J Biosci Vol. 42, No. 3, September 2017, pp. 459–468 Ó Indian Academy of Sciences DOI: 10.1007/s12038-017-9688-3

Can embryonic skipper ( cyanophlyctis) learn to recognise kairomones in the absence of a nervous system?

, SWAPNIL CSUPEKAR and NARAHARI PGRAMAPUROHIT * Department of Zoology, Savitribai Phule Pune University, Pune 411 007,

*Corresponding author (Email, [email protected])

MS received 29 June 2016; accepted 28 March 2017; published online 12 June 2017

In this study, we used larval to determine the predator recognition mechanism. We conducted a series of experiments to determine if larval E. cyanophlyctis have the innate ability to recognise predatory odour (kair- omones) as a threat or if they learn to do so during ontogeny. In the case of learning, we wanted to determine the developmental window during which learning is accomplished. Further, we tested the antipredator response of predator- naı¨ve as well as predator-experienced tadpoles to chemical cues of different origins in order to assess if they exhibit differential responses. Our results clearly indicate that predator-naı¨ve tadpoles of E. cyanophlyctis do not reduce their activity against predatory cues of dragonfly nymphs, suggesting that they lack the innate ability to recognise kairomones. However, they could learn to do so when trained to perceive kairomones simultaneously along with alarm cues. Sur- prisingly, larval E. cyanophlyctis could learn to recognise kairomones through association during embryonic stages even before the development of a nervous system. Although larval E. cyanophlyctis lack the innate ability to recognise kair- omones, they were able to recognise conspecific alarm cues on the first encounter, indicating that they have the innate ability to recognise alarm cues as a potential threat.

Keywords. Alarm cues; associative learning; E. cyanophlyctis; embryonic learning; kairomones; predator recognition.

