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Resumo
A ocorrência de sinalização de alarme por meio de pistas químicas liberadas por dano tecidual pode ser influenciada por diversos fatores, entre os quais o microhabitat ocupado e o tamanho ou idade do organismo. Entre os ofiuróides, Ophiactis savignyi faz parte de um sistema perfeito para se testar esse tipo de influência, uma vez que pode ser encontrado associado a diferentes espécies de algas e esponjas. O reconhecimento intraespecífico do sinal de alarme foi testado em diferentes concentrações para ofiuróides de tamanho semelhante coletados na alga Amphiroa beauvoisii e na esponja Amphimedon viridis. Dentre os indivíduos coletados em alga foi observada uma resposta intraespecífica para a maioria das concentrações testadas (100 a 12,5%), enquanto que nos ofiuróides coletados em esponjas não foi observado reconhecimento do sinal. Testes interespecíficos realizados com ofiuróides coletados em algas demonstraram um amplo reconhecimento frente a todos os estímulos (Amphipholis squamata, Ophiothrix angulata e Ophionereis reticulata), e total ausência de resposta para aqueles coletados em esponjas. O amplo reconhecimento em indivíduos associados a algas parece resultado de uma maior pressão de predação nesse tipo de hospedeiro, provavelmente devido a maior exposição dos epibiontes quando comparado aos associados a esponjas, além de uma maior diversidade de ofiuróides. Isto resultaria em um maior número de oportunidades de aprendizagem, com os indivíduos capazes de reconhecer indicadores de risco sendo selecionados. Este tipo de plasticidade de resposta, frente às pressões de predação, pode ser uma das características de O. savignyi que torna possível sua ampla distribuição, uma vez que indivíduos dispersos para diferentes localidades seriam capazes de se ajustar a novas guildas de presas, apresentando uma vantagem frente a novos predadores.
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Abstract
The signaling by damage release alarm cues may be influenced by several factors among these, the occupied microhabitat and age or body size of the organism. In brittle stars, Ophiactis savignyi provides the perfect system to test this influence, since it can be found associated with several types of host in different stages of its development. Considering that, the conspecific recognition of this kind of signal was tested in algae and in similar size sponge sampled individuals, for several concentrations. In alga sampled brittle stars recognition was observed in dilutions 100 to 12.5% while sponge sampled individuals presented no response. Since individuals of similar sizes collected in algae and in sponges presented different responses, it was possible to demonstrate that recognition was not size related. Subsequent heterospecific tests demonstrated a broad recognition, to all species signals (Amphipholis squamata, Ophiothrix angulata and Ophionereis reticulata), in alga sampled brittle stars while sponge sampled individuals presented none. Both conspecific and heterospecific alarm signal recognition in alga sampled O. savignyi may be the result of a higher predation pressure in this host, due to their higher exposition when compared with those associated with sponges, added with the occurrence of other brittle stars species. This would result in learning opportunities for recognition of the signal, with animals that were able to react to alarm signals being selected. The same conditions are not truth in the sponge host, where other brittle stars do not co-occur close with O. savignyi and the principal predators group is usually physically separated from it. This plasticity could be one of the species characteristics that makes possible its broad distribution, since dispersing individuals would be able to adjust to new guild members and cope with new predators.
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Introduction
Among echinoderms, the defenses against predation may base in several traits, such as morphology, chemistry and behavior (Bryan et al., 1997; Sköld, 1998). These adaptations can be categorized as: (1) avoidance adaptations that limit the potential number of encounters with predator; and (2) escape adaptations that reduce the risk of consume when a predator has been detected or encountered (Legault & Himmelman, 1993). In this last category are the damage released alarm substances that provide an early warning about predation risk. These cues result in increased vigilance, during which the animal may take fewer risks by altering its microhabitat use or behavior, therefore increasing its chance of survival, what represents a clear benefit for the prey (Kiesecker et al., 1999; Chivers et al., 2002; Pollock et al., 2003).
