Plasticity of the Phonotactic Selectiveness of Four Species of Chirping Crickets (Gryllidae): Implications for Call Recognition Benjamin Navia

Andrews University

From the SelectedWorks of Benjamin Navia

Winter January 5, 2010

Plasticity of the phonotactic selectiveness of four species of chirping crickets (Gryllidae): Implications for call recognition Benjamin Navia

Available at: https://works.bepress.com/benjamin-navia/14/ Physiological Entomology (2010) 35, 99–116 DOI: 10.1111/j.1365-3032.2009.00713.x

Plasticity of the phonotactic selectiveness of four species of chirping crickets (Gryllidae): Implications for call recognition

JOHN STOUT1, BENJAMIN NAVIA2, JASON JEFFERY1, LESLIE SAMUEL1, LAURA HARTWIG1, ASHLEY BUTLIN1, MARY CHUNG1, JESSICA WILSON1, ERICA DASHNER1 andGORDON ATKINS1 1Biology Department, Andrews University Berrien Springs, Michigan, U.S.A. and 2Department of Human Biology, Kettering College of Medical Arts, Kettering, Ohio, U.S.A.

Abstract. Earlier studies of phonotaxis by female crickets describe this selective behavioural response as being important in the females’ choices of conspecific males, leading to reproduction. In the present study, moderate (30+) to very large data sets of phonotactic behaviour by female Acheta domesticus L., Gryllus bimaculatus DeGeer, Gryllus pennsylvanicus Burmeister and Gryllus veletis Alexander demonstrate substantially greater plasticity in the behavioural choices, as made by females of each species, for the syllable periods (SP) of model calling songs (CS) than has been previously described. Phonotactic choices by each species range from the very selective (i.e. responding to only one or two SPs) to very unselective (i.e. responding to all SPs presented). Some females that do not respond to all SPs prefer a range that includes either the longest or shortest SP tested, which fall outside the range of SPs produced by conspecific males. Old female A. domesticus and G. pennsylvanicus are more likely to be unselective for SPs than are young females. Each species includes females that do not respond to a particular SP when responding to CSs with longer and shorter SPs. The results suggest that the plasticity of phonotactic behaviour collectively exhibited by the females of each species does not ensure that choices of a male’s CS effectively focus the female’s phonotactic responses on CSs that represent the conspecific male. The phonotactic behaviour collectively exhibited by females of each species does not readily fit any of the models for selective processing by central auditory neurones that have been proposed to underlie phonotactic choice. Key words. Call recognition, L3/AN2 neurone, neuronal filtering, phonotaxis, plasticity, selective behaviour.

Introduction several species and has been described as a rather consistent, species-specific behaviour that precedes mating (Popov Female crickets locate and walk towards (phonotactic behav- & Shuvalov, 1977; Moiseff et al., 1978; Pollack & Hoy, 1981; iour) the source of model calling songs (CSs) when their Thorson et al., 1982; Doherty, 1985a; Stout & McGhee, 1988). carrier frequencies (4–5 kHz) and their temporal structures In many species, syllable period (SP) is the most important are similar to that produced in the CSs of conspecific males temporal feature of the CS used by females to discriminate (Popov & Shuvalov, 1977; Stout et al., 1983; Jeffery et al., between calls of males of different species. Thorson et al. 2005). Phonotaxis by female crickets has been examined in (1982) report that female Gryllus campestris respond with SP- selective phonotaxis to CSs with SPs in the range 40–60 ms, Correspondence: John Stout, Biology Department, Andrews Uni- which is characteristic of the CS of conspecific males. Jeffery versity Berrien Springs, Michigan 49104, U.S.A. Tel.: +1 269 471 et al. (2005) demonstrate that female G. pennsylvanicus are 3261; fax: +1 269 471 6911; e-mail: [email protected] most attracted to CSs with SPs in the range 35–60 ms. Female

G. bimaculatus (Doherty, 1985a) show an attractive range of Louisiana). The crickets were raised to adults in 40-L plastic SPs similar to that reported for G. campestris and exhibit little containers under an LD 12 : 12 h photoperiod (lights on ◦ variability that can be attributed to the order of presentation of 06.00 h) at 21–22 C. Cricket chow (Fluker’s Cricket Farm), the stimuli. water, and egg cartons (for shelter) were provided in each In an early comparative study of phonotactic responses of container. Each day, the containers were checked for adults. female crickets to the CS of conspecific males, Popov & Newly-moulted females (0 days old) were isolated from the Shuvalov (1977) first suggest plasticity in the phonotactic males each day and transferred to 16-L containers, containing responses of female crickets from five different species. The paper towels for cover, and were housed in a separate study concludes that the phonotactic response of females is environmental chamber. Adult males were discarded. Cricket not a fixed, automatic reaction but a rather complex behaviour chow and water were provided daily. that can be modified by internal and external conditions. In a more recent study, Stout et al. (2007) show that phonotaxis by female G. bimaculatus is variable; some females respond Gryllus pennsylvanicus and G. veletis. Nymphs of these selectively to a range of SPs that included the conspecific species were caught in the field in Berrien and Crawford male’s CS, whereas others are much less selective. counties in Michigan, placed in 16-L containers as nymphs Phonotaxis in female Acheta domesticus is also originally and transferred to 1-L containers on the day of the imaginal described as a rather consistent behaviour: female A. domesti- molt. Otherwise, they were cared for in the laboratory cus are most attracted to CSs with syllable periods in the range using procedures that were identical to those described for 50–70 ms, comprising the range of SPs of the natural CS of A. domesticus. this species (Stout et al., 1983). However, a degree of plasticity is described by Walikonis et al. (1991), who demonstrate Gryllus bimaculatus. All experiments were performed at that, as virgin females age, they became less selective for SP the Department of Neurobiology, Institute of Zoology, Univer- (i.e. exhibit phonotaxis to most or all SPs tested). Gray (1999) sity of Goettingen, using their culture of this species. Methods confirms the age-related change in phonotactic selectiveness for caring for this species were very similar to those used in our by this species. Walikonis et al. (1991) also demonstrate that facility, described above. Temperatures were approximately ◦ during periods of maximal juvenile hormone III (JHIII) produc- 25 C under an LD 12 : 12 h photoperiod. Nymphs were raised tion, younger females are most selective to SP, whereas during from eggs in containers (60 × 40 × 40 cm). Adult females periods of lower production of JHIII, older females are unse- were isolated each day from the rest of the colony on the day of lective. The addition of JHIII causes females that are attracted their imaginal molt and placed together in smaller containers. to a broad range of SPs to become as selective for SP as young females (Walikonis et al., 1991; Atkins et al., 2008a). Ongoing studies of the mechanisms that control phonotactic Behavioural testing selectiveness and the underlying bases for recognition of conspecific CSs include extensive work with female A. domesticus Sound stimuli. Computer-generated model calling songs as well as recent studies of the phonotactic behaviour and pro- (5 kHz carrier frequency) were produced with either three cessing by auditory interneurones in female G. pennsylvanicus syllables with a chirp period of 667 ms (A. domesticus)or (Jeffery et al., 2005) and female G. bimaculatus (Jeffery, 2003; 450 ms (G. veletis) or four syllables and a chirp period Stout et al., 2007). Work also has been initiated with female of 500 ms (G. pennsylvanicus, G. bimaculatus), which were G. veletis (Stout et al., 2004). As part of these studies, phono- within the natural range of the conspecific male’s CSs taxis is tested repeatedly, using a standard protocol as a basis (Desutter-Grancolas & Robillard, 2003). The intensity, syllable for additional tests resulting in data sets that range from very duration (20 or 25 ms; 5 ms rise and fall times) and syllable large (500+ experiments) for A. domesticus to moderate (e.g. period (25–90 ms) could be varied. Model CSs were made 30+ experiments for female G. pennsylvanicus).Thesizeof using SoundEdit 16, version 2 (Adobe Corp., San Jose, the data sets for each of the four species allows an evalua- California), generated using an Apple computer (Macintosh tion of both the individuality and plasticity that underlies the Performa 6110; Apple Corp., Cupertino, California) and played many different behavioural choices by females of each species through an amplifier (Technics VC- 4; Panasonic Corp., during phonotaxis. They also provide an important resource Secaucus, New Jersey) and loud speaker (model 40–1221; for evaluating proposed mechanisms for call recognition and Radio Shack, Fort Worth, Texas). CSs were presented in a the underlying plasticity and characteristics that recognition standard nonsequential order with SPs in the range 30–90 ms mechanisms should demonstrate. or 25–90 ms (50, 90, 70, 40, 60, 30 and 80 ms SPs for A. domesticus females and 40, 70, 25, 50, 90, 35, 60, 80 for G. bimaculatus females). Because some G. bimaculatus and G. Materials and methods pennsylvanicus females responded preferentially to CSs with 35 or 40 ms SP and discriminated between CSs with SPs of 35 Animal care and 40 ms, and because the male’s call used syllable durations in the range 20–25 ms (Doherty, 1985a), CSs with SPs of 25, Acheta domesticus. Four-week-old nymphal A. domesticus 35, 40, 50, 60, 70, 80 and 90 ms and a syllable duration of were purchased from Flukers’ Cricket Farm (Baton Rouge, 20 ms were used for these two species.