1. Introduction provide information on the type of predator as well as the intensity of predation (Lima and Dill 1990; Ferrari et al. 2010). In the ‘arms race’ of prey–predator interaction, both the prey For instance, prey can recognise predators even before and predator are under selection to become ever more effi- the predation event by using predators’ signature odours cient and effective in recognising each other so as to prevail (kairomones) and can thus evade them prior to an encounter over one another (Ferrari et al. 2010). Successful recognition (Kats and Dill 1998; Ferrari et al. 2010). Second, pre-attack of the predator increases the chances of survival of the prey, chemicals, in the form of urinary ammonia, released by star- while failure to do so increases the prey’s chances of being tled or disturbed prey, act as disturbance cues that serve as eaten (Lima and Dill 1990). Because of the intense need for warning signals to other prey (Wisenden et al. 1995; Kiesecker early predator recognition, prey animals inhabiting diverse et al. 1999). Third, alarm cues, chemicals released by prey ecosystems have evolved various sensory mechanisms of animals when attacked by a predator, can serve as accurate recognition that vary depending on the type and complexity indicators of predation event (Wisenden 2000). Related to of habitat, and include the use of visual and/or chemical cues alarm cues is another class of chemicals referred to as dietary (Lima and Dill 1990; Chivers et al. 2001; Kelley and cues, chemically similar to alarm cues or their metabolic Magurran 2003). Chemical cues are the most reliable source derivatives, but released from predators’ digestive system of communication in a wide variety of prey as they are quick during digestion and defecation (Ferrari et al. 2010). Dietary to diffuse and efficient to rely on, even at night and in murky cues can potentially reveal the type of food that has been habitats (Dodson et al. 1994). These chemical cues are also ingested recently by predators (Mathis and Smith 1993; Wil- crucial for prey organisms that encounter cryptic or sit-and- son and Lefcort 1993; Mirza and Chivers 2003; Vilhunen and wait predators (Mathis and Smith 1993; Chivers et al. 1996). Hirvonen 2003; Schoeppner and Relyea 2005). Chemical cues of diverse origins are used for the recogni- Irrespective of the type of chemical cue used, prey ani- tion of potential predators (Kats and Dill 1998). Myriad mals are known to have two mechanisms of chemoreception chemical cues such as kairomones, disturbance cues, alarm for the recognition and discrimination of their potential cues and dietary cues can serve as potential signals of preda- predators: innate (Griffiths et al. 1998; Laurila 2000; tion risk at various stages of the predation event as they can Batabyal et al. 2014) and/or learned (Chivers and Smith http://www.ias.ac.in/jbiosci 459 460 Swapnil C Supekar and Narahari P Gramapurohit 1998; Wisenden 2003; Batabyal et al. 2014). An innate temporalis. Hylarana temporalis was considered as a single predator recognition mechanism helps prey to recognise species distributed throughout the Western Ghats until the certain predators on the first encounter without any prior revision of the genus Hylarana by Biju et al. in 2014. With experience, while a learned recognition mechanism needs extensive surveys and sapling from different localities of the prior experience of a predation event that helps prey to Western Ghats and , the authors established that Hy- recognise predators through associative learning (Epp and larana temporalis never existed in India and it was a case of Gabor 2008; Batabyal et al. 2014). However, recent studies wrong identification which persisted over long time. The have shown that learning is a critical component of predator distributed in the Koyna region of the Western Ghats was recognition regardless of the presence or absence of an identified as Hylarana caesari). Another important component innate recognition mechanism as it helps in updating infor- of learned predator recognition that has received little attention mation about a wide spectrum of predators at different stages is the developmental stage at which the learning to recognise of ontogeny (Epp and Gabor 2008; Brown et al. 2013). predators is accomplished. Studies reporting the ability of Alarm cues are known to mediate such associative learning to learn to recognise their predators during when a naı¨ve individual simultaneously perceives con- embryonic stages have not focused on determining the window specific alarm cues along with cues of a novel predator, such of embryonic development that is sensitive to learning as its image, odour or sound (Ferrari et al. 2010). The (Batabyal et al. 2014). Similarly, information on the preferential concentration of chemical cues may provide information use of different chemical cues by prey to recognise their such as the distance to a predator or the number of predators potential predators during ontogeny is scarce (Batabyal et al. nearby (Horat and Semlitsch 1994; Ferrari et al. 2010). 