Co-occurring species that share predators and that are able to recognize both conspecific, as heterospecific alarm substances, will be better informed about the predation risks. This represents the predicted scenario for the prey guild hypothesis, where different prey species are considered as the shared resource of some local predators (Mirza & Chivers, 2001). Considering prey guild hypothesis and given the generalized diet of many predators it is reasonable to predict that prey would be selected to utilize any information that indicates elevated level of predation. One of the factors that determine this kind of recognition is the phylogenetic relationship of the species (Hazzlet & McLay, 2005). The other one is learning, and since that, it is directly influenced by the potential opportunities of association (Mirza et al., 2003, Pollock et al., 2003).
However, the intensity of the response may be influenced by the age or size of the emitter animal. Animals of the same age/size of the emitter can respond strongly to their cues than to those released by older conspecifics, although still
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recognizing their alarm signal (Mirza & Chivers, 2002). The occurrence of this recognition, as the resulting behavioral response, may also be affected since the experienced predation pressure may be different in the subsequent life stages. Many species undergo ontogenetic shifts in habitat and/or diet during their life cycle. This alteration is particularly clear in fishes that change their diet based, for example, initially on invertebrates, to piscivory. In this case, it has been verified that the same signal identified by young fishes as alarm signal can become foraging cues for adults (Brown et al., 2001). This shift are expected when any survival advantage gained from antipredator response are outweighed by potential foraging benefits (Golub & Brown, 2003).
Among brittle star it has been verified alarm signaling (chapter 2), being the conspecific and heterospecific recognition apparently influenced by the occupied microhabitat and the interactions among species. However, to ensure that microhabitat, and the consequent evolutionary pressures associated with it, are influencing the observed occurrence or absence of alarm signal recognition it is necessary the use of different populations of a same species occurring in different microhabitat and therefore, exposed to distinct interactions and predation pressures. This is the case of Ophiactis savignyi that can be associated both with sponges and algae (Hendler et al., 1995). Sponges may provide both a physical and chemical refuge, especially from fishes. Additionally, in this host O. savignyi usually occurs alone in the internal folds of the sponge’s body, with other brittle star species occurring eventually only in the outer layer. On the other hand, in algae this brittle star usually co-occurs with several other species, even if only as juveniles. Apparently, algae are also more exposed refuges for brittle stars than sponges, especially for small sized predators that also occur associated to these organisms, as
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many species of small Xanthidae crabs (personal observation). However, the size structure of this brittle star in these different groups of hosts is profoundly distinct in the sampled area. In sponges it is observed a wide variance of sizes while in algae only small and medium sized brittle stars are collected (unpublished data).
Large adults of O. savignyi sampled in sponges has already been shown not to respond to conspecific damage release alarm signal. However, since other species of brittle star in the area present conspecific and/or heterospecific chemical alarm recognition, this lack of response may be size dependent, or even result of the lack of learning opportunities, caused by a reduced or null predation pressure in sponges. If the prey guild hypothesis is applicable for this brittle star, those animals collected in algae will present both conspecific and heterospecific recognition of damage release alarm signal. However, if the recognition is size-dependent and genetic based, it would be expected that small to medium sized animals sampled in both kinds of hosts would present an escape response to alarm cues. To elucidate these questions, it was investigated the applicability of the prey guild members hypothesis for O. savignyi and the influence of size in the alarm signal recognition.
Methodology
Sampling of brittle stars was performed in São Sebastião Channel, São Paulo, Brazil (S 23º50’ - W 45º24’). Individuals of Ophiactis savignyi (Ophiactidae) were collected in the sponge Amphimedon viridis and in the alga Amphiroa beauvoisii, being separated according to the host. After sampling, all brittle stars had their longest arm measured and those with sizes between 3.5 mm and 13 mm were selected for the
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experiments. They were kept separated in aquaria with aerated seawater for at least three days prior the experiments.
The stimulus used to test the occurrence of damage released alarm signal was prepared through homogenization of brittle stars. For each 0.1 g of animal 1mL of filtered sea water was added. The homogenized was then centrifuged for 20 minutes at 2000 rpm. The supernatant was removed and this stimulus (referred as D0) was diluted in five additional concentrations: D1 – 25.00%, D2 – 12.50%, D3 – 6.25%, D4 – 3.12% and D5 – 1.56%. These stimuli, as the control filtered seawater, were aliquoted and frozen to -20ºC until the tests.
