Acta Herpetologica 14(1): 15-19, 2019 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-22855 Experience of predacious cues and accessibility to refuge minimize mortality of Hylarana temporalis tadpoles Santosh Mogali*, Bhagyashri Shanbhag, Srinivas Saidapur Department of Zoology, Karnatak University, Dharwad-580 003, Karnataka State, India. *Corresponding author. E-mail: santoshmoga- li@rediffmail.com Submitted on: 2018, 8th March; revised on: 2018, 29thOctober; accepted on: 2018, 31st October Editor: Adriana Bellati Abstract. We explored the effect of a prior experience of predation threat and access to the refuge sites on larval mor- tality in Hylarana temporalis with a 2 × 2 factorial design. The design included predator-naïve or predator experienced prey subjects, and presence or absence of a natural shelter. Water scorpion adult individuals (Laccotrephes species) provided predation threat and also an opportunity to the prey to experience a direct danger of predation. Both previ- ous experience with predators and availability of shelters affected larval survival independently and not conjointly. A prior encounter with predators enabled prey tadpoles to escape predation more effectively with a significant increase in their survival in comparison to the predator-naïve subjects. Similarly, access to refuge sites significantly increased survival of predator-naïve as well as predator experienced tadpoles compared to that in the absence of shelters. Clear- ly, ability to sense water borne predacious cues in the vicinity and use refuge sites plays a key role in escaping from predation in the bronze frog tadpoles. Keywords. Hylarana temporalis, Laccotrephes sp., predator, refuge availability, tadpole mortality. Predator-prey interactions invariably lead to an evo- lutionary arms race in which early detection of either party is often the key for success. Progressive elimination of prey individuals from the ecosystem by the coexisting predators can have far reaching impact on their popula- tion dynamics. Most anuran amphibians show a biphasic mode of life cycle involving aquatic larval phase. In the aquatic phase of life the larval mortality is often due to desiccation of the ephemeral ponds before completion of the metamorphosis, infection, and to predation by the coexisting aquatic invertebrate and vertebrate preda- tors. Therefore, adoption of phenotypic plasticity and diverse strategies become essential for their survival. Many anuran larvae including Hylarana temporalis are known to alter their behavior and the life-history traits under predation risk (Relyea, 2007; Sharma et al., 2008; Saidapur et al., 2009; Mogali et al., 2012, 2015, 2016; Mogali, 2018). For example, H. temporalis tadpoles meta- morphosed early and at a larger body size under preda- tion threat, when provided by a caged dragonfly larva (Pantala flavescens), compared to those reared under predation threat-free environment (Mogali et al., 2016). They also decreased swimming activity, by remain- ing still for longer times, to avoid being detected and increased burst speed on encounter with the predacious cues in the vicinity (Mogali et al., 2012). Taking shelter under the leaf litter, aquatic vegetation and other objects that provide refuge and utilization of benthic habitat can help tadpoles by reducing the risk of being detected by predators (Lima and Dill, 1990; Eklov and Persson, 1996; Hossie and Murray, 2010). Besides, predators may find difficult to capture a prey residing inside a refuge; at the same time, prey may have a better opportunity to assess the actual risk of predation by the predators residing in close vicinity (Hemmi and Zeil, 2005) and, consequently, evoke defensive behaviors. Nevertheless, hiding and stay- 16 Santosh Mogali, Bhagyashri Shanbhag, Srinivas Saidapur ing still by prey involve trade-offs. These strategies may reduce encounter rates with predators but also tend to reduce time dedicated to foraging activity. Indeed, often the habitats that are energetically very profitable are also the most dangerous, since the distribution of predators tends to match their prey’s resource distribution (Lima, 1998). Besides, the refuge may not always be the best place for feeding. For instance, inadequate time spent in feeding leads to alteration in the metamorphic traits in Bufo melanostictus (Mogali et al., 2011). The bronze frog (Hylarana temporalis) tadpoles are found along the gently flowing streams and iso- lated pockets of water along sides of the streams during the post-monsoon season (October-January) in South- Western Ghats of India (Mogali et al., 2012). They are bottom dwellers and thrive on detritus and algal matter (Hiragond and Saidapur, 2001). In these water bodies visibility is low due to shadows of the dense vegetation, brownish-dark color of