2014). Therefore, in the present study, we used tadpoles of Several species of prey have an innate mechanism Euphlyctis cyanophlyctis to address these questions. to recognise their potential predators (Vilhunen and Hirvonen The Indian skipper frog, E. cyanophlyctis, is widely dis- 2003;Hawkinset al. 2004; Dalesman et al. 2007;Galland tributed throughout Asia and is abundant throughout its range Mathis 2010;Batabyalet al. 2014). Likewise, several others (Daniels 2005). It is a continuous breeder, and the tadpoles can have evolved experience-mediated associative learning for be seen throughout the year in all types of ephemeral as well as predator recognition (Woody and Mathis 1998;Eklo¨v 2000; permanent waterbodies including streams and rivers (Daniels Mirza and Chivers 2001;Griffin2004;Mirzaet al. 2006; 2005). In such ecologically diverse habitats, larval E. Gonzalo et al. 2007; Ferrari and Chivers 2008; Ferrari et al. cyanophlyctis are prone to predation risk by both native as well 2008; Smith et al. 2008;Batabyalet al. 2014). While both as non-native predators. However, the wide distribution of the mechanisms have advantages and limitations, their evolution species associated with its abundance indicates that its tadpoles appears to be species-specific and depends on the type and might have novel strategies to counter potential native as well as complexity of habitats (Gonzalo et al. 2007;EppandGabor exotic predators, thus flourishing in large numbers to reach 2008;Batabyalet al. 2014). For instance, the innate predator adulthood. Hence, the rationale of the present investigation was recognition mechanism that confers a high speed of response to to understand the following: (1) Do predator-naı¨ve and preda- a limited spectrum of predators is expected to be favoured in tor-experienced tadpoles of E. cyanophlyctis exhibit similar habitats where the predator community is constant over evo- antipredator responses? (2) Do larval E. cyanophlyctis have an lutionary time and the predator diversity is quite low (Wisenden innate mechanism of predator recognition? (3) Do they learn to 2003; Brown and Chivers 2005;Brownet al. 2013). On the recognise their predators during ontogeny? (4) If they learn, do other hand, learned predator recognition, which offers an they use conspecific alarm cues to label novel predatory odours adaptive response to predation risk against a wide range of through associative learning? (5) Are antipredator responses of species over time, is believed to evolve in complex habitats with tadpoles the same against different chemical cues? spatiotemporal variation in predator communities (Wisenden 2003; Brown and Chivers 2005). While some ecosystems may favour the evolution of either of the two, others may favour both 2. Methods and materials mechanisms in concert for efficient predator recognition and avoidance (Gonzalo et al. 2007; Epp and Gabor 2008). Prey that Five pairs of E. cyanophlyctis in amplexus were collected can recognise certain predators innately and can also learn to from a semi-permanent pond located on the Savitribai Phule ° 0 ° 0 recognise novel predators through associative learning would Pune University campus (18 55 N, 73 82 E), Pune, India, be at a selective advantage in complex, dynamic habitats. Sur- between 2130 and 2230 h and were quickly transported to prisingly, information on such experience-mediated risk-sen- the laboratory where each pair was kept separately in a glass 9 9 sitive adjustments to innate avoidance responses by larval aquarium (60 cm 40 cm 45 cm) for spawning. Spawned amphibians is scarce (Gonzalo et al. 2007; Epp and Gabor eggs from all the pairs were collected the next morning and 2008; Batabyal et al. 2014). Recently, Batabyal et al. (2014) maintained in another glass aquarium with aged tap water 9 9 have demonstrated such a dual mechanism of predator recog- until they hatched (45 cm 30 cm 10 cm) while the nition in larval Hylarana caesari (formerly, Hylarana adults were returned to nature. The hatchlings were mixed Predator recognition in Euphlyctis cyanophlyctis 461 thoroughly and maintained in the glass aquaria (60 cm 9 45 d) Heterospecific alarm cues (CH) – Ten tadpoles (0.41 g) of cm 9 15 cm) at a density of 4/L with aged tap water. F. syhadrensis at stage 27–28 were frozen to deep Aquarium water was renewed every third day, and the tad- hypothermia at 0°Cto-1°C for 10–15 min and then poles were fed partially boiled spinach ad libitum. Devel- quickly crushed with a mortar and pestle. The crushed opmental stages of the embryos/tadpoles were identified tissues were mixed in 500 mL aged tap water. The solution according to Gosner (1960). The embryos, hatchlings and was filtered and 5 mL of this filtrate was used as CH. tadpoles (predator-naı¨ve) from this stock were used for e) Kairomones ? Conspecific alarm cues (K ? CC) – K and experiments depending upon the stage of predator exposure. CC prepared using the above-described protocol were Tadpoles of E. cyanophlyctis (stage 29–33) were also mixed thoroughly (2.5 mL each) and used as stimulus. collected from the same pond (from where the adults were f) Kairomones ? Heterospecific alarm cues (K ? CH) – K collected) and quickly transported to the laboratory. They and CH prepared using the above-described protocol were were acclimated to the laboratory conditions for 48 h and mixed thoroughly (2.5 mL each) and used as stimulus. then used for experiment I. g) Dietary cues of conspecific origin (DCC) – Six dragonfly Fourth instar nymphs of the dragonfly (Bradinopyga gem- nymphs were allowed to feed on 0.32 g of larval inata) collected from the same pond (X=23.01±0.14 mm) E. cyanophlyctis. Nymphs collected after ingestion of the were used as predators. Since dragonfly nymphs are known to prey were washed thoroughly (to make sure the predator feed voraciously on amphibian tadpoles at different stages of is devoid of any chemical cue attached to it) and their development, they are the model predators of choice in maintained in a beaker with 400 mL aged tap water for various laboratory studies. Nymphs were maintained in per- 24 h. Subsequently, water was collected and filtered. Five forated plastic cups (10.5 cm diameter) and fed with tadpoles mL of this water was used as DCC. of either E. cyanophlyctis or Fejervarya syhadrensis daily h) Dietary cues of heterospecific origin (DCH) – Six until they were used for experimentation. During experi- dragonfly nymphs were allowed to feed on 0.32 g of mentation, nymphs were either starved or fed with a constant larval F. syhadrensis. Nymphs collected after ingestion amount of conspecific (E. cyanophlyctis) or heterospecific (F. of the prey