Arena. Phonotaxis was evaluated for female A. domesticus for 3 min before presentation of CSs began, and each test and G. veletis in a circular, sand covered, arena (diameter lasted 5 min. Model CSs with varying SPs were presented in 152 cm), which was contained inside a square chamber lined a standard, nonsequential order (see above) with 1–2 min of with dense fiberglass material developed for absorbing sound silence between each CS presentation. and reducing echoes. An omnidirectional speaker (Radio Shack 40–1221) was placed in the center of the arena facing upward and was isolated from the arena floor. Dense acoustic Classification of behaviour absorption material (thickness 10 cm, diameter 20 cm) was placed above the speaker so that sound projecting upward was Because some females of each of the four species tested absorbed. Sound did not vary more than ±2 dB around the responded to all SPs presented, it is important to classify edge of the arena. White cloth covered the speaker, preventing these females separately and to describe their behaviour as the cricket from reaching the speaker. Females (one to five at unselective. Females responding to all but one of the SPs a time) placed in the arena remained along the edge (bordered presented (six of seven or seven of eight SPs presented) by a plastic boundary strip; hight 10 cm, inclination inward of were also included. All of the other females were classified ◦ 45 ) until an attractive call was presented at which time they as exhibiting selective phonotaxis, although differences in the walked towards the loud speaker in the center of the arena. degrees of selectiveness were recognized. It was also important Phonotaxis to model CSs was tested in the lighted arena at to recognize that some selective females skipped SPs, whereas ◦ 22–24 C at an intensity of 85 dB [root mean square (rms), others responded to a contiguous range of SPs. Responding to a calibrated ±2 dB using a Heath real-time spectrum analyzer, contiguous range of SPs and responding to the same number of AD 1308] (Heath Company, Saint Joseph, Michigan) at the SPs distributed over a broader range when skipping SPs within edge of the arena. Each test lasted 5 min unless all the crickets that range also makes different predictions about the way that reached the speaker in less time, in which case the test was the underlying nervous system mechanisms function. Thus terminated. Each test was followed by at least 1–2 min of contiguous responses and responses that involved skipping SPs are included in separate categories. Although all of these no sound. When the presentation of the CS was terminated, categories are somewhat arbitrary, they recognize important the animals usually returned to the edge of the arena within differences in the strategies that females use in choosing the 1 min (occasionally, a female was gently prodded). Positive model CSs (and by extension, calling males) they respond to phonotaxis was identified when the cricket reached the speaker phonotactically. in a path that continuously approached the speaker [i.e. no ◦ If the female’s behaviour met the criteria described above turning away from the speaker (more than 90 ) circling or for orientation either in an arena or on the noncompensating walking back and forth]. Female phonotaxis was observed and treadmill, it was classified as phonotaxis. Responses by the recorded using a video camera directly above the arena that was females to the location of the source of model CSs presented connected to a computer, thus keeping the experimenter out of that were at a rather constant angle with respect to the sound sight of the orienting crickets and providing orientation tracks source with little wandering were not distinguished from the that could be carefully evaluated after the experiment was over. responses in which the female’s angular orientation was more Control experiments were also run in which young females varied, resulting in more wandering. (n = 41) were placed in the arena in groups of one to five females and treated identically, except that no sound was played back. Statistical analysis

The purpose in statistically evaluating the phonotactic Treadmill. Phonotactic responses to model CSs were mea- choices female crickets made is only to determine the sured for female G. bimaculatus and G. pennsylvanicus walk- probability that, for each separate experiment, the choices made ing in the dark on a noncompensating treadmill apparatus (open by the group of crickets tested were nonrandom. For these loop, the female was free to turn toward or away from the purposes Pearson’s chi-square test is used, with rejection of sound source; Walikonis et al., 1991). For female A. domesti- the null hypothesis that the females’ SP choices were random cus, phonotaxis on a treadmill was compared with phonotaxis basedonaP ≤ 0.05. Possible differences between the SP of this same species in an arena. Computer-generated CSs were choices females of the four species included in the present presented through a single speaker (Optimus 3 3/4 inch Radio study were not statistically evaluated. Shack Exponential Horn Tweeter, #40–1233) and were calibrated ±2 dB (rms) at the location of the tethered cricket on the styrofoam ball using either a Heath real-time spectrum ana- Results lyzer (AD 1308) or a Bruel & Kjaer measuring ampliﬁer (type 2610; Bruel¨ & Kjær, Denmark) and 1/2 inch microphone. Acheta domesticus All experiments were performed at room temperature ◦ (21–24 C). Phonotaxis was described as positive when the Control experiments. Of the 41 females placed in the arena distance walked toward the speaker was at least two-fold (in groups of one to ﬁve females) and observed without greater than away with an average angular error of no more playback of a CS, only three females walked to the centrally ◦ than ±60 . Females placed on the treadmill were left in silence located loudspeaker.