the benthic area that is typically covered by the leaf litter and detritus matter (Veerana- goudar et al., 2004). The water bodies are also inhabited by several types of invertebrate predators that include aquatic beetles, dragonfly larvae, crabs and water scor- pions that prey upon anuran tadpoles (our personal observations). During field visits we have observed water scorpions (Laccotrephes sp., Nepidae) actively feeding on bronze frog tadpoles. They detect their prey visu- ally and by mechanical cues. The H. temporalis tadpoles have poor vision (Veeranagoudar et al., 2004) as in most anuran tadpoles (Hoff et al., 1999). However, they pos- sess an innate ability to detect predators by chemorecep- tion (Mogali et al., 2012) and evoke antipredator behav- ior. Further, predator-experienced H. temporalis tad- poles show enhanced defensive behaviors on subsequent encounter with predators (Mogali et al., 2012). The natural habitats in which H. temporalis tadpoles live is full of detritus material and leaf litter. Therefore, we hypothesized that tadpoles of the bronze frog profit- ably use these shelters (leaf litter) to reduce predation risk of the coexisting predators. An additional assumption was that tadpoles having prior experience of predacious cues will be more successful in escaping predation by using shelters compared to the those facing the predators for the first time (predator-naive). To test our hypotheses, tadpoles of H. temporalis were exposed to free hunting water scorpion (Laccotrephes sp). The prey subjects were either predator-naïve or predator experienced. They were exposed to predator in laboratory set ups with or without the refuge sites (leaf litter) for a predetermined period to record survival/mortality of prey. Eight clutches of H. temporalis were collected in November, 2013 from a stream in the Western Ghats near Anmod village (15.430888°N, 74.373601°E), Karnataka State, India. Each clutch was individually reared in plastic tub (32 cm diameter and 14 cm deep) containing 5 L of aged tap (dechlorinated) water. Hatching time occurred six days later, at Gosner stage 19 (Gosner, 1960). The tadpoles of different clutches were mixed to normalize genetic differences among the groups. After mixing, tad- poles were reared in two separate glass aquaria (90 cm L × 30 cm W × 15 cm H) each containing 25 L of aged tap water. Upon reaching stage 25, tadpoles were supplied with boiled spinach ad libitum. The water scorpions (Lac- cotrephes sp.) were collected from the same location from where the eggs of H. temporalis were obtained and were reared individually, to avoid cannibalism, in small plas- tic bowls (14 cm diameter and 7 cm deep) with 500 mL of aged tap water. Prey tadpoles served as food for water scorpions. The tadpoles born in laboratory, and never exposed to predator, served as the predator-naïve subjects. To obtain predator-experienced tadpoles we exposed groups of 30 tadpoles to a 48 h starved predator for 8 h (09:00- 17:00 h). After the 8 h trial period the predator and injured tadpoles were removed from the test bowl. On an average, predator ate 3 ± 0.3 tadpoles and injured 4 ± 0.5 (x ± SE) during the trial period. Predator-threat expe- rienced and uninjured tadpoles obtained from 80 trials were used in the experiments the following days, in order to assess their performance against predators on the sub- sequent encounter with them. The leaves of Aporosa lindleyana were collected from the same sites from which H. temporalis eggs were col- lected. Structural refuge was made using water soaked (2 days) leaves (dry mass 15 g ± 1.8; x ± SE) chopped to have ~ 1 cm2 pieces. These were spread at the bottom of the testing bowls to serve as shelters/ refuge site). In all trials, prey tadpoles were of comparable siz- es (length 20.48 ± 0.09 mm, width 5.31 ± 0.07 mm, and weight 62.0 ± 0.64 mg; x ± SE; n = 30) and developmental stages (stages 28-30). The water scorpions used in various trials were comparable in size (length 31.0 ± 0.08 mm, width 10.11 ± 0.05 mm, and weight 622.0 ± 5.2 mg; x ± SE; n = 30). The size of prey and predators was measured using a digital caliper (accuracy 0.01 mm) and weight was recorded using an electronic balance (accuracy 0.001 g). The experiment involved using a 2 × 2 factorial design with the following groups. 