were washed thoroughly and maintained in a syhadrensis) tadpoles as required, and 5 mL of cue prepared separate beaker with 400 mL aged tap water for 24 h. water was used as a stimulus for the behavioural assay. Five mL of this water was used as DCH. Thirty tadpoles were used for testing their antipredatory response to each chemical cue. Each tadpole was tested 2.1 Experiment I: Responses of predator-naı¨ve separately and only once. The behavioural assay was carried and predator-experienced tadpoles of E. cyanophlyctis out using an established protocol (Batabyal et al. 2014). Test to different chemical cues trials were conducted in a ventilated room between 1000 h and 1730 h. The mean temperature (24±1°C) and pho- This experiment was designed to assess the antipredator toperiod were maintained constant during all tests. The responses (in terms of activity reduction) of predator-naı¨ve behavioural assay used to assess the antipredator response of (laboratory-reared) and predator-experienced (nature-col- tadpoles was as follows: lected) tadpoles to different chemical cues. The tadpoles A specially designed glass aquarium (50 cm 9 6cm9 6 cm) between stages 29 and 33 were tested for their antipredator was used as the testing chamber. The chamber was marked response using a behavioural assay. The chemical cues from outside at the bottom with lines at an interval of 5 cm. At used as stimulus for the behavioural assay were as follows: the beginning of each trial, the test chamber was filled with 1 L a) Control – 5 mL of aged tap water (since the use of aged tap water to allow free movement of tadpoles. A test distilled water resulted in an increase in the activity of tadpole was introduced at one end of the chamber and allowed tadpoles, it was not used as a control). to acclimatise for 10 min. Subsequently, as a measure of b) Predator odour/kairomones (K) – Six starved dragonfly activity, the number of lines crossed by the test tadpole before nymphs were maintained in 400 mL aged tap water for and after the addition of stimulus cue was recorded. The test 24 h without any food. Subsequently, water was tadpole was considered to have crossed the line when its entire collected and filtered. Five mL of this water conditioned body was on the other side of a line. If the tadpole did not move with predator odour was used as a stimulus cue. in the test chamber for more than 2 min during the acclima- c) Conspecific alarm cues (CC) – Five tadpoles (0.41 g) of tisation period, it was discarded (\3%). The total time period E. cyanophlyctis at stage 27–28 were frozen to deep of each trial was 10.5 min (4 min pre-stimulus activity hypothermia at 0°Cto-1°C for 10–15 min and then recording followed by 2.5 min during which the added cue quickly crushed with a mortar and pestle. The crushed diffused throughout the tank and 4 min of post-stimulus tissues were mixed in 500 mL aged tap water. The solution activity recording). An equal volume of the stimulus cue (2.5 was filtered and 5 mL of this filtrate was used as CC. mL) was added at either end of the testing chamber. 462 Swapnil C Supekar and Narahari P Gramapurohit 2.2 Experiment II: Recognition of kairomones through The embryos/hatchlings/pre-feeding and feeding stage associative learning tadpoles were exposed to predators feeding on conspecific tadpoles (table 1). The treatment groups were as follows: Since the antipredator response of predator-naı¨ve and Group 1 – Rearing of embryos/tadpoles in cue-free water predator-experienced tadpoles to various chemical cues was from stage 1–33 (Control) different, particularly against kairomones, this experiment Group 2 – Exposure to K ? CC from stage 1–12 was designed to determine whether or not these differences (cleavage – gastrulation, CG) are due to associative learning. Predator-naı¨ve tadpoles Group 3 – Exposure to K ? CC from stage 13–20 (stage 29–33, N = 30/ aquaria) were transferred to three glass (neurulation – hatching, NH) aquaria (45 cm 9 30 cm 9 10 cm) with 6 L aged tap water. Group 4 – Exposure to K ? CC from stage 21–24 They were subjected to three treatments as follows: (hatching – feeding, HF), Group 1 – Conditioning of the predator-naı¨ve tadpoles Group 5 – Exposure to K ? CC from stage 25–33 with aged tap water (control). Tadpoles were allowed to (feeding onwards, FO) perceive 5 mL/L of aged tap water added to the aquarium Group 6 – Exposure to K ? CC from stage 1–33 during conditioning. (cleavage through hatching, feeding until tested for Group 2 – Conditioning of predator-naı¨ve tadpoles with K behavioural assay, Continuous). alone. Tadpoles were allowed to perceive 5 mL/L of K The embryos/hatchlings/pre-feeding and feeding stages during conditioning. were maintained in glass aquaria (45 cm 9 30 cm 9 10 cm) Group 3 – Conditioning of predator-naı¨ve tadpoles with with 6 L of aged tap water at a density of 5/L. Each CC and K simultaneously (tadpoles were allowed to aquarium was equipped with a perforated transparent cage perceive chemical cues from dragonfly nymphs feeding (10.5 cm diameter) housing a dragonfly nymph. A nymph on a conspecific tadpole during a caged encounter). was allowed to feed on conspecific tadpoles in the cage. Kairomones were also added at a concentration of 5 mL/L After the exposure, developing embryos and hatchlings were to facilitate the perception of both K and CC washed thoroughly with aged tap water to ensure that no simultaneously. chemical cues remain attached to the jelly/egg surface. After 24 h of conditioning, the tadpoles were maintained Subsequently, they were maintained in the aquaria under in cue-free water for 24 h. Subsequently, they were tested for laboratory conditions until they were tested for behavioural their antipredator response to K alone using the behavioural response. The response of tadpoles to K alone was tested assay mentioned above. between stages 29 and 33.