= A Old females (21–31 days, n 318; Fig. 1B) were much more likely to be unselective for CS SPs (47%, n = 148) 120 than were young females. However, 53% (n = 170) of old 110 females were selective for SP using the criteria deﬁned above. 100 Old, selective females chose the longest (90 ms, n = 61) and 90 the shortest (30 ms, n = 52) SPs with approximately equal 80 frequency. 70 Of the young A. domesticus females classiﬁed as selective, 60 91 responded only to a contiguous range of SPs (Fig. 2A), 50 whereas 80 of the 171 skipped one or more SPs, one or more

No. of Females 40 times within the range of the shortest to the longest SPs that 30 they responded to (Fig. 2B). 20 10 0 Young, selective females. The data resulting from the 30 40 50 60 70 80 90 phonotactic choices of females that responded to a contiguous Syllable Period (ms) range of five or fewer SPs (91 of 170; Fig. 2A) demonstrate Selective, n=171, 90% that the greatest number of females were most selective (one = Unselective, n=19, 10% SP, n 30) with decreasing selectiveness occurring with progressively lower frequencies (i.e. responded to progressively more SPs: two SPs, n = 22; three SPs, n = 17; four SPs, B n = 14; five SPs, n = 8). Of the 91 females that responded to a contiguous range of SPs, only three females chose SPs 160 of 40 ms with no response to SPs of 30 ms (Fig. 2A). For the contiguous females as a group, an SP of 70 ms was chosen 140 most frequently followed by 60 and 50 ms. It is also clear that 120 females that responded to progressively more (three to five) SPs did so by more frequently increasing their responses to 100 the longest SPs of 80 and 90 ms than to the shortest SPs of 30 80 and 40 ms. Of the 190 young females, 80 selective (responded to five 60 or fewer SPs) females ‘skipped’ one or more SPs (Fig. 2B). No. of Females 40 Of these 80 females: (i) 63 responded to a range of three to five SPs and skipped an average of 1 (range = three SPs) 20 to 1.8 (range = five SPs), resulting in a positive phonotactic 0 response to an average range of 2–3.2 SPs and (ii) 17 30 40 50 60 70 80 90 females that included a range of 6–7 SPs responded to an Syllable Period (ms) average of 4.5 SPs; Thus, skipping SPs clearly results in increased selectiveness, as demonstrated by the choices for Selective, n=170, 53% only two or three SPs by 52 of the 80 females in this group. Unselective, n=148, 47% However, only 21 of these females responded to SPs of both 60 and 70 ms that most characterize the CSs of conspecific Fig. 1. (A) The phonotactic selectiveness of 190 young (5–7 days males. old) female Acheta domesticus tested in an arena. The females were Females that responded to a contiguous range of three to five classified into two groups: Selective, responded to no more than five SPs, most frequently included SPs of 70, 60, 80 and 50 ms (in of seven syllable periods (SP) presented, and Unselective, responded that order, all above 50%). For females that skipped SPs, 50, to six or seven of the SPs presented. (B) The phonotactic selectiveness 70 and 90 ms SPs were most frequently chosen, followed by of 318 old (21–31 days old) female A. domesticus tested in an arena. SPs of 60 and 80 ms. Both groups were more likely to respond The females were classified into selective and unselective groups as to the longer SPs of 80 and 90 ms than they were to respond defined above. to the shortest SPs of 30 and 40 ms.

Selective versus unselective females: age correlations. Evaluation of the phonotactic selectiveness of 190 young Young, unselective females. Eleven young females chose to (5–7 days old) females revealed that 171 (90%) were selective exhibit phonotaxis to six of the seven SPs presented and eight using the criteria deﬁned above (Fig. 1A), whereas 19 (10%; responded to all seven SPs presented (data not shown). Eight of Fig. 1A) females were unselective. Somewhat more than twice the 11 females that responded to six SPs responded to the full as many selective young females (n = 52) were attracted to the range of 30–90 ms SPs presented, with no pattern of choosing longest SP (90 ms) than chose the shortest SP (n = 23, 30 ms). to skip a particular SP.

Fig. 2. The phonotactic choices made by individual female Acheta domesticus. (A) The syllable periods (SP) responded to by 91 selective young female A. domesticus that chose only contiguous SPs. The females are ranked (from top to bottom) by the number of SPs they responded to. The histograms summarize the SPs responded to by females that responded to only one SP, two SPs, etc. For these, and all subsequent histograms, tests of phonotaxis were performed at each of the SPs indicated on the abscissa, including the SPs not associated with a female response where the value would be 0.0. The probability that the choices these females made were random is P < 0.005. (B) The SPs responded to by 80 selective young female A. domesticus that skipped one or more SPs within the range of SPs chosen for response. The females are ranked by the number of SPs included in the female’s range of responses. The histograms summarize the SPs responded to by females that responded to a range of three SPs, four SPs, etc. The probability that the choices these females made were random is P < 0.005. (C) The histogram compares the percentage that responded to each SP for females that responded to three to ﬁve contiguous SPs or skipped an SP(s) within a range of three to ﬁve SPs.

A 40 1SP,n=11 35 2 SPs, n=8 30 3 - 4 SPs, n=25

25 5 SPs, n=36

15 No. of Females 10

0 30 40 50 60 70 80 90 Syllable Period (ms)

B 35 3 - 4 SP Range, n=17 30 5 SP Range, n=34

25 6 - 7 SP Range, n=39

No. of Females 10

0 30 40 50 60 70 80 90 Syllable Period (ms)

Fig. 3. (A) The syllable periods (SP) chosen for response by 80 selective (responded to no more than ﬁve of the seven SPs presented) old female Acheta domesticus that responded to the indicated range of contiguous SPs. (B) The SPs chosen for response by 90 selective old females that skipped SPs within the indicated range of SPs responded to.

Old females. As noted above, 148 old females were Of the old females that skipped SPs, 73 out of 90 included unselective, responding to six or seven of the seven SPs a range of five to seven SPs in their response with the largest presented (Fig. 1B). Evaluation of the responses of the 170 group (n = 39) responding to a range of six or seven SPs old A. domesticus females classified as selective demonstrated (Fig. 3B). There was no clear difference in the likelihood that that 80 responded only to a contiguous range of SPs, whereas females would respond to the longest (90 ms) or shortest SP in 90 of these females skipped one or more SPs, one or more any of these groups. Females that responded to a range of four times within the range of the shortest to the longest SPs they or five SPs skipped, on average 1.6 of two or three possible responded to (Fig. 3). SPs. The large majority of old, selective females (61 of 80) responded to three to five contiguous SPs and only 19 responded to one SP or two contiguous SPs (Fig. 3A). In Phonotactic choices by females tested serially. Female general, the females that were selective for four or five SPs A. domesticus that were tested sequentially over a period were most likely to respond to SPs in the range 50–80 ms. of days (one phonotactic test on any 1 day; Fig. 4) also The old females that responded to five contiguous SPs showed demonstrated changes in their phonotactic choices of SPs to a clear preference for 90 ms SPs compared with 30 ms SPs. respond to. This was true whether the females were tested

Fig. 4. The syllable periods chosen for response by: (A) four young female Acheta domesticus tested sequentially on the days indicated; (B) three old females tested sequentially on the days indicated. In each case, the first test on a female was performed on the day shown as the age of the female, and each female was retested sequentially on the days indicated after the first test. The seven females shown accurately represent the sequential behaviour of the 24 females tested. every day (Fig. 4A), every other day, or on a more variable frequently to SPs of 35, 40 and 50 followed by 25 and 60 ms, schedule. The variability in response was similar for both and an unselective group (responded to seven or eight SPs, young and old females (Fig. 4A, B). Some individual females n = 20) that responded essentially equally to all SPs presented. demonstrated a degree of consistency as seen for the 6-day and The phonotactic choices made by the 99 selective females 11-day-old females (Fig. 4A) that never responded to SPs of (Fig. 6) included those that responded to a range of one to six 30 and 40 ms. contiguous SPs (above the dashed line) and those that skipped SPs (below the dashed line). The most selective females responded to two contiguous SPs Gryllus bimaculatus (n = 18) in the range 35–50 ms. As the number of contiguous = Of the 119 female G. bimaculatus females tested, on a SPs responded to increased to 3 (n 25), 25 ms SPs were treadmill 32 were young (5–12 days of age) and 57 were included by some females. Females that responded to four or old (24 days or older; the remaining 30 females were either five contiguous SPs all chose SPs of 35–50 ms, most included between 12 and 24 days old or were of unknown age). There the 25 ms SP and one half also responded to a 60 ms SP). were no differences between the phonotactic selectiveness All but three of the 31 females that skipped SPs responded between the groups of young and old females (Fig. 5A). The to a range of at least six SPs (Fig. 6). As the range broadened 119 females were separated into a selective group (responded to eight SPs, there were many more responses to the shorter to one to six of eight SPs, n = 99) that responded most SPs in the range 25–60 ms than there were to 70 ms SPs.

