Parental females of a nest-brooding cichlid improve and benefit from the protective value of young masquerading as snails

Animal Behaviour 124 (2017) 75e82

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Parental females of a nest-brooding cichlid improve and benefit from the protective value of young masquerading as snails Shun Satoh a, *, Tetsumi Takahashi b, Shinya Tada c, Hirokazu Tanaka a, Masanori Kohda a a

Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Osaka, Japan Institute of Natural and Environmental Science, University of Hyogo Prefecture, Hyogo, Japan c Graduate School of Science and Engineering, Ehime University, Ehime, Japan b

a r t i c l e i n f o Article history: Received 17 March 2016 Initial acceptance 10 May 2016 Final acceptance 14 November 2016 MS. number: 16-00239R Keywords: Lake Tanganyika cichlid masquerade Neolamprologus furcifer removal experiment Reymondia horei

Masquerade is a strategy whereby prey animals resemble environmental objects (e.g. twigs, bird droppings and stones) or inedible animals to avoid predatory attack. However, most studies of this strategy have been restricted to only a few animal groups. Therefore, novel examples are required to elucidate the diversity of masquerade strategies. Neolamprologus furcifer is a maternal nest-brooding cichlid inhabiting shaded areas of large rocks in Lake Tanganyika. In contrast to the cryptic, dark brown adults, the small young have well-defined white stripes on their brown trunk in nest territories where many whitestriped snails Reymondia horei are present. We hypothesized that young N. furcifer masquerade as the model snail R. horei. We found four results consistent with our hypothesis. (1) The size, coloration, shape and posture of the young fish resembled those of the snail. (2) Experimental removal of the model snail from territories caused females to attack predators more frequently than before the removal, indirectly suggesting the protective value of the young fish's coloration. (3) Parental females selectively removed nonmodel snails from their territory, probably causing the higher density of R. horei observed at nest sites. (4) Guarded young had black/white coloration only in populations where the model snails were present. Therefore, we suggest that young N. furcifer masquerade as the model snail R. horei. This study describes a novel masquerade pattern in which the protective value is improved by, and beneficial to, a third party. © 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Animals have evolved a number of survival strategies using coloration (Caro, 2005; Skelhorn, 2015; Skelhorn, Rowland, & Ruxton, 2010; Skelhorn, Rowland, Speed, & Ruxton, 2010; Stevens & Merilaita, 2009), including crypsis (avoiding detection by a predator), aposematism (warning coloration) and mimicry (resembling a defended organism), which have been studied intensively for many years (Edmunds, 1981; Ruxton, Sherratt, & Speed, 2004; Vane-Wright, 1980). Some animals closely resemble environmental objects (e.g. stones, dead leaves, twigs and bird droppings) or other living organisms that are free from the risk of predation, thereby avoiding predation due to misidentification as inedible objects, in a strategy known as a masquerade (Endler, 1981; Skelhorn, Rowland, Speed, & Ruxton, 2010). Masquerade differs from crypsis or camouflage strategies, such as background matching and disruptive coloration, in which

* Correspondence: S. Satoh, Department of Biology and Geosciences, Graduate School of Science, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi, Osaka City, Osaka, 558-8585, Japan. E-mail address: [email protected] (S. Satoh).

predators fail to detect the prey (Cuthill et al., 2005; Ruxton et al., 2004; Skelhorn, Rowland, & Ruxton, 2010; Stevens & Merilaita, 2009). In some cases, however, masquerade is not easy to distinguish from camouflage. For example, it is considered masquerade when predators detect a prey item that resembles a stone and misidentify it as such, whereas when predators fail to detect the same prey in stony habitats, it is deemed camouflage. The benefits of resemblance for masquerading prey animals are intuitively obvious in many examples, but there is scant experimental evidence for masquerade functions, i.e. reduced risk of predation (Skelhorn, 2015; Skelhorn, Rowland, Speed, & Ruxton, 2010). Well-studied examples of prey masquerades include moth caterpillars that resemble bird droppings (Liu, Blamires, Liao, & Tso, 2014; Wagner, 2005) and twigs (Skelhorn, Rowland, Speed, & Ruxton, 2010; Skelhorn & Ruxton, 2010, 2011, 2012). These masquerading animals resemble their model objects not only in coloration but also their size (Skelhorn, Rowland, Speed, & Ruxton, 2010; Skelhorn, Rowland, Speed, Wert et al., 2010; Valkonen et al., 2014) shape and position (Skelhorn, 2015; Suzuki & Sakurai, 2015). After a study identifies a masquerade, its protective value should be