2.3 Experiment III: Determining the sensitive stages 2.4 Statistical analysis for associative learning Activity data were converted into a relative activity using the Since experiments I and II showed that larval E. formula: cyanophlyctis learn to recognise K as a threat through associative learning, this experiment was designed to Relative activity ¼ determine the sensitive window for associative learning Number of lines crossed in the post-stimulus period during ontogeny. the number of lines crossed in the pre-stimulus period

Table 1. Various embryonic and post-embryonic (pre-feeding and post-feeding) stages of E. cyanophlyctis during which they were exposed to a mixture of kairomones, alarm and dietary cues as well as the duration of exposure.

Exposure to K ? CC Time interval between exposure Sr. No. Treatment groups Stage of development Duration in h/day and testing (day) 1. Control – – 43 2. Cleavage to gastrulation (CG) Up to stage 12 12 h 41 3. Neurulation to hatching (NH) Stage 13–20 4 day 41 4. Hatching to feeding (HF) Stage 21–24 5 day 36 5. Feeding to onwards (FO) Stage 25–33 37 day 2 6. Continuous Cleavage–stage 33 49 day 1 Predator recognition in Euphlyctis cyanophlyctis 463 The data were confirmed for normality using probability when tested against different chemical cues such as aged tap plots and the Anderson-Darling test before subjecting to water (Control), K, CC, CH, K ? CC, K ? CH, DCC, and statistical analyses. A few outliers (\2%) from some DCH (table 2; figure 1). Further, both prey-type and cue- groups were excluded from the analyses to meet the type had a significant impact on the antipredator responses assumptions of normality. Responses of predator-naı¨ve of tadpoles (table 2). Interestingly, the interaction between and predator-experienced tadpoles against different chem- prey-type and cue-type was also significant (table 2). ical cues were analysed using a two-way analysis of Predator-naı¨ve tadpoles reduced their activity significantly variance (ANOVA) with prey-type and cue-type as inde- against CC by 40% and 30% in comparison to that against pendent variables and antipredator responses as dependent aged tap water and kairomones respectively (table 2). Sim- variable, followed by univariate tests and Tukey’s multiple ilarly, when presented with CH, predator-naı¨ve tadpoles pair-wise comparison test. Responses of predator-naı¨ve reduced their activity significantly by 32% and 22% than tadpoles conditioned with aged tap water, kairomones or their activity against aged tap water and kairomones kairomones ? CC were analysed using one-way ANOVA respectively (table 2). Further, responses of predator-naı¨ve followed by Tukey’s multiple pair-wise comparison test. tadpoles against K ? CC (31% and 21%), K ? CH (64% Ontogenetic changes, if any, in the response of tadpoles and 54%), DCC (73% and 63%) and DCH (82% and 72%) to the kairomones was analysed using one-way ANVOA were also significant in comparison to that against aged tap followed by Tukey’s test. All the tests were two-tailed water and K (table 2; figure 1A). However, the responses of and the significance level was set at 0.05. All the sta- predator-naı¨ve tadpoles against CC, CH, K ? CC, K ? CH, tistical analyses were performed using SPSS ver 19.0 and DCC and DCH were comparable with each other (table 2). XLstat. Interestingly, the response of predator-naı¨ve tadpoles against K was similar and comparable to that against aged tap water (table 2; figure 1A). Predator-experienced tadpoles reduced their activity sig- 3. Results nificantly against K (74%), CC (120%), CH (88%), K ? CC (74%), K ? CH (73%), DCC (41%) and DCH (39%) in 3.1 Experiment I: Response of predator-naı¨ve comparison to that against aged tap water (table 2; fig- and predator-experienced larval E. cyanophlyctis ure 1A). Further, the responses of these tadpoles were to different chemical cues comparable among K, CC, CH, K ? CC, K ? CH, DCC and DCH (table 2; figure 1A) Overall, a significant difference was observed in the relative Comparison of relative activity between predator-naı¨ve activity of predator-naı¨ve and predator-experienced tadpoles and predator-experienced tadpoles showed a significant

Table 2. Results of two-way ANOVA followed by univariate tests and Tukey’s multiple pair-wise comparisons test depicting the antipredator response of predator-naı¨ve and predator-experienced tadpoles of E. cyanophlyctis