A 100 Young, n=32 90 Old, n=57 80 70 60 50 40 % Phonotaxis 30 20 10 0 25 35 40 50 60 70 80 90 Syllable Period (ms) B

100 Selective, n=99 90 Unslelective, n=20 80 70 60 50 40

No. of Females 30 20 10 0 25 35 40 50 60 70 80 90 Syllable Period (ms)

Fig. 5. The phonotactic selectiveness of female Gryllus bimaculatus tested on a treadmill. (A) The responses of young and old females. (B) All of the females (n = 119) were classiﬁed into two groups: selective [responded to no more than six of the eight syllable periods (SP) presented]; unselective (responded to seven or eight of the eight SPs presented). Fig. 6. The phonotactic choices made by 99 individual selective female Gryllus bimaculatus. The heavy dashed line separates the The responses to 80 and 90 ms increased to approximately the females that responded to only a contiguous range of syllable periods same levels as the shorter SPs (with the exception of 35 ms). (SP) (n = 68, ranked from top to bottom by the number of SPs they Individual female G. bimaculatus that were tested sequen- responded to) from those that skipped SPs (n = 31, ranked by the tially over a period of several days, including some tested twice range of SPs they responded to). The histograms summarize the SPs on the same day (Fig. 7) also demonstrated changes in their responded to by females that responded to a contiguous range of two phonotactic choices of SPs when tested twice on the same day SPs, three SPs or four to ﬁve SPs (above the dashed line), or to a range and/or on sequential days. of three to eight SPs for those females that skipped SPs (below the dashed line). The probability that the choices made by both contiguous females and females that skipped SPs were random is P < 0.005. Gryllus veletis

Young (4–11 days, n = 18) adult female G. veletis,tested (Fig. 8A). Most old females (30–60 days) were also selective in an arena, were very selective for CS SP, and responded (responded to up to ﬁve of seven SPs, n = 15; Fig. 8A) but primarily to SPs in the range of 40–60 ms with the peak included a wider range of SPs in their responses, especially at (approximately two-thirds of the females) at 50 and 60 ms 30 and 70 ms SPs. Four (one young and three old; data not

Fig. 7. The syllable periods chosen for response by six female Gryllus bimaculatus tested sequentially on the same day or sequential days as indicated. In each case, the first test on a female was performed on the day shown as the age of the female, and then each female was retested sequentially after the first test as shown (1 + 3h= retested 3 h after the first test on the same day). The six females shown in this figure accurately represent the sequential behaviour of the 13 females tested. shown) of the 37 females tested responded to six of the seven selective (responded to six or fewer of eight SPs), contiguous SPs presented and thus were classified as unselective. females included SPs of 25, 80 or 90 ms in their phonotaxis, The phonotactic behaviour of the 33 selective females and thus did not extend their range to include the shortest or (young and old were grouped together) demonstrated for the longest SPs tested. Although only a single selective, contiguous females responding to either a single SP or a contiguous range female responded to five SPs (and none to six SPs), all but of SPs (n = 22) that the largest group was highly selective, one of the females that skipped SPs (n =10) included a range responding to only one SP (n = 8) in the range 5–80 ms with of five to eight SPs in their responses (Fig. 9C). For the ten 50 and 60 ms SPs being chosen most frequently (Fig. 8B). females that skipped SPs in their response, the SPs chosen Females that responded to two or three SPs (n = 8) most fre- peaked at 40 and 70 ms. quently included SPs of 40–60 ms. Females responding to four or five contiguous SPs (n = 6) extended their range of SPs to 30 ms. Only two of these 22 females included five SPs in their Comparison of behavioural methods response. Conversely, of the 11 females that skipped SPs, eight females included a range of five to seven SPs in their responses. Because phonotactic selectiveness was measured in the SPs in the range 40–60 ms were most frequently included in present study using orientation either in an arena (where their responses and an SP of 80 ms was least frequently chosen. the female experiences different CS intensities) or orientation on a treadmill (in a much more constant sound field), it is relevant to compare the results of these different measurement Gryllus pennsylvanicus techniques on the same species and age group of females. Old female A. domesticus (80 females) were tested on a treadmill Young (8–12 days) adult female G. pennsylvanicus,tested and their orientation to CSs with 30, 50, 70 and 90 ms SPs on the treadmill, included both those that were selective for was evaluated. As a base for comparison, the phonotactic CS SP (up to six SPs, n = 26; Fig. 9A) and those that were orientation of the 318 old females in an arena, in response to unselective (seven to eight SPs, n = 5; Fig. 9A). The young, the same SPs (Fig. 2), was categorized in an identical manner. selective females responded most frequently to CSs with SPs On a treadmill, 20% of old females responded to either or of 40 ms but also included SPs of 35, 50, 60 and 70 ms both of the CSs with SPs of 50 and 70 ms, but not 30 or with approximately equal frequency (Fig. 5A). The seven 90 ms, whereas, in the arena, 23% chose these SPs (Fig. 10A, unselective older females (30–34 days) responded to at least B). Fifty-three percent of old females orienting on a treadmill seven of the eight SPs presented (Fig. 5B). Three older females included responses to both 30 and 90 ms SPs and 42% of were selective (one responded to SPs of 40, 50 and 60 ms, and old females made these choices in an arena (Fig. 10A, B). A two responded to SPs of 25 and 40–90 ms, not shown). similar percentage of old females included the 90 ms SP but Females that responded to a contiguous range of SPs most not the 30 ms SP in their responses in both the arena (21%) frequently included SPs of 35–60 ms. The largest number (11 and the treadmill (22%). The smallest number of old females of 16) included three to four SPs in their response. None of the responded to CSs with 30 ms SPs, but not 90 ms SPs in both

Fig. 8. The phonotactic selectiveness of 33 female Gryllus veletis tested in an arena. (A) The responses of young and old females. (B) The syllable periods (SP) chosen by 33 individual female G. veletis that responded to no more than ﬁve of the seven SPs presented. The heavy dashed line separates the females that responded to only a contiguous range of SPs (n = 22, ranked from top to bottom by the number of SPs they responded to) from those that skipped SPs (n = 11, ranked by the range of SPs they responded to). The histograms summarize the SPs responded to by females that responded to a contiguous range of SPs as indicated (above the dashed line) or to a range of three to seven SPs for those females that skipped SPs (below the dashed line). The probability that the choices made by contiguous females were random is P < 0.005 and the same measure for females that skipped SPs is P < 0.02.