http://dx.doi.org/10.1016/j.anbehav.2016.12.001 0003-3472/© 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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S. Satoh et al. / Animal Behaviour 124 (2017) 75e82

examined by experimental manipulation; however, such studies have been limited mostly to a few animal groups such as caterpillars (Skelhorn, 2015; Skelhorn, Rowland, Speed, & Ruxton, 2010; Suzuki & Sakurai, 2015). Other aspects of masquerades, such as the frequency of both masquerading animals and model objects in microhabitats, have not been well studied. For example, masquerading animals may occur more frequently in microhabitats where model objects are abundant. This correspondence may occur by the active microhabitat selection of the masquerading animal (Skelhorn & Ruxton, 2013, 2014), but the process inducing the phenomenon is not well documented. Studies of these aspects are still limited to a few types of animals, and novel examples of masquerade strategies are required to elucidate these phenomena. In nest-brooding cichlids in Lake Tanganyika, young fish are vulnerable to predators while in the nest and often develop disruptive coloration (Maan & Sefc, 2007; Stevens & Cuthill, 2006) and camouflage (Kuwamura, 1997). The nest-brooding cichlid Neolamprologus furcifer inhabits flat surfaces of shaded rocks on which females guard the young (Yanagisawa, 1987). However, young N. furcifer possess well-defined white stripes that contrast with their dark brown body (Fig. 1a and b), which seems easier for predators to detect on shaded rocks than an entirely dark brown coloration; this is at least true for the human eye. Interestingly, in and around nesting sites we found the snail Reymondia horei (Bandel, 2006; Konings, 1998), which has similar white stripes on its brown shell (Fig. 1c and d). These young cichlids thus resemble

R. horei when stationary on the nest surfaces. Therefore, we hypothesized that young N. furcifer fish may masquerade as the snail R. horei, which are not preyed on by predatory fishes. In this study, we tested this hypothesis by removing the model snails to examine their protective value. If young fish under parental care benefit from their visual appearance by masquerading, then parents that guard their broods against predators will also benefit from this effect via reduced costs for brood guarding. Therefore, it is plausible that parental fish will try not only to defend their brood physically against predators, but also to increase the density of models in the nest to improve the efficacy of the hypothesized masquerade. To the best of our knowledge, this would be the first example of parents both improving the protective value of masquerading young and benefiting from that masquerade as a third party. If masquerade coloration is restricted only to populations where the models are present, this will provide further evidence of the masquerade (e.g. Skelhorn, Rowland, & Ruxton, 2010; Suzuki & Sakurai, 2015). We found that young of N. furcifer assumed an entirely dark brown coloration in some localities along the shore of Lake Tanganyika. Therefore, we studied local variation in coloration of the young of N. furcifer and the presence of the model snail R. horei at five localities. METHODS Studied Species

(a)

(c)

(b)

(d)

i

5 mm 1 mm

(e)

ii

5 mm

5 cm (f)

iii

5 mm

Neolamprologus furcifer (female parent standard length, SL 7e10 cm), a substrate-brooding cichlid, inhabits shallow rocky shores ubiquitously in Lake Tanganyika (Brichard, 1979; Hori, Yamaoka, & Takamura, 1983). The cichlids dwell on shaded vertical or overhanging flat surfaces of large rocks, on which they breed and forage for small shrimps. They have a harem polygyny mating system and females care for the young in the nest (breeding territory, 1e1.5 m in diameter) on the flat surfaces of dark shaded rocks (Yanagisawa, 1987). Parental females spawn on the nest surface, where the hatched young are scattered and defended by the female against approaching predators for 2.5 months until the young become independent (Yanagisawa, 1987). Before independence, the young have well-defined white stripes on the dark brown body (SL 6e30 mm, Fig. 1a and b). Young on the nest surfaces feed on copepod nauplii when they are 10e20 mm SL, but shift their diet to small benthic shrimps when they measure 20e30 mm SL (Yanagisawa, 1987). When the young become independent (30e35 mm SL), they lose the white stripes on the trunk (S. Satoh, personal observation). The snail R. horei possesses five or six whorls with white stripes (Bandel, 2006; Fig. 1c,d) and is common on rocky surfaces covered with fine silt at the southern end of Lake Tanganyika (Konings, 1998). We observed them on the nest surfaces of female N. furcifer, as well as small numbers of other snail species: Lavigeri grandis, Lavigeri spinulosa, Lavigeri nassa and three unknown species. These other species were beige to brown and overlapped in size with the model snail R. horei. None of the snails appeared to be food items for predatory fishes that feed on young fish.