Source of variation F df P Overall 11.079 15, 452 0.000 Prey-type 5.919 1, 452 0.015 Cue-type 16.686 7, 452 0.000 Prey-type * Cue-type 6.118 7, 452 0.000 Tukey’s highest significant difference for multiple pair-wise comparisons

Predator-naı¨ve Predator-experienced

Cues KCCCHK?CC K?CH DCC DCH K CC CH K?CC K?CH DCC DCH C 1.000 0.001* 0.002* 0.005* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* K – 0.001* 0.002* 0.006* 0.000* 0.000* 0.000* – 0.940 1.000 1.000 1.000 0.996 0.992 CC – 1.000 1.000 1.000 0.997 0.969 – 1.00 0.951 0.936 0.167 0.131 CH – 1.000 0.999 0.979 0.899 – 1.000 1.000 0.761 0.696 K ? CC – 0.997 0.955 0.836 – 1.000 0.997 0.993 K ? CH – 1.000 1.000 – 0.998 0.995 DCC – 1.000 – 1.000 * Indicates significant difference. 464 Swapnil C Supekar and Narahari P Gramapurohit

Figure 2. Pre- and post-conditioning relative activity (M ± SE) of predator-naı¨ve tadpoles of E. cyanophlyctis when presented with kairomones (K). Tadpoles were conditioned with aged tap water, K and K ? CC. * Indicates significant difference.

3.3 Experiment III: Determining the sensitive stages for associative learning

When exposed to K clubbed with CC during embryonic, post-hatching and post-feeding stages, tadpoles of all groups (CG, NH, HF, FO and continuous) reduced their activity Figure 1. (A) Relative activity (M ± SE) of predator-naı¨ve and significantly against K in comparison to that of the control predator-experienced tadpoles of E. cyanophlyctis when presented group (F = 10.540, p = 0.000; figure 3). Further, ? ? 5,170 with aged tap water (control), K, CC, CH, K CC, K CH, DCC reduction in the activity of tadpoles exposed continuously to and DCH. Dissimilar alphabets over the bars indicate significant K ? CC was significantly higher than that of CG (81%, difference among predator-naı¨ve (a, b) and predator-experienced (x, = = y) groups and (B) relative activity (M ± SE) of predator-naı¨ve and p 0.012), HF (87%; p 0.004) and FO (88%; = predator-experienced tadpoles of E. cyanophlyctis when presented p 0.006) except the NH group (60%) with which it was with aged tap water (control), K, CC, CH, K ? CC, K ? CH, DCC, comparable (p = 1.00; figure 3). DCH. * Indicates significant difference. difference in their response against K. Predator-experienced tadpoles reduced their activity significantly (54%) against kairomones in comparison to predator-naı¨ve tadpoles (p = 0.000; figure 1B).

