The túngara frog is a neotropical species in which reproduction occurs during the rainy season (approximately May to December). Females will lay multiple clutches of eggs during a single breeding season with gravid periods separated by approximately 4 to 6 weeks (Ryan, 1985
). It has been noted, however, that female túngara frogs will oviposit during the dry season months if an adequate rainfall occurs (S. Rand, personal communication).
All female túngara frogs used in this study were from a colony maintained at the University of Texas at Austin. Frogs in the colony were maintained in 5- or 10-gal aquariums with damp moss in groups of five and fed 1-week old crickets three times per week. The frogs were housed with simulated, but clock-shifted, equatorial light/dark cycles so that dusk began at 14:00 and dawn began at 02:00.
All females had previously mated with a male (or released eggs) at least once before used for phonotaxis tests in this study, which allowed us to be sure that all females were reproductively able. Phonotaxis tests were conducted in July 2003. The mean weight of females was 2.45 g, and the mean snout–vent length was 29.36 mm.
During the phonotaxis test, the subject was placed into an acoustic chamber measuring 1.8 × 2.7 m with acoustic foam on the walls to reduce reverberation. Two speakers were placed 2.7 m apart at equal distances from the center of the chamber. The peak intensity of the acoustic stimulus was set at 80 dB SPL (re 20μP) in the center of the chamber where the female was initially released. All acoustic stimuli were synthesized on a Dell computer with unpublished software produced by J. Schwartz. Phonotaxis tests were conducted from approximately 13:00 to 19:00.
To initiate the phonotaxis test, the female was placed under a funnel in the center of the chamber for three minutes while acoustic stimuli are antiphonally broadcast from opposing speakers with a 0.5-s interval between the stimuli. The side on which each stimulus is presented was alternated to control for side bias. After the funnel was lifted, the female was allowed 15 min to approach either speaker. If the female came within 10 cm of a speaker, it was considered a response to the stimulus being broadcast from that speaker. If the female remained stationary for at least 2 consecutive min, failed to move from the release site within 5 min or did not approach a speaker within 15 min, she was considered unresponsive to the acoustic stimuli. In total, each female completed four phonotaxis tests during each trial: the first receptivity test, a permissiveness test, a discrimination test and a final receptivity test.
Most of these tests were repeatedly used with wild-caught females to determine how each of these behaviors changed as they progressed through different reproductive stages. Lynch et al. (2005)
reported that receptivity and permissiveness fluctuated in unison so that the frequency of these behaviors was low in the unamplexed stage (i.e., unmated stage) but increased during the amplexed stage (i.e., mated stage that is near the time of ovipositing). Both receptivity and permissiveness simultaneously declined in the post-mated stage (i.e., after egg release). The increase in permissive mate choices during the amplexed stage was not due to a decrease in discrimination because females maintained discriminatory responses during this stage. Discrimination, however, did decline in the post-mated stage. Here, we use similar phonotaxis tests in order to determine whether hormone fluctuations may play a role in such behavioral flexibility.
Receptivity phonotaxis tests
The female’s receptive state was examined in the first phonotaxis test. In this test, the female heard two conspecific mate signals, a whine alone and a whine–chuck. A response to either of these signals is sufficient to consider the female as receptive to mate signals. That is, this test did not measure which conspecific signal the female responded to, only whether she responded to one or the other. The same stimuli were repeated in the last phonotaxis test, and the female needed to respond to a conspecific call in both the first and last test in order to be considered fully receptive. Females that approached a speaker in only one of the two tests were not considered receptive, as we could not be sure that an apparent response in only one test indicated receptivity or was simply a random movement toward one of the speakers.
Permissiveness phonotaxis test
Immediately following the first receptivity test, the female was re-tested to determine if she would approach an artificial hybrid call. The artificial hybrid whine was synthesized by varying spectral and temporal components of the P. pustulosus
whine so that the call would be intermediate between the calls of P. pustulosus
and P. enesefae
, a closely related species. Details describing the synthesis of this call are discussed in Ryan et al. (2003)
. This artificial hybrid whine has previously been shown to elicit a response from only 25% of females (Ryan et al., 2003
), indicating this call is less attractive to P. pustulosus
females than are conspecific mate calls. The hybrid whine was paired against white noise with equal amplitude and duration. Females that respond to white noise were excluded from statistical analyses because it is not possible to interpret the meaning of this response. This single-choice design was used because a female túngara exhibits strong discrimination in favor of a conspecific signal when present, yet displays some degree of recognition toward heterospecific calls when the conspecific call is not available (Ryan et al., 2003
). This type of design allowed us to examine how hormonal state influences the strength of female preferences.