5 mm Field Observations Figure 1. Photographs of a young Neolamprologus furcifer and snails found from breeding territories. (a) Neolamprologus furcifier young on the nest surface, (b) sketches of young N. furcifer at different sizes, (c) the white-striped snail Reymondia horei on the nest surface and (d) a representative R. horei individual. (e) The other snail species near the territories of N. furcifer. (f) Lavigeri grandis, the snail species removed by females from their territories. Photographs of fish and snails on shaded nest surfaces were taken under artificial lighting.

To test our hypothesis that young N. furcifer masquerade as the snail R. horei, we conducted field observations using SCUBA and collected samples at Wonzye near Mpulungu, Zambia, from September to December 2014. At our study sites, the water depth was 3e8 m with large, scattered rocks, which were inhabited by

S. Satoh et al. / Animal Behaviour 124 (2017) 75e82

many N. furcifer individuals. Numerous breeding territories with young were easily observed on the vertical and overhanging shaded surfaces of flat rocks. We randomly selected nine territories with young, each isolated >4 m from the nearest neighbouring territory. We recorded the numbers and SLs of the young, which were estimated underwater to the nearest 1 mm by comparing them to a ruler attached to the substrate (N ¼ 184; one 36 mm SL fish was considered exceptionally large and omitted from the statistical analyses). To consider the relationship between masquerading young and model animals, we placed quadrats (50  50 cm2) in nine territories to examine the snail species composition and the density of individuals at three sites: the centre of the territory, near the territory (on plots 1 m from the territory border) and well away from female territories (on shaded rock surfaces >4 m from the territories). All snails inside the quadrat were collected using mesh nets. The specimens were identified and counted, and the shell height (length from bottom to top) was measured to 0.1 mm in the laboratory. Photographs of young fish and snails were acquired using a digital camera (HDRGWP88; Sony, Tokyo, Japan) with a light (FIX WaterShot V1800, Fisheye, Tokyo, Japan). In the early period of the field study, we observed parental females picking up and removing snails, and depositing them 1e3 m from the territories using their mouths. The removal of snails by Tanganyikan cichlids is associated with their foraging ecology, e.g. to remove food competitors and to find small prey under large snails on sandy bottoms (Karino, 1998; Yuma, 1994). However, the snail removal by N. furcifer females appeared to be unrelated to their feeding ecology. Therefore, we recorded all these unique behaviours while observing 22 females for a total of 645 min. We collected the snails that were deposited by the females, and identified them in the laboratory. Field Experiments Experiment 1: effect of snail removal on guarding behaviour To determine the protective value of masquerading as the model snail R. horei, we experimentally removed this snail from female territories. If removal incurs a higher predation risk for the young, then the aggression level of the females against predators should increase immediately to compensate for the reduction in protection afforded by the masquerade. Otherwise, the number of young would decrease significantly. Therefore, in this experiment, we focused on the reactions of female parents to the predators of young fish. The attacks by females against approaching fish were video-recorded by a camera (HDR-GWP88; Sony, Tokyo, Japan) set 2 m in front of a vertical rock surface to cover the whole territory. Before snail removal, we video-recorded the attacks by females for 10 min in 15 territories where the young were visually estimated as measuring 22e25 mm SL. After these video recordings, all R. horei in and around nine territories were removed using tweezers. We predicted that the removal of the model snails would decrease the protective value of the masquerade and increase the attack frequencies of female parents. On the next day, we again recorded the attacks by the females for 10 min. In a control experiment, we pretended to pick up snails from the rock surfaces using tweezers in the six remaining territories for 5 min, which resembled the experimental removal process. On the next day, we recorded the attacks by females in these territories in the same manner (N ¼ 6). Experiment 2: effect of snail removal on survival of young We performed a removal experiment in 18 other territories to examine how the presence of model snails affected the survival rates of young fish (and therefore the protective value of the masquerade). Reymondia horei were removed after counting the

