insects
 accepting a tastant/Total number of insects tested. The effective
 concentration (EC<sub>50</sub>) for each tastant was obtained from
 dose-response curves using this assay. To evaluate EC<sub>50</sub> for
 glucose in the recombinant lines, 4-day-old ♀ (30 F14 WT_aa were
 non-starved and 30 F14 GA_aa were starved for 1 day without water) were
 tested with a concentration series of glucose (0, 10, 30, 100, 300 and
 1000 mmol l<sup>-1</sup>). The EC<sub>50</sub> values of WT♀ and GA♀ were obtained from
 previous work [29]. To evaluate the EC<sub>50</sub> of female feeding acceptance for nuptial
 secretions from WT♂ and GA♂, each nuptial secretion was diluted with
 HPLC-grade water (Fisher Scientific) to 0.001, 0.01, 0.03, 0.1, 0.3 and 1
 male-equivalents/µl, and 20 non-starved 4-day-old WT♀ and GA♀ were
 tested. (d) Consumption assay
 To quantify the amount of 0.1
 male-equivalents/µl of nuptial secretion females ingested, non-starved
 5-day-old females were observed until they stopped drinking, and we
 considered this a single bout of feeding (n = 10 ♀ for
 each). (e) Effect of female saliva on acceptance of
 nuptial secretion Freshly collected saliva of WT♀ and GA♀ was
 immediately used in experiments. Assays were prepared as follows: Nuptial
 secretion (1 µl representing 10 male-equivalents) was mixed with 1 µl of
 either HPLC-grade water or saliva from WT♀ or GA♀, and 8 µl of HPLC-grade
 water was added to the mix. The final concentration of the nuptial
 secretion was 1 male-equivalent/µl in a total volume of 10 µl. The mix of
 saliva and nuptial secretion was incubated for 300 sec at 25°C, then the
 <strong>Acceptance-rejection assay</strong> was carried out
 with 5-day-old WT♀ and GA♀ (n = 20–33). Saliva alone does not affect
 acceptance or rejection of stimuli [29]. Additionally, to confirm the
 contribution of salivary glucosidases in salivary digestion of nuptial
 secretion, the effect of the glucosidase inhibitor acarbose wwas tested in
 the acceptance-rejection assay. Test solutions were prepared as follows: 1
 µl of either HPLC-grade water or saliva from 5 days-old GA♀ was mixed with
 0.5 µl of either 250 µmol l<sup>-1</sup> of acarbose or
 HPLC-grade water. This mixture was added to 0.5 µl of 10 male-equivalents
 of nuptial secretion. HPLC-grade water was added for a total volume of 10
 µl and a final concentration of 1 male-equivalent/µl. Salivary incubation
 was done with 300 sec at 25°C. Test subjects were 5-day-old GA♀ (n =
 20). (f) Nuptial secretion and saliva
 collections The nuptial secretion of males was collected by
 the following method: Five 10–12-days-old males were placed in a container
 (95 × 95 × 80 mm) with 5-day-old GA♀. After the males displayed
 wing-raising courtship behavior toward the females, individual males were
 immediately decapitated and the nuptial secretion in their tergal gland
 reservoirs was drawn into a calibrated borosilicate glass capillary
 (76 × 1.5 mm) under the microscope. The nuptial secretions from 30 males
 were pooled in a capillary and stored at -20 °C until use. Saliva from
 5-day-old WT♀ and GA♀ was collected by the following method: individual
 females were briefly anesthetized with carbon dioxide under the microscope
 and the side of the thorax was gently squeezed. A droplet of saliva that
 accumulated on the mouthparts was then collected into a microcapillary
 (10 µl, Kimble Glass). Fresh saliva was immediately used in
 experiments. (g) Sugar analysis using
 GC-MS We focused the GC-MS analysis of nuptial
 secretion on glucose, maltose and maltotriose of WT♂, GA♂, WT_aa♂ and
 GA_AA♂. Details of the methods are described elsewhere [29], but
 additional details are provided in the electronic supplementary material.
 D-(+)-maltose (Fisher Scientific), D-(+)-glucose and maltotriose
 (Sigma-Aldrich) were used as calibration standards. Sorbitol was used as
 an internal standard for each sample.
 N-methyl-N-(trimethylsilyl)trifluoroacetamide
 (MSTFA; Sigma-Aldrich) was used for derivatization of sugars, and samples
 were analyzed by GC-MS, as detailed in [29] and in the electronic
 supplementary material). To quantify the time-course of saliva-catalyzed
 hydrolysis of WT♂ and GA♂ secretion to glucose, 1 µl of GA♀ saliva was
 mixed with 1 µl of 10 male-equivalents/µl HPLC-grade water. We incubated
 the mixtures for 0, 5, 10 and 300 s at 25 °C, and added 4 µl of methanol
 to stop enzyme activity (n = 5 each treatment). Each sample contained the
 nuptial secretions of 5 males to obtain enough detectable amounts of
 sugars. For the statistical analysis, the amounts of sugars were divided
 by 5 to obtain the amounts of sugars in 1 male (1
 male-equivalent). (h) Statistical analysis
 The sample size and number of
 replicates for each experiment are noted in the respective section
 describing the experimental details. In summary, the sample sizes were:
 Mating bioassays, n = 24–80; Feeding assays using females, n = 20–30;
 Sugar analysis, n = 5–7. All statistical analyses were conducted in Prism
 (GraphPad Software, San Diego, CA). For bioassay data and sugar analysis
 data, we calculated the means and standard errors, and we used the
 Chi-square test with Holm’s method for post hoc comparisons, t-test, and
 ANOVA followed by Tukey’s HSD test (all α = 0.05), as noted in each
 section describing the experimental details, results, and in the
 electronic supplementary material, tables S1–8. 
 <strong>Reference</strong>
 <strong>[29]</strong>  Wada-Katsumata A,
 Hatano E, McPherson S, Silverman J, Schal C. 2022 Rapid evolution of an
 adaptive taste polymorphism disrupts courtship behavior. Commun Biol. 5,
 450. doi: 10.1038/s42003-022-03415-8.