Fig. 9. The phonotactic selectiveness of 36 female Gryllus pennsylvanicus tested on a treadmill. (A) The responses of selective [responded to no more than six of eight syllable periods (SP) presented] and unselective (responded to seven or eight of eight SPs presented) young females. (B) The responses of seven unselective (responded to seven or eight of eight SPs presented) old females. (C) The SPs chosen by 26 individual selective young female G. pennsylvanicus that responded to no more than six of the eight SPs presented. The heavy dashed line separates the females that responded to only a contiguous range of SPs (n = 16, ranked from top to bottom by the number of SPs they responded to) from those that skipped SPs (n = 10, ranked by the range of SPs they responded to). The histograms summarize the SPs responded to by females that responded to a contiguous range of SPs as indicated (above the dashed line) or to a range of three to seven SPs for those females that skipped SPs (below the dashed line). The probability that the choices made by contiguous females were random is: P < 0.005 and the same measure for females that skipped SPs is: P < 0.025.

the arena (14%) and the treadmill (5%). The percentages of females making each of these classes of choices in an arena or on a treadmill were so similar that the demonstration of their preferences was not dependent on using one or the other of these two test procedures.

Discussion

Phonotaxis by female crickets to models of the CSs of conspeciﬁc males is reported in earlier studies as a consistent, species-typical behaviour (Teleogryllus oceanicus:Moiseff et al., 1978; Pollack & Hoy, 1981; G. campestris: Thorson et al., 1982; A. domesticus: Stout et al., 1983; G. bimaculatus: Doherty, 1985a). However, because some of the formative studies use small (frequently less than ten) to very small samples of preselected females, which are in some cases tested repeatedly over several days, the degree of variability demonstrated would almost certainly be limited (Thorson et al., 1982). Although the sample sizes employed in the present study for A. domesticus (Figs 1 and 2) and G. bimaculatus (Figs 5 and 6) are substantially larger than those used in many other studies of female gryllid phonotaxis, the smaller sample sizes for female G. veletis and G. pennsylvanicus (Figs 8 and 9) are also larger than those in most other studies of the phonotaxis of these two species (Doherty & Storz, 1992) and for similar work with other gryllids, and also reveal more phonotactic plasticity than is typically described in studies using small numbers of females. Although the differences in sample sizes for the four species included in the present study limit to some degree a detailed quantitative comparison of the phonotactic choices exhibited by the four species included, the smallest samples used (n = 41 for G. pennsylvanicus, n = 33 for G. veletis) are large enough to document substantial differences in their choices (Figs 8 and 9). Substantial differences in the phonotactic choices made by G. bimaculatus females are also demonstrated by Verburgt et al. (2008) using a large sample (n = 130). Hedrick & Weber (1998), in a study of G. integer based on moderate sample sizes (n = 14–20), also demonstrate individual variability in the females’ phonotactic choices. Another cause for the consistent phonotactic behaviour shown in earlier studies could result from the preselection of females, such as in the study by Thorson et al. (1982) in which the ﬁve female G. campestris are chosen as a result of pretests demonstrating that they are ‘good trackers’. Doherty (1985a) also describe a study using preselected female G. bimaculatus, in which an ‘ideal’ CS is presented and then the females responding to this optimized CS are used for further evaluation. In the present study, females are often selected by age (young or old). Otherwise, females are not pretested or preselected in any way. Only females that do not respond to any of the test Fig. 10. Comparison of the phonotactic choices of 398 old female CSs presented are excluded. Acheta domesticus tested in either (A) an arena (n = 318) or (B) on a treadmill (n = 80). To compare the two experimental procedures the Most previous studies (e.g. Stout et al., 1983; Doherty, females were divided into the response groups: responded to SPs of 1985a) do not identify the differences in the phonotactic 50 and/or 70 ms; responded to an syllable periods (SP) of 90 ms but choices exhibited by individual females, possibly as a result not 30 ms; responded to both 90 and 30 ms SPs; responded to 30 ms of pooling the data into single graphics that obscure their but not 90 ms. individual behaviour. In the present study this is readily

© 2010 The Authors Journal compilation © 2010 The Royal Entomological Society, Physiological Entomology, 35, 99–116 Plasticity of phonotactic selectiveness 111 observed by comparing the data presented in the histograms or improve the females’ phonotactic selectiveness over the (i.e. that subdivide the data into separate curves that are based presentation of single CSs (Doherty, 1985a). Doherty (1991), on the number of contiguous SPs responded to) in Figs 2A, using choice experiments, reports results that fall within the 6, 8B and 9C with the results shown for individual females. range of the responses of the substantially larger groups shown The histograms do not reveal the variety of choices made for this species in Figures 5 and 6. by individuals. Rather, they suggest that individual females Several studies of phonotactic choice by female gryllids may centre their responses around the SPs produced by the use more quantitative measures of phonotaxis than the criteria conspecific males and differ mainly in the number of SPs in the present study, which classify a female’s behaviour as chosen. Even very selective individual contiguous females do phonotactic or not phonotactic based on their response to a not necessarily centre their phonotactic responses around the stimulus presentation that would model a call from a single mean SPs produced by conspecific males but may include, for male in a single location. It is quite possible that quantitative example, a single SP that is within the conspecific range and measurements such as the female’s steering velocity toward one or two that are outside of that range (Figs 2, 6, 8 and 9). an attractive CS (Poulet & Hedwig, 2005) or the relative As also demonstrated by Verburgt et al. (2008) for female distance walked (Hedrick & Weber, 1998) during phonotaxis G. bimaculatus (field caught as nymphs), the variability of could demonstrate a female’s relative preferences for one SP phonotactic choice by a large majority of selective females of over another when she responds phonotactically to both SPs all four species is seen most frequently in response to SPs in single presentations. outside of the conspecific range of SPs (Figs 2A,B, 4, 6, 7, 8B Within the procedural boundaries discussed above, it is and 9C). concluded that the emergent similarities in the types of phonotactic choices made by the four species in the present study represent important themes in the individual choices Rearing conditions and experimental procedure these females make when determining how to respond to a calling song, rather than simply resulting from different raising By accumulating relatively large numbers of experiments or experimental conditions. with these four species that are not designed for interspecific comparisons of their behavioural responses, striking similarities in the types and ranges of their phonotactic behaviour Comparison of the phonotactic choices made by female emerge. It can be argued that, despite some differences in A. domesticus, G. bimaculatus, G. veletis and the origin and raising conditions for each species, the result- G. pennsylvanicus ing similarities in their behaviour transcend these differences and thus offer important insights into the underlying function Phonotactic selectiveness. Approximately 17% of virgin and control of the behaviours, and more clearly suggest what female G. bimaculatus are unselective (i.e. equally likely to be the controlling nervous system mechanisms might be. In these young or old). In an evaluation of the reliability of phonotaxis respects, this synthesis is very similar to comparative stud- by female G. bimaculatus (raised from field caught nymphs) ies of gryllids or other related species that may be found in by Verburgt et al. (2008), unselective females are not identified very different habitats and face different selective pressures, specifically. They clearly show that the phonotactic response yet exhibit important similarities as well as some differences. to model CSs with SPs that are near the conspecific values A comparison of phonotactic behaviour and its plasticity is consistent, whereas the more extreme SPs are individually within or between species might also be complicated by dif- responded to more variably, suggesting that some females ferences in the experimental procedure, including the measure- are much less selective for SP than others. Approximately ment of phonotaxis and presentation of stimuli. For example, 25–30% of young and old virgin female A. domesticus Stout et al. (1983) and Stout & McGhee (1988) evaluate the (assuming about equal sample sizes of young and old female phonotaxis of female A. domesticus in an arena, whereas A. domesticus) are unselective and the combined incidence Walikonis et al. (1991) use a noncompensating treadmill. How- of unselective behaviour by young and old virgin female ever, based on evaluations in an arena and on a treadmill, Stout G. pennsylvanicus was approximately 29% (12 of 41 young et al. (1991) present measurements of phonotactic threshold and old females). Thus, these data support the occurrence and its change in very young females for which the data are of unselective behaviour in these three species that was not so similar that they lead to identical conclusions about the greatly different and did not result from being raised from pre- changes and values for phonotactic thresholds with age. In existing cultures (A. domesticus and G. bimaculatus)rather the present study, strikingly similar results are demonstrated than being field caught as nymphs (G. pennsylvanicus). The on testing the phonotactic choices of large numbers of old incidence of unselective phonotaxis by female G. veletis,is females using either an arena or a noncompensating treadmill lower (four of 37 young and old virgin females–11%, field (Fig. 10A, B). Studies evaluating the phonotactic choices of caught as nymphs). female G. bimaculatus using a Y-maze (Popov & Shuvalov, Hedrick & Weber (1998) also report different degrees of 1977) or a compensating treadmill (Doherty, 1985a) also lead phonotactic selectiveness by female G. integer, noting that to very similar descriptions of the females SP preferences. some females respond to different ranges of SPs, whereas other Doherty (1985b) concludes that offering female G. bimacula- females are unselective, responding to most or all of the SPs tus choices between two different model CSs does not change presented.