The methodology used to test the stimuli was described in chapter 2. The differences in time spent during the escape response were tested through an one-way ANOVA for each species. A Dunnet test was performed to compare the control with the additional treatments when ANOVA was significant with p ≤ 0.05. Since data did not meet normality or homoscedasticity, a logarithmic transformation was applied.
Results
In the concentration tests for alga sampled O. savignyi it was observed a fastening response from D0 to D2 (Table 1 and Figure 1a). However, beside the significant result of the ANOVA, no difference between response to dilutions and control was observed in the tests performed with sponge sampled individuals (Table 1 and Figure 1b). Being so, the additional heterospecific tests were performed in alga sampled brittle stars and the selected concentration was the smaller one with a significant response, in this case D2 (12.50%).
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Table 1. Result of the one-way ANOVA comparing the time of response (seconds - log transformed) of O. savignyi individuals sampled in alga (A. beauvoisii) or sponge (A. viridis) to tested conspecific dilutions and heterospecific stimuli.
Source of variation Df MS F p
Dilution and conspecific test Alga sampled brittle stars
Intercept 1 1228.71 8487.66 <0.0001
Stimuli 6 0.70 4.831 0.0001
Error 203 0.15
Sponge sampled brittle stars
Intercept 1 1216.64 6436.32 <0.001
Stimuli 6 0.44 2.33 0.04
Error 168 0.19
Heterospecific tests
Alga Sampled brittle stars
Intercept 1 891.39 4113.64 <0.0001
Stimuli 3 2.46 11.37 <0.0001
Error 96 0.22
Sponge sampled brittle stars
Intercept 1 1160.50 4066.49 <0.0001
Stimuli 3 0.38 1.32 0.273
Error 96 0.29
In the heterospecific tests, alga sampled O. savignyi presented a positive response for all tested stimulus (Table 1 and Figure 2a). On the contrary, sponge sampled brittle stars did not respond to any of the tested stimuli (Table 1 and Figure 2b).
72 Figu re 1 - T im e of res pon se i n s ec o n d s of O. sa vigny i to da ma g ed rel eas ed al ar m sig n al (c ontr o l and tr ea tm e n ts at d iff erent c onc entr ati o ns of co nspecif ic sti m u lus - D 0 = 1 00%, D 1 = 2 5 % , D 2 = 12.5 % , D3 = 6. 25 %, D 4 = 3. 12%, D 5 = 1.5 6 %). a - al ga sa m p le d bri ttle stars, b - sp ong e sa m p led b rittle st ars . Figu re 2 - Ti me of re sp onse in second s of O. sa vigny i to heter o specific d a m age released al arm si g n al. a - alga sa m p le d b rittle st ars, b - spong e sa m p le d b rittl e star s. Bar s r epr es ent 0. 95 c onfi d e n ce int erval s, * * p ≤ 0 .001 .
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Discussion
The absence of an alarm signal response by sponge brittle stars while it occurred in those collected in algae shows that the recognition is influenced by the occupied host. The alga A. beauvoisii and the sponge A. viridis have several differences and these concerns also the associated fauna. Therefore, in each host, brittle stars experienced different interactions with both predators and guild members. For example, in the sampled alga O. savignyi co-occurs with other brittle stars, as Amphipholis squamata, Ophiactis lymani and juveniles of Ophiothrix angulata. In this host, these species usually present more similar sizes (unpublished data) and since its principal predators use to be generalists, in the area fishes and crabs (Santos, 2005 and see in Hendler et al., 1995) these brittle stars are exposed to the same predators. Therefore these species belong to the same prey guild (Mirza & Chivers, 2003) and those individuals that are able to perceive alarm signals will clearly have an advantage since they will have more time to escape an imminent danger. If this recognition occurs to both conspecific and heterospecific signals the brittle star will have even more information to assess predation risk, allowing a flexible antipredator response (Mirza & Chivers, 2001).