3.2 Experiment II: Associative learning of kairomones through conspecific alarm cues

Overall, a significant difference was observed in the relative activity of predator-naı¨ve tadpoles following their condi- tioning (F2,87 = 14.739, p = 0.000). Following their con- ditioning with K mixed with CC for 24 h, tadpoles responded to K alone by reducing their activity significantly in comparison to the tadpoles conditioned with aged tap ± = Figure 3. Relative activity (M SE) of predator-naı¨ve (control) water (69%) or kairomones (49%; p 0.000; figure 2). and predator-experienced (embryos of Gosner stage up to stage 12, Once it was clear that the tadpoles learn to recognise kair- stage 13–20, stage 21–24, stage 25–33, and up to stage 33) tadpoles omones by associative learning, embryos, hatchlings and of E. cyanophlyctis when presented with kairomones. Dissimilar tadpoles were used for determining the sensitive stages for alphabets over the bars indicate significant difference. associative learning. Predator recognition in Euphlyctis cyanophlyctis 465 4. Discussion 2007). Apparently, species-specific differences in the ability of prey animals to recognise kairomones, conspecific alarm cues Successful coexistence of prey animals with their predators or both may depend on the time they spend in aquatic habitats depends on the prey’s ability to recognise and discriminate and the complexity of habitats. various predatory threats and evolve novel strategies to Findings of the present investigation indicate that tadpoles avoid/deter them (Lima and Dill 1990; Kats and Dill 1998; of E. cyanophlyctis could acquire the ability of recognizing Ferrer and Zimmer 2007; Ferrari et al. 2010). Studies using kairomones through associative learning as the tadpoles col- various model organisms including larval amphibians have lected from nature (predator-experienced) could recognise revealed that the goal of predator recognition and discrimi- kairomones. Studies using various model systems including nation in prey species inhabiting diverse ecosystems can be larval amphibians have emphasised the importance of con- achieved by the evolution of two mechanisms: innate and/or specific alarm cues in associative learning (Chivers and Smith acquired predator recognition depending on the species and 1998; Ferrari et al. 2010). For instance, predator-naı¨ve tad- the complexity of habitats (Epp and Gabor 2008; Ferrari poles of H. caesari could use alarm cues for recognising novel et al. 2010; Batabyal et al. 2014). predator cues through associative learning (Batabyal et al. Results of the present study indicate the absence of innate 2014). Similarly, larval R. perezi and adult Notopthalmus predator recognition mechanism in E. cyanophlyctis as evi- viridescens could use alarm cues to recognise predatory cues denced by the failure of predator-naı¨ve tadpoles to reduce through associative learning (Woody and Mathis 1998; their activity against kairomones on the first encounter. In Gonzalo et al. 2007). Although recent studies have focused on contrast, tadpoles of Bufo americanus, H. caesari and Rana the embryonic learning of predators, the importance of perezi and adult salamanders (Eurycea nana) have the innate olfactory and nervous systems in perception, learning and ability to recognise kairomones on the first encounter (Gallie memory have received less attention (Mathis et al. 2008; et al. 2001;Gonzaloet al. 2007; Epp and Gabor 2008;Mogali Ferrari and Chivers 2009; Batabyal et al. 2014). For instance, et al. 2012; Batabyal et al. 2014). Prey can have innate ability embryonic wood frogs (Rana sylvatica) conditioned to per- to recognise kairomones and/or alarm cues. Some authors ceive salamander odour along with alarm cues could asso- consider the recognition of alarm cues as a measure of innate ciatively learn to recognise predatory salamanders as a threat predator recognition mechanism. However, we feel that when tested during tadpole stages (Mathis et al. 2008). Fur- recognition of kairomones rather than alarm cues should be ther, embryos of R. sylvatica could also learn to distinguish considered as a measure of innate predator recognition, and in between predators and non-predators based on their experi- this context larval E. cyanophlyctis lack the innate predator ence during embryonic development (Ferrari and Chivers recognition mechanism. Further, tadpoles of E. cyanophlyctis 2009). However, in both the studies embryos were condi- have an innate ability to recognise alarm cues as a threat on tioned during neurulation (Gosner stage 13–15) when the their first encounter as evidenced by the reduction in the neural tube is formed. In amphibians, the olfactory system is activity of predator-naı¨ve tadpoles against alarm cues. Simi- known to develop between Gosner stages 18 and 21 or after larly, predator-naı¨ve tadpoles of R. perezi reduce their activity neurogenesis (Spaeti 1978). In the present study, embryonic against conspecific alarm cues on the first encounter (Gonzalo E. cyanophlyctis conditioned with kairomones along with et al. 2007). In contrast, predator-naı¨ve tadpoles of H. caesari conspecific alarm cues before neurulation could learn to did not reduce their activity against conspecific alarm cues, associate kairomones with alarm cues in the absence of indicating the lack of an innate ability to recognise conspecific functional olfactory and nervous systems. In contrast, condi- alarm cues as a threat (Batabyal et al. 2014).Thelackof tioning of embryonic H. caesari to a mixture of cues (K ? CC innate predator recognition mechanism in larval ? DCC) before neurulation failed to elicit any antipredator E. cyanophlyctis might be due to the diversity and complexity response when tested later during larval stages (Batabyal et al. of habitats associated with simultaneous exposure to fluctu- 2014). Hence, species-specific differences could exist in their ating predatory communities. In such complex habitats with abilities to learn through an association, which, in turn, may spatiotemporal variation in predatory communities, innate determine