Correlation of selectiveness with age. Young female include the complete range of SPs produced, whereas others G. pennsylvanicus (Fig. 9) and A. domesticus (Fig. 1) are that are less selective exceed that range, responding to SPs that, much less likely to be phonotactically unselective than are old depending on the species, are either longer or shorter than is females of these species. Gray (1999) notes that old female found in the conspecific male’s CS, or both. A. domesticus are more likely to be unselective for SP than Some females of all four species skip SPs within the range are young females. Only three of 18 old female G. veletis are of SPs they respond to (Figs 2, 6, 8 and 9), and such a response classified as unselective. However, the 15 old females shown has not been clearly described for the chirping species of in Figure 8(A) respond to a total of 55 SPs (3.67 per female), gryllids (Hedrick & Weber, 1998). For all of these species whereas the 18 young females respond to 37 SPs (2.06 per except A. domesticus, skipping is most likely to occur when female). Thus, old females are more likely to be less selective the range of SPs equals or is longer than the longest range of and include SPs of 30 and 70 ms much more frequently than SPs commonly responded to by contiguous selective females young females (Fig. 8A). Female age does not influence the (Figs 2B, 6, 8B and 9C). Although responding to longer ranges selectiveness of female G. bimaculatus, as also reported by of SPs would increase the probability of skipping SPs, these Verburgt et al. (2008). females very frequently respond to a smaller number of SPs Because the three species with old females that are less that fell within the range of SPs produced by conspecific selective or unselective include one species raised from pre- males than females whose response is to a contiguous range existing cultures and two species raised from field caught of three or more SPs. Because SPs that are characteristic nymphs, the differences in age related selectiveness are not of the conspecific male are frequently skipped, skipping is based on the rearing methods or origins of these three species. not simply the result of using criteria for phonotaxis that Unselective female G. pennsylvanicus and G. bimaculatus are are too permissive. Taken together, these results suggest that, not recognized in earlier studies of their phonotactic behaviour by skipping SPs, some females reduce their probability of and age related changes in phonotactic selectiveness are not responding to conspecific males, whereas other females, by described for female G. pennsylvanicus or G. veletis. responding over a wider range of SPs, may also increase the probability of responding to a heterospecific male cricket.

The phonotactic choices of individual females. Individual Females retested serially. The changes in phonotactic females of all four species that respond to a contiguous range behaviour exhibited by females that are retested serially of SPs differ in their degree of selectiveness, as measured by demonstrates that changes in phonotactic choices are also seen the number and range of contiguous SPs that they respond to when testing single females serially over a period of hours (Figs 2A, 6, 8B and 9C). It is clear that individual females (G. bimaculatus; Fig. 7) or days (young and old A. domes- differ in the values and number of SPs chosen and also how ticus;Fig.4A,B;G. bimaculatus;Fig.7).Formanyofthe their phonotactic choices relate to the SPs in the conspecific females, the range of SPs responded to remains more con- male’s CS. sistent than the response to individual SPs within that range. The differences exhibited by contiguous females of all four Some are quite selective on the first day of testing and this is species that are less selective for the SPs of model CSs are followed by a sequential variability in their responses, which expressed in somewhat different ways. Female A. domesticus includes broadening of the range of SPs responded to, skipping are most likely to include SPs that are longer than those SPs or responding contiguously (Figs 4A and 7). However, for produced by the conspecific male (Fig. 2A; male SPs are both species, responses to SPs that are within the conspecific in the range 50–70 ms at experimental temperatures; Stout range are more likely. It is not possible to determine whether et al., 1983; Desutter-Grancolis & Robillard, 2003; Burden, this serial change in phonotactic choices results from repeated 2009) as are female G. veletis (Fig. 8B; SPs are in the range changes in the female’s selectiveness that are independent of 30–50 ms; Desutter-Grancolis & Robillard, 2003; present the serial retesting, or are caused, at least in part, by the retest- study). Female G. bimaculatus are more likely to extend the ing procedure. However, it is apparent that these serial changes range of SPs responded to by choosing SPs that are shorter in responsiveness represent a strategy that, when exhibited than the conspecific range (25 ms; Fig. 6), although they also under more natural conditions, would bring the females into include SPs that are longer. Although Desutter-Grancolis & contact with a larger group of more variable males of the same Robillard (2003) indicate a very limited range of 33–34 ms or different species. for the SPs of conspecific males, Ferreira (2006) demonstrates mean SPs for this species in the range 34–40 ms and Evidence that these differences/changes in SP-selectiveness further document substantial within individual variability that result from behavioural/neuronal plasticity. Thedatareviewed extends the range of SPs produced by males to approximately above include large differences between the SP-selectiveness 30–42 ms. Contiguous female G. pennsylvanicus increase of individual females of all four species that range between their range of SP responses by adding longer and shorter responding to only one or two SPs and responding to all SPs (Fig. 9; SPs in the range 43–63 ms; Desutter-Grancolis & SPs tested. It would be possible to interpret these results as Robillard, 2003; or approximately 25–60 ms at experimental simply demonstrating variability in the female’s responses to temperatures; Burden, 2009). In summary, although almost all models of the male’s calls. The factors indicating that these contiguous females of these four species include some of the behavioural changes result from plasticity in the control of SPs that characterize the conspecific male’s CS, many do not phonotactic behaviour are described below.

