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.

(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 Backcross assay 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 Acceptance-rejection assay (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 Two-choice feeding assay. 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 (EC₅₀) for each tastant was obtained from dose-response curves using this assay. To evaluate EC₅₀ 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^-1). The EC₅₀ values of WT♀ and GA♀ were obtained from previous work [29]. To evaluate the EC₅₀ 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 Acceptance-rejection assay 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^-1 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.

Reference [29] 

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.