77

young N. furcifer in and around nine territories. We then counted the young every other day for the 8 days following the removal. In a control experiment, we counted young as in the other nine territories, but the model snails were not removed. We predicted that the number of young in broods with experimentally removed model snails would not be different from the control because guarding females might invest significantly more effort in defending broods from predators. The size and number of young were not different between the experimental and control groups; i.e. the mean SL ± SE of the young was 21.8 ± 1.4 mm in the nine experimental territories versus 21.3 ± 1.7 mm in the nine control territories (ManneWhitney U test: U ¼ 39, N1 ¼ 167, N2 ¼ 185, P ¼ 0.97). The number of young did not differ between the experimental territories (18.6 ± 2.2) and control territories (13.8 ± 1.4; U ¼ 42, N1 ¼ N2 ¼ 9, P ¼ 0.92). We tried to perform an experiment by increasing the number of model snails in the female territories, but it was difficult to artificially place the snails on vertical or overhanging surfaces, and, therefore, this trial ended in failure. Experiment 3: posing of young in response to predators Posing as model animals may have a high protective value for masquerading animals (Suzuki & Sakurai, 2015). Thus, we experimentally introduced the major predator, Lepidiolamprologus elongatus (N ¼ 1, SL ¼ 124 mm, Hori et al., 1983) to three female territories with young present. This fish was placed in a plastic bag (400  280 mm) with two floats about 0.6 m in front of female territories. The predator hovered in the bag. In a control experiment, a plastic bag of the same size with floats attached was presented to young fish in a similar manner and we observed the behaviour of the young fish. In these experiments, we captured movies for 5 min, with a ruler set on the rock surface (HDR-GWP88; Sony, Tokyo, Japan). We randomly selected young fish measuring 22e25 mm (SL) from the movie data and observed their movement distance on the surface for 1 min (experiment, N ¼ 30; control, N ¼ 32). We predicted that the young would remain motionless in the presence of the predators, whereas they would continue moving when a plastic bag was presented. Coloration in Young Fish and the Model Snail in Five Localities We examined the coloration of young N. furcifer and the model snail R. horei at four other sites (Katoto, Mutondwe, Kasenga and Isanga; each within 2e10 km of Wonzye) as we did at Wonzye and compared the data between the five localities. At the four additional localities, the guarding behaviours of female N. furcifer were similar to those of the Wonzye population. There were two types of R. horei: black-shelled and white-striped. However, although all small (<8 mm SL) N. furcifer fry had a black/white coloration similar to the fry of many black/white Tanganyikan cichlid species (regarded as disruptive coloration; Maan & Sefc, 2007; Stevens & Cuthill, 2006), the presence of white stripes in larger young (>10 mm SL) varied between populations. We examined the size at which the white coloration disappeared in young fish and compared the sizes between the five localities (see Statistical Analyses). Statistical Analyses The data from the experiments (removal of model snails, young mortality and experimental presentation of a predator fish) were analysed using generalized linear mixed models (GLMMs) with Poisson and Gaussian distributions; the territory site identity was included as a random factor. The error distributions of each model were determined by KolmogoroveSmirnov tests. The snail

S. Satoh et al. / Animal Behaviour 124 (2017) 75e82

composition was analysed using GLMM with Poisson error distribution and Bonferroni correction; the territory site was included as a random factor. The error distributions of each model were determined by KolmogoroveSmirnov tests. Differences in snail coloration were analysed using a GLMM with a binomial distribution, and the differences in young coloration between localities (Yes ¼ 0, No ¼ 1) were analysed using a GLMM with a binomial distribution, in which the territory site identity was a random factor. To elucidate the relationship between snail and young fish coloration, we fitted the presence of white stripes in young fish as a binomial response term (Yes ¼ 0, No ¼ 1) in a GLMM. In this model, because we detected significant differences in the coloration of snails and young fish between localities (see Results), territory site identities and localities (Katoto, Wonzye, Mutondwe, Kasenga and Isanga) were included as random factors in the GLMM. All explanatory terms were entered and dropped stepwise until only those terms that explained significant variation remained. Each dropped term was put back into the optimal minimum model to determine their significance levels. All twoway interactions were tested but the results are only presented if significant. We fitted the following potential explanatory terms: the trunk length of the young, the number of R. horei with white bands in a nest territory, and the frequency of R. horei with white bands in a nest territory (Vertical point ¼ 0, Overhanging point ¼ 1). The significance level was set at 0.05. All statistical analyses were two-tailed and performed using R (R Core Team, 2014) with the glmer function in the lme4 package. Results are reported as means ± SE. Ethical Note All experimental protocols were approved by the Animal Care and Use Committees at Osaka City University for Advanced Studies and adhered to the ASAB/ABS guidelines for the treatment of animals in behavioural research. Our field research was conducted with the Permission for Fish Research in Lake Tanganyika from the Zambian Ministry of Agriculture, Food and Fisheries and complied with the current laws in Zambia.