◦ First, age-dependent reduction in selectiveness documented thermometer)mayvarybyasmuchas±10 C over a 10-m for three of the four species in the present study indicates path that a female might traverse in responding to a male. that progressive changes in selectiveness occur in individual The tuning of female phonotaxis to SPs is also influenced females as they age (earlier demonstrated for individual female by temperature (G. bimaculatus; Shuvalov & Shchekanov, A. domesticus; Stout et al., 1991). 2004) and females would likely walk through environmental Second, daily retesting of female A. domesticus (Fig. 4) or ‘patches’ with different temperatures when responding to a G. bimaculatus (Fig. 7; also within 3 h) indicates that changes male’s CS. Atkins et al. (2008b) show changes in male A. in SP-selectiveness occur repeatedly resulting (reversibly) in domesticus calling and female SP-selective phonotaxis that ◦ decreased or increased selectiveness. change at rates of 2–3 ms C−1. Third, applying JHIII to the body surface of old, unselective These results, by using more carefully executed procedures female A. domesticus (Walikonis et al., 1991) makes them and many more individuals, support and reinforce the results of as SP-selective as young, selective females. Nanoinjecting the earlier study by Alexander & Meral (1967) indicating that, ◦ JHIII or picrotoxin into the prothoracic ganglion of female within the range 10–38 C, the average SP produced by male A. domesticus (Atkins et al., 2008a) and G. bimaculatus (Stout G. veletis changes from approximately 60 ms to approximately et al., 2008) reduces the number of SPs that they respond to. 30 ms. Alexander & Meral (1967) also note that some males Fouth, photoinactivating the paired prothoracic ON1 neu- may be singing in the direct sun at relatively high temperatures, rones of female A. domesticus results in immediate transforma- whereas others at the same time sing from shaded, protected tion of the female’s behaviour from unselective to SP-selective spots at considerably cooler temperatures, providing females (Atkins et al., 2008a). These observational and experimental with a potentially large range of CSs with different SPs. outcomes demonstrate that changes in SP-selective phonotactic Thus, for the four species (A. domesticus, G. bimacula- behaviour occur progressively over shorter or longer time peri- tus, G. veletis and G. pennsylvanicus) included in the present ods and are not simply an illustration of behavioural variability. study, the demonstrated flexibility of the female’s phonotactic Rather, they are under the immediate and longer-term con- choices, given the documented variability in conspecific male’s trol of the female’s selective auditory processing. Thus, these calling songs, increases the probability that more sexually results represent an important demonstration of the behavioural responsive females will find sexually ready males. For indi- plasticity resulting from the neuronal processing that con- viduals that are not reproducing sympatrically and synchron- trols the female’s decision to respond or not respond to an ically with other gryllid species with similar calling songs, individual CS. the demonstrated plasticity in the female’s phonotactic choices would be accompanied with little cost and could offer important selective advantages. Of the species included in the present Possible biological roles for phonotactic plasticity. Because study, only G. veletis and G. pennsylvanicus are sympatric. of the plasticity of phonotaxis demonstrated for the four species However, there is almost no seasonal overlap in their reproduc- in the present study, Burden et al. (2007) undertook an inten- tion (Alexander & Meral, 1967) and thus they are allochronic. sive study of the variability shown in the male’s CSs for three For three of these four species, older virgin females are, of the four species (A. domesticus: laboratory recordings; G. on average, less selective for SPs, suggesting that the risk veletis and G. pennsylvanicus: field recordings). A brief sum- of mating heterospecifically might be overridden by increased mary demonstrates that: for 135 separate field recordings (i.e. opportunities to find and reproduce with a conspecific male as little chance of recording the same male twice), the SPs of male the virgin females age. G. pennsylvanicus are largely in the range 30–60 ms, with a Clearly, studies of these four species, in geographical areas ◦ ◦ mean of approximately 44 ms at 18–22 C. At 28–32 C, the also populated by gryllids with similar calling songs, need to SPs are largely in the range 25–50 ms with a mean of approx- be carried out that are focused on character displacement in imately 32 ms. For 144 separate field recordings, the SPs of species-specific calling songs as found for G. fultoni (Jang & male G.veletis are largely in the range 22–53 ms with a mean Gearhardt, 2007) or other potential isolating mechanisms. ◦ ◦ of approximately 38 ms at 18–22 C. At 28–32 C, the SPs are largely in the range 20–47 ms, with a mean of approximately 30 ms. For 67 laboratory recordings of 30 male A. domesti- Implications for recognition of CSs cus, the SPs are largely in the range 52–72 ms with a mean ◦ ◦ of 60 ms at 20–22 C. At 29–32 C, the SPs are largely in the Although there are substantial quantitative differences range 30–60 ms with a mean of approximately 40 ms. between the phonotactic choices made by individuals of each Atkins et al. (2008b) further demonstrates that, in the field, species and between the four species included in the present where calling males of G pennsylvanicus and G. velities study, females of each of the species (under the present exper- are located in a 60 × 60 cm2 area in the sun, temperatures imental conditions) share several features of phonotactic plas- measured with an infrared ‘spot’ thermometer vary by ticity. The choices made by very selective individual females ◦ approximately ±10 C, depending on the amount of cover, vary by species: A. domesticus and G. veletis respond most compared with the air temperature measured 60 cm above the frequently to single SPs (Figs 2A and 8B) and G. bimacula- substrate. Atkins et al. (2008b) also demonstrate that, in natural tus and G. pennsylvanicus respond to two or three contiguous microclimates where G. pennsylvanicus and G. veletis are SPs (Figs 6 and 9C). Many contiguous, less selective females found, the temperature differences (measured with an infrared of each species respond beyond the boundaries described for

© 2010 The Authors Journal compilation © 2010 The Royal Entomological Society, Physiological Entomology, 35, 99–116 114 J. Stout et al. the SPs of the conspecific CS. Females of each species are cannot be excluded by the present data, filtering plasticity unselective (i.e. respond to all or all but one of the SPs pre- is not addressed and the substantial behavioural plasticity sented). Finally, substantial numbers of females of each species demonstrated for G. bimaculatus (Fig. 6) would require that skip SPs. the stored information to be in a form that could support the It is especially striking that the features of phonotactic demonstrated plasticity. Thus, recognition solely based only plasticity shared by all four species emerge, despite differences on cross correlation with stored information about the SPs with respect to the source and raising of the females noted contained in the conspecific male’s CS would require a wide above, as well as differences in the testing apparatus (i.e. on range of stored information about SPs that could be recovered a treadmill or in an arena). Verburgt et al. (2008) describe a selectively. similar variability in the response of G. bimaculatus,which A more recent model for recognition of SPs by bush crickets were field caught as nymphs and raised in the laboratory, to (Bush & Schul, 2005) depends on matching the responses SPs outside of the conspecific range. to the various SPs with an oscillator (or other mechanism) Any model of the selective processing of the CS’s syllable that causes an auditory neurone’s excitability to resonate with period by chirping gryllids should include the mechanisms and a periodicity that matches the SPs of the male’s CSs. One plasticity that results in the phonotactic choices made by the prediction made by this model is that SPs that were half females of these four species (Figs 2, 6, 8 and 9). the normal, ideal SP would also frequently lead to positive Several models proposed for recognition of the SPs that phonotaxis. This model has predictive value for the bush characterize the CSs of conspecific male gryllids depend on cricket Tettigonia cantans and could help explain the tendency ‘storage’ in a retrievable form of information regarding the by the species of female gryllids included in the present study SPs of the conspecific males CSs. Because the most selective to skip SPs. However, a pattern of skipping that is focused on females in the present study most frequently respond to only female gryllids responding to SPs that are twice as long as the SPs that are within or very near to the conspecific CS’s SP most attractive SPs is only seen occasionally (Figs 2, 6, 8 and range, and procedures applied to the prothoracic ganglion (see 9) and thus does not offer a likely mechanism for these species. above) of female A. domesticus (Atkins et al., 2008a) and G. bimaculatus (Stout et al., 2007) cause relatively unselective females to narrow their phonotactic responses to a range of A possible filter mechanism in the prothoracic ganglion SPs that match the SPs of the conspecific CS, the importance of having the information about the male’s CS available in the As noted above, Atkins et al. (2008a) demonstrate that female’s nervous system is reinforced. manipulations of neuronal processing in the prothoracic gan- Schildberger (1984, 1985) provide data on the responses of glion (killing both ON1s or nanoinjecting either JHIII or picro- three different classes of SP-filtering neurones in the brain of toxin) changes phonotaxis by relatively unselective female A. female G. bimaculatus: a ‘low pass’ neurone (BNC1d); a ‘high domesticus causing an immediate narrowing of the range of pass’ neurone (BNC2b); and two ‘band pass’ neurones (BNC2a attractive SPs to a range of SPs that much more closely approx- and BNC2c) whose outputs are proposed to be dependent on imates those produced by the conspecific male. Nanoinjection ‘ANDing’ the output of the low pass and high pass neurones, of JHIII or picrotoxin into the prothoracic ganglion of female thus responding tonically to only the SPs that are found in the G. bimaculatus has very similar effects on their phonotac- male’s CS. The present data for contiguous female G. bimac- tic choices (Stout et al., 2008). Samuel (2008), for the first ulatus in Figure 6 indicate that the behavioural choices are time in crickets, reports recorded SP-selective processing by an consistent (within the range of SP testing) with both a band auditory interneurone subsequent to (within 0.5 h) an evalua- pass filter and, for some females, a high pass filter, suggesting tion of the same female’s SP-selective phonotactic choices. In the possibility that more than one selective filter may con- Samuel’s (2008) study, the SP-selective responses of the pro- trol the female’s behaviour. With the very limited amount of thoracic AN2 auditory interneurone are significantly correlated data about the properties of these filter neurones, including (P<0.005) with the same female’s (G. bimaculatus) decision possible plasticity in their selective responses, it is not pos- to respond (or not) to a specific SP. Thus, it is likely that infor- sible to evaluate whether the proposed filtering mechanisms mation about the male’s SPs resides in a prothoracic neural could underlie the necessary degree of plasticity of phonotac- network, and might represent a default range for phonotaxis in tic behaviour demonstrated for females of this species (Fig. 6). response to conspecific CSs, as suggested by Hennig (2003). Without important modifications such as addition of a notch These results reinforce the demonstration by Stout et al. filter(s), Schildberger’s model of neuronal filtering would not (1997) that CS stimuli could be configured in such a deal effectively with the behavioural choices made by the 31 way (through amplitude modulation of the syllables) that females that skipped SPs (Fig. 6). Schildberger’s model also a female would respond phonotactically to an SP that had does not include any selective processing in the prothoracic just been demonstrated to be phonotactically unattractive ganglion demonstrated for both female A. domesticus (Atkins before modulation. This modulated stimulus also ‘fools’ et al., 2008a) and G. bimaculatus (Stout et al., 2007, 2008). the prothoracic L3 neurone, causing it to respond to this Hennig (2003) proposes a model for SP-selective filtering by SP in the same way as it responds to phonotactically female Teleogryllus commodus that results in recognition based attractive SPs (Henley et al., 1992). The reverse experiment, on cross correlation between a stored ‘copy’ of responses to the in which females do not respond phonotactically to an SP ‘ideal’ CS and the just encoded response. Although this model that had just been demonstrated to be attractive, results