On the other hand, in the sponge host O. savignyi will not really co-occurs with other species, since when these are present, they are usually found in the outer layer of the sponge and not in the internal folds as this brittle star. The same occurs with crabs, brittle stars predators (Boffi, 1972), that despite are also found associated with sponges, usually occur in the area where its basal lamina attach with the rock (unpublished data). Therefore, sponges appear to be an efficient refuge for O. savignyi and since that, these animals will not be so exposed to predation unless its association is temporary or
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occasional, what do not seem to be the case. Although there are no studies about the host fidelity of O. savignyi this fact has been verified for Ophiothrix lineata in association with the sponge Callyspongia vaginalis (Henkel & Pawlik 2005). Assuming the fidelity to the host, O. savignyi could be physically sheltered from predators even during its entire lifetime. This would represent an extreme reduction in the predation pressure and could explain the lack of alarm signal recognition. Additionally, the escape behavior to alarm signals or kairomones has an associated cost that may affect survival, reproduction and growth (Peckarsky et al., 1993). Since this kind of recognition would not be expected unless it provided a survival improvement or at least were neutral. That’s why a flexible mechanism of recognition would be advantageous, since the costs of associated defense would only incur when the threat of predation was high, in this case in alga host and not in sponge.
Since the sampled sponge and algae occurs in the same area, and not unusually as a continuum, and considering that O. savignyi reproduces sexually with a larval phase, due to the proximity it seems improbable that the gametes of sponge and alga hosts don’t fertilize each other. This cross fertilization would result in individuals with similar response to alarm signal if this kind of recognition was genetic based, what is not the case. An alternative mechanism usually associated with alarm signal recognition is learning and this seems to be the responsible for the observed response in O. savignyi. In this case the occurrence of alarm signaling will depend on the learning opportunities that will be directly influenced by predation pressure (Pollock & Chivers, 2004). On the other hand, considering the close co-occurrence of predators as crabs in the utilized alga, O. savignyi collected in this host would be exposed to plenty learning opportunities, as other prey guild members. This would explain the broad recognition of both conspecific and heterospecific signals. A similar result, but not in terms of
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intensity, has been observed by the fish Culea inconstans however towards a kairomone. In this case, individuals from populations that frequently encountered predator typically showed stronger antipredator response than individuals from populations that rarely experienced predators (Gelowitz et. al, 1993).
The only signal recognition that does not perfectly fit in guild members hypothesis is that presented towards O. reticulata signal. This brittle stars do not co- occurs with O. savignyi and is really larger than those individuals associated with algae (O. reticulata arms can grown over 120 mm (Hendler et al., 1995) while in the area, the arm of the larger algae sampled O. savignyi measured 19.8 mm) therefore they cannot be considered as belonging to the same prey guild. However, O. reticulata is an occasionally brittle stars predator (Yokoyama, 2005), and it could be considered that the response to its signal may represent the recognition of a known predator, a kairomone. However, it is not possible to exclude that the recognition of the signal of more than one species may result in the recognition of all others, due to some similar compound. To elucidate this point more tests are necessary.
Another interesting point is the concentration spectrum to which a response was observed. In alga sampled O. savignyi this recognition was observed until the dilution of 12.5% while in other tests performed with A. squamata and O. reticulata this happened until 3.12%. This difference may be due to the frequent asexual reproduction of O. savignyi that happens, as in other fissiparous brittle stars, through the middle of the disc, separating the brittle star in two halves. The healing of the torn parts in Ophiocomella ophiactoides, another fissiparous brittle star, proceeds while fission is underway. This separation apparently happens in 24 hours, however it may takes longer if the animal is perturbed (Mladenov et al., 1983). This process seems similar in O. savignyi and during it is probable that at least some internal contents are released to the
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exterior and this would probably be similar to the tested stimulus. Therefore, since asexual reproduction happens throughout the year and will be constantly releasing a small amount of these compounds, it may be that fissiparous species respond only to higher concentrations of alarm signal than those with other reproductive strategies.
Several characteristics in O. savignyi, as its dual capacity for sexual and asexual reproduction and the dispersion ability by larvae or rafting, are considered as primordial to its widespread distribution (Hendler et al., 1999 and see in Hendler et al., 1995). It is generally assumed that species that more readily extend their ranges tend to be more flexible in many aspects of their biology (Schweitzer & Larson, 1999). It would be this feature that would make this species able to cope with new environmental features, including new predators (Hazlett et. al, 2003). With our results it was possible to observe the flexibility of O. savignyi in alarm signal recognition according to the surrounding characteristics, in this case the interactions experience by brittle star in its chosen host. This ability may be one of O. savignyi characteristics that allows or at least facilitates the occupancy of new areas.
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