their spatiotemporal distribution and abundance in predator recognition could be costly to evolve and maintain different ecological conditions. To our knowledge, this is the (Wisenden 2003). first report on the associative learning by embryonic Although larval E. cyanophlyctis lack the innate ability of amphibians in the total absence of a nervous system. perceiving kairomones as a potential threat, they can perceive Information on the mechanisms involved in associative conspecific alarm cues on the first encounter as a potential learning in the absence of a functional nervous system is threat. In contrast, H. caesari tadpoles with the innate ability of very limited. Although some information on the embryonic perceiving kairomones as a potential threat cannot recognise learning of odours is available for R. temporaria and conspecific alarm cues (Batabyal et al. 2014). Interestingly, R. sylvatica, developmental stages that are sensitive to such R. perezi have the innate ability to recognise both kairomones associative learning were not defined (Hepper and Waldman and conspecific alarm cues as a potential threat (Gonzalo et al. 1992). Similarly, embryos of R. lessonae exposed to the 466 Swapnil C Supekar and Narahari P Gramapurohit odours of the native pond during stages 18–21 could respond ‘phylogenetic-relatedness theory’ proposes that response of to those odorants later (Ogurtsov and Bastakov 2001). prey to alarm/dietary cues of closely related heterospecifics Generally, learning and memory, critical for the survival of should be stronger than such cues from distantly related ones organisms, are associated with a functional brain and ner- (Parker and Shulman 1986; Mathis and Smith 1993; Sullivan vous system or at least neural activity (Ball 2008). Surpris- et al. 2003; Schoeppner and Relyea 2005). However, empir- ingly, embryonic E. cyanophlyctis could associatively learn ical evidence supports the phylogenetic relatedness hypothe- to recognise novel predatory odours in the total absence of a sis. For instance, R. catesbeiana tadpoles exhibit an nervous system. Moreover, embryonic E. cyanophlyctis antipredator response to heterospecific cues of their close could achieve associative learning in a very short time (12 relative Rana septentrionalis (Raymond and Murray 2008). h). Information perceived in such a short time could be Similarly, P. promelas (fathead minnows) exhibit an memorised and used for making decisions about their antipredator response to heterospecific cues of brook stickle- antipredator responses when tested 41 days after condition- backs (Mirza and Chivers 2001). Further, larval H. caesari ing. Previously, Ferrari et al. (2009) have shown that exhibit an antipredator response to heterospecific cues of its embryonic R. sylvatica require 4–5 days for learning tem- close relative Clinotarsus curtipes (Batabyal et al. 2014). In poral information about their predators. Further, embryonic studies using R. catesbeiana and P. promelas, prey animals R. sylvatica were able to memorise this information to make were conditioned with dietary cues before testing for their decisions during later larval life when tested 15 days after response to heterospecific cues. Surprisingly, larval E. conditioning. Cellular reception, interpretation and adjust- cyanophlyctis and H. caesari not only reduced their activity ments to environments are well known (Ball 2008). How- against heterospecific cues but they did so in the total absence ever, the ability of cells to learn and memorise information of any prior experience. Hence, species-specific differences about environmental challenges is shown only in a unicel- could exist in the prey animals’ response to heterospecific lular amoeba Physarum polycephalum (Ball 2008). Possibly, dietary cues. embryonic cells of E. cyanophlyctis may have some mech- In conclusion, larval E. cyanophlyctis have the innate anism that could help in learning and memory in the absence ability to recognise conspecific alarm cues as a potential of olfactory and nervous systems. Alternatively, the chemi- threat, while they learn to recognise kairomones through cal cues attached to the egg jelly or embryonic surface could their association with alarm cues. Further, embryonic skipper remain to be available for learning later after the develop- frogs learn to recognise kairomones through associative ment of olfactory and nervous systems. Future studies are learning even before the development of functional olfactory required to clarify this component of prey–predator and nervous systems. interaction. Interestingly, tadpoles of E. cyanophlyctis that had been conditioned (K ? CC) as embryos/post-hatchlings and post- Acknowledgements feeding stages reduced their activity when tested against kairomones alone, suggesting behavioural plasticity in asso- This work was supported by UGC-CAS Phase III and DRDP ciative learning. Moreover, increase in the intensity of activity to Department of Zoology, Savitribai Phule Pune University. reduction by tadpoles conditioned during feeding stages sig- SCS is grateful to Savitribai Phule Pune University for a nifies the importance of continuous exposure in eliciting a research fellowship. Thanks are also due to Neelesh Dha- maximum antipredatory response by larval E. cyanophlyctis. hanukar for his help in statistical analysis. This study was The findings of the present study indicate that tadpoles of E. carried out following the guidelines of Departmental Com- cyanophlyctis can recognise alarm cues of heterospecific ori- mittee for Ethics (In India, animals other than gin as evidenced by the reduction in the activity of predator- mammals do not come under the purview of the institutional naı¨ve tadpoles against alarm and dietary cues of F. syha- committee for animal ethics, No. 538/CPCSEA). drensis. This observation is consistent with those reported for H. caesari, Rana catesbeiana, E. nana and Pimephales promelas (Mirza and Chivers 2001; Raymond and Murray References 2008; Epp 2013; Batabyal et al. 2014). 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