© 2010 The Authors Journal compilation © 2010 The Royal Entomological Society, Physiological Entomology, 35, 99–116 Plasticity of phonotactic selectiveness 115 from manipulating the amplitude modulation of the stimulus References (without changing its SP) in such a way that L3 responds as if the SP should not be attractive. Nabatiyan et al. (2003) Alexander, R. & Meral, G. (1967) Seasonal and daily chirping cycles also suggest that SP-selective processing takes place in in the northern spring and fall field crickets, Gryllus veletis and the prothoracic ganglion (based on SP-dependent differences G. pennsylvanicus. Ohio Journal of Science, 67, 200–209. in the instantaneous firing rates of the prothoracic ON1 Atkins, G., Burden, C., Greene, C. et al. (2008b) Temperature cou- pling between the male’s calling song, the female’s phonotac- neurone of G. bimaculatus). These SP-selective prothoracic tic response, and the SP-selective responses of the L3 auditory auditory neurones might provide processed information to interneuron in Acheta domesticus. Proceedings of the 12th Inver- neurones, such as the BNC2a or c (Schildberger, 1984), tebrate Sound and Vibration Meeting, p. 19. Tours, France. for final processing, leading to the female’s phonotactic Atkins, G., Kilmer, J., Scalfani, M. et al. (2008a) Modulation of response. syllable period-selective phonotaxis by neural processing in the The results of the present study, together with those of prothoracic ganglion of female crickets (Acheta domesticus): Atkins et al. (2008a) and Stout et al. (2008), suggest that juvenile hormone III, picrotoxin and photoinactivation of the ON1 SP-selective processing by female crickets should exhibit sub- neurons. Physiological Entomology, 33, 322–333. stantial plasticity, involve neuronal networks that include the Burden, C. (2009) Variability in the calling songs of two field prothoracic ganglion and be influenced by neurochemicals such cricket species (Gryllus veletis and G. pennsylvanicus). MS Thesis, Andrews University, Berrien Springs, Michigan. as picrotoxin and JHIII. These results do not suggest how the Burden, C., Purvis, S., Magispoc, M. et al. (2007) Phonotaxis by mechanisms of SP-selective filtering in either the prothoracic female crickets is more variable than the males’ calling song. Eighth ganglion or brain might operate. Navia (2005) and Samuel Congress of the International Society for Neuroethology, PO57. (2008) evaluate SP-selective processing by the homologous L3 Vancouver, Canada. (A. domesticus) or AN2 (G. bimaculatus) prothoracic auditory Bush, S. & Schul, J. (2005) Pulse-rate recognition in an insect: interneurones in females, suggest mechanisms for SP-selective evidence of a role for oscillatory neurons. Journal of Comparative filtering and demonstrate plasticity in the filtering properties Physiology A, 192, 113–121. of these neurones that parallels the plasticity in the females’ Desutter-Grancolas, L. & Robillard, T. (2003) Phylogeny and the phonotactic behaviour. evolution of calling songs in Gryllus (Insect, Orthoptera, Gryllidae). Zoological Script, 32, 173–183. Doherty, J. (1985a) Tradeoff phenomena in calling song recognition and phonotaxis in the cricket, Gryllus bimaculatus (Orthoptera, Conclusions Gryllidae). Journal of Comparative Physiology A, 156, 787–801. Doherty, J. (1985b) Phonotaxis in the cricket, Gryllus bimaculatus The results of the present study suggest that the differ- DeGeer: comparisons of choice and no-choice paradigms. Journal ences in phonotactic behaviour exhibited by individual females of Comparative Physiology A, 157, 279–289. of all four species and by single females tested serially Doherty, J. 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MSc Thesis, University of Pretoria, South behavioural roles discussed above, strongly suggests impor- Africa. tant reproductive functions for the behavioural diversity docu- Gray, D. (1999) Intrinsic factors affecting female choice in house mented. For female A. domesticus, G. bimaculatus, G. veletis crickets: time cost, female age, nutritional condition, body size, and and G. pennsylvanicus, the plasticity of phonotactic selec- size-relative reproductive investment. Journal of Insect Physiology, tiveness results from CS recognition mechanisms comprising 12, 691–700. Hedrick, A. & Weber, T. (1998) Variance in female responses to the dynamic and changing processes that are probably influenced fine structure of male song in the field cricket, Gryllus integer. by the female’s experience (Wagner et al., 2001; Shuvalov Behavioral Ecology, 9, 582–591. et al., 1990) as well as environmental and other internal fac- Henley, J., Greenwood, J., Stout, J. & Atkins, G. (1992) Age- tors. 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(2003) Selective processing of calling songs by auditory grant IBN 98-08834 and Andrews University faculty grants. interneurons in two species of female crickets: possible roles

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