1. Predator-naïve tadpoles were exposed to predator in the absence of refuge 2. Predator-naïve tadpoles were exposed to predator in the presence of refuge 3. Predator-experienced tadpoles were exposed to predator in the absence of refuge 17Experience of predators and accessibility to refuges minimize larval mortality of bronze frog 4. Predator-experienced tadpoles were exposed to predator in the presence of refuge Each treatment consisted of 30 trials (overall 120 tri- als). For each trial the test tadpoles were released (n = 30) into the plastic tub (32 cm diameter and 14 cm deep) containing 3 L of aged tap water and with or without the structural refuge and allowed to familiarize themselves to the tub for 15 min. Then one water scorpion starved for 48 h was introduced gently into the tub and left there. The trials were terminated after 24 h. The number of sur- viving tadpoles in various trials was recorded to compute the number of tadpoles lost due to predation. Data were analyzed using two-way analysis of variance for assess- ing the overall consequence of a prior exposure to preda- ceous cues and the access to the refuge and their interac- tion on prey survival. Data on number of tadpoles con- sumed between the two treatment groups were analyzed by independent-samples t test. Two-way ANOVA showed significant main effect of predator exposure (P < 0.001, Table 1) and refuge avail- ability (P < 0.001, Table 1) but not of their interaction (P = 0.253, Table 1). Accessibility to the refuge significantly reduced the larval mortality in predator-naïve tadpoles when com- pared to that without refuge (t58 = 4.402, P < 0.001, Fig. 1A). A similar trend was also evident in the case of pred- ator-threat experienced tadpoles (t58 = 5.648, P < 0.001, Fig. 1B). The larval mortality was significantly lower in predator-experienced tadpoles compared to predator- naïve tadpoles regardless of the availability (t58 = 4.144, P < 0.001) or unavailability (t58 = 4.353, P < 0.001) of ref- uge sites (Fig. 1). The present study shows the importance of avail- able shelters and of a prior experience with predator in evoking defensive behaviors and enhances escape pre- dation in H. temporalis tadpoles. Both these factors independently and not conjointly affect the larval sur- vival following their encounter with water scorpion. The results reveal that accessibility to refuge sites and prior experience of predation threat are key determinants of survival from the aquatic insect predators in H. tempo- ralis tadpoles. Previous studies have shown that bronze frog tadpoles exhibit antipredator defense behaviors in response to water borne chemical cues of predator by staying away from the predator and reducing their swim- ming activity (Mogali et al., 2012). Our study revealed a heightened defense response in predator-experienced tadpoles in the subsequent encounters with the predator (Mogali et al., 2012) in conformity with the observations reported on other anuran species (Semlitsch and Reyer, 1992; Hettyey et al., 2011). The results of the present study show that predator-experienced tadpoles learn to escape predation and become less vulnerable to predator compared to the predator-naive individuals regardless of the accessibility to structural refuge. This clearly dem- onstrates that prior experience with predator plays a key Table 1. Results of two-way ANOVA for exposure and refuge avail- ability and their interactions. The response variable is mean number of Hylarana temporalis tadpoles lost due to predator. Significant val- ues are bolded. Source df MS F P Exposure 1 108.300 35.382 <0.001 Refuge availability 1 132.300 43.222 <0.001 Exposure × refuge availability 1 4.033 1.318 0.253 Fig. 1. Number of predator-naïve and predator-experienced Hylar- ana temporalis tadpoles consumed by the predator, Laccotrephes sp., in relation to accessibility to refuges in trials of 24 h. An asterisk over the bar indicates a significant difference between two treat- ments. Data represents x ± SE. Each trail consists of 30 tadpoles of either predator-naïve or predator-experienced. 30 trials were carried out for each treatment and total all together 120 trials. 18 Santosh Mogali, Bhagyashri Shanbhag, Srinivas Saidapur role in enhancing survival chances of tadpoles by escap- ing predation. Healey and Reinhardt (1995) made simi- lar observation on Coho salmon against the predacious Rainbow trout. Álvarez and Nicieza (2006), who studied Rana temporaria tadpoles, showed that 48 h associa- tion with the predator was a sufficient period of time to enhance their ability to escape predation. The present study on H. temporalis shows that even mere 8 h expo- sure to predatory cues is sufficient to evoke a stronger effective defense behavior on subsequent encounters with the predator. Possibly, even a shorter time expo- sure might be sufficient for prey tadpoles to evoke their defensive behavior on following encounters with preda- tors. However, additional studies are needed to establish the minimum period of exposure to predators that is required to condition the prey and modify their future performance of defensive behaviour. Further, releasing predacious cues that remain effective for longer periods may not be in the interest of the predator. 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