Discrimination phonotaxis test
Immediately following the permissiveness test, we examined the female’s ability to discern the difference between the conspecific whine and the hybrid whine. A response to a conspecific whine over the hybrid whine indicates that the female is able to discern the difference between the two whines; that is, she is maintaining her discrimination. Conversely, a response to a hybrid whine indicates that the female has not maintained normal discriminatory responses.
Effects of HCG administration on mate choice behaviors
Females completed phonotaxis assays (pre-HCG tests) and were randomly placed into one of five dose groups in which they received human chorionic gonadotropin (HCG; Sigma). HCG is a ligand for luteinizing hormone (LH) receptors, which causes gonadal activation and therefore, the production of gonadal hormones. Doses of HCG include: 0 (control; N = 8), 10 (N = 8), 100 (N = 8), 500 (N = 16) or 1000 IU (N = 20). HCG was dissolved in 0.9% saline solution and given in a subcutaneous injection in a volume of 50 μl. The same phonotaxis tests described above were repeated approximately 20–24 h after HCG administration (post-HCG tests). The observer scoring the behavior was unaware of the treatment each subject received.
Egg laying and mate choice response
HCG induces egg laying in approximately 24 h in most females that are treated with high doses (i.e., 500 or 1000 IU). In the 500 IU group, 75% of females released eggs after injection with HCG and in the 1000 IU, 68.75% of females released eggs after injection with HCG. Only 8% of females in the 0, 10 and 100 IU groups laid eggs. We used Chi-squared goodness of fit to compare mate choice behavior between egg layers (N = 25) and non-egg layers (N = 8) to determine whether mate choice flexibility depended on the presence of mature oocytes.
Upon completion of phonotaxis tests, blood samples were collected via the orbital sinus from five subjects in each dose group. These procedures were approved by the University of Texas IACUC. The blood was centrifuged, and the plasma layer was stored at −20°C until assayed. Plasma volumes ranged from 5 μl to 20 μl. Twenty μl of tritiated estrogen or testosterone (approximately 1000 cpm) was added to each plasma sample for recovery determination. Plasma samples were extracted using 3 ml of diethyl ether. The mean recovery after extraction was 71 ± 0.09% and 95 ± 0.16% for estrogen and androgens respectively. Hormone assays were conducted with enzyme immunoassay (EIA) kits purchased from Caymen Chemical. These kits were validated prior to use in this study by extracting hormone from a pooled sample of frog plasma. We then diluted the pooled plasma to be three or four different concentrations that were within the most sensitive portion of the standard curve. The assay kit estimated the hormone concentration at each of the dilutions to be within 20% of each other. Each sample was assayed in duplicate, and each sample was measured at a minimum of two dilutions. Inter-assay variation was 12% for estrogen whereas androgens were measured on a single plate. Intra-assay variation was 9.9% and 9.5% for estrogen and androgens respectively. Cross-reactivity in the estrogen kit was 0.1% for testosterone and 5 α-DHT, 0.07% for 17 α-estradiol and 0.03% for progesterone and the detection limit is 8 pg/ml. The detection limit for testosterone EIA kits is 6 pg/ml. Testosterone EIA kits have a 27.4% and 18.9% cross-reactivity with 5α-dihydrotestosterone and 5β-dihydrotestosterone respectively. Therefore, we refer to testosterone measurements simply as androgens.
We used two analyses to assess variation in receptive and permissive behaviors. First, we tested whether the probability of a response to a mate signal changed between the pre- and post-HCG tests using Chi-squared goodness of fit. We used the frequency of responses in the post-HCG condition as expected and the frequency of response in the pre-HCG condition as observed. These tests were not used in the lower dose groups (0, 10 and 100 IU) because low response frequencies yielded low expected values. Second, we tested whether individual females changed their responses between the pre- and post-HCG phonotaxis tests by using a Kruskal–Wallis test to analyze the difference in the female’s response time before and after HCG treatment. Response time was measured as the time between when the funnel was lifted, allowing the female freedom to move and the time at which she reached the 10 cm point around a speaker. These scores were recorded in seconds, and non-responsive females received the maximum number of seconds (900 s). These analyses were done for both the receptivity and permissiveness phonotaxis tests.
We tested responses of females after treatment with HCG to determine whether hormonal manipulation influenced discrimination. We used a binomial test to examine whether the probability of a discriminatory response was significantly different from the probability of a random response.
We examined whether HCG administration significantly altered the plasma concentration of estrogen and androgens using one-way ANOVA with Tukey’s post hoc comparisons. Alpha values were set at 0.05 for all statistical tests, and all reported values are mean ± SE.