***

(a)

**

80

60

40

20

0 Number of individuals per 2500 cm2

78

**

(b)

*

20

15

10

5

0

80

(c)

***

60

***

40 ***

RESULTS Clear, contrasting black-and-white colours were observed in young fish measuring 6.0e31.0 mm SL (9.4 ± 5.2 mm, N ¼ 184). These fish had five white stripes on their trunk like those on the shell of R. horei, although the stripes were not clear in small fry (Fig. 1a and b). The shell heights of R. horei in female territories were 2.4e13.5 mm (9.2 ± 0.2 mm, N ¼ 244). The fish were larger than the snails, but the length of the trunk (trunk length) with the banded area (Fig. 1a and b) was 3.6e18.9 mm (SL, 6.6 ± 0.32 mm), which overlapped the shell height of the snail. There were significant differences in density of snail individuals between the centre of the territory, near the territory and areas well away from the territory (GLMM: R. horei: c2 ¼ 444.6, P < 0.001; other six snail species: c2 ¼ 32.86, P < 0.001). The density of R. horei in the centre of territories of female N. furcifer (44.0 ± 7.1 per 2500 cm2, N ¼ 9) was significantly higher than near the territory (26.0 ± 4.6 per 2500 cm2, N ¼ 9, GLMM with Bonferroni correction: c2 ¼ 444.6, P ¼ 0.003) and much higher than areas well away from territories (1.77 ± 1.16 per 2500 cm2, N ¼ 9; c2 ¼ 435.83, P < 0.001; Fig. 2a). In contrast, the other six snail species were less numerous at the centre of territories than near the territory (c2 ¼ 24.336, P ¼ 0.022; Fig. 2b), and

20

0 Inside territory

Near territory

Well away

Figure 2. Abundance of (a) model snails Reymondia horei and (b) all other snails inside the territory, near the territory and well away from the territory. Both appear in (c). The box plots show the median and 25th and 75th percentiles; the whiskers indicate the values within 1.5 times the interquartile range and the circles are outliers. GLMM: *P < 0.05; **P < 0.01; ***P < 0.001.

their densities near the territory and in areas away from territories did not differ significantly (c2 ¼ 0.096, P ¼ 0.76). Numbers of R. horei were much higher than those of all other snails inside the territories (c2 ¼ 373.53, P < 0.001) and near the territories (c2 ¼ 99.683, P < 0.001), but were lower in areas away from territories (c2 ¼ 51.735, P < 0.001; Fig. 2c). The total numbers of snails were much higher inside territories than outside (GLMM: c2 ¼ 215.58, P < 0.001); R. horei was abundant only inside territories (93% of snails inside territories).

S. Satoh et al. / Animal Behaviour 124 (2017) 75e82

Number of attacks per 10 min

10

150

Distance (cm/5 min)

Snail removal by female parents was observed 12 times during 645 min of observations; i.e. 1.12 removals/h per fish. The removed snails were L. spinulosa (N ¼ 6), L. grandis (N ¼ 2) and unknown species (N ¼ 4) (Fig. 1e and f). The model snail R. horei was not removed despite its high abundance inside territories. That is, females selectively removed nonmodel snails from territories (Fisher's exact probability test: V ¼ 0.678, P < 0.001). In experiment 1, after the removal of R. horei, female parents attacked approaching predators much more frequently than they did before removal (before removal: 3.8 times ± 0.4; after removal: 6.8 ± 0.6; N ¼ 9; Fig. 3). In contrast, after the control treatment, the frequency of attacks did not increase from that before manipulation (before: 3.8 ± 0.4; after: 3.6 ± 0.2; N ¼ 6). Therefore, there were significant differences (GLMM: c2 ¼ 2.472, P ¼ 0.013) in these interactions (c2 ¼ 2.116, P ¼ 0.034, Fig. 3). Following experiment 2, which examined the effect of removing the model snail on the mortality of the young, the mean number of young before snail removal (18.6 ± 2.2, N ¼ 9) decreased after 8 days (13.9 ± 1.4, N ¼ 9). The mean number of young in the control territories (20.6 ± 2.7, N ¼ 9) also decreased after 8 days (15.0 ± 1.6, N ¼ 9). The young in both cases decreased significantly (c2 ¼ 8.825, P ¼ 0.003), but there were no significant differences between them (c2 ¼ 0.284, P ¼ 0.594). Therefore, the mortality rates were not different between the experimental and control groups. The young N. furcifer foraged on small benthic animals and moved actively on nest rock surfaces inside the territories. In experiment 3, when we presented their major predator L. elongatus, the young immediately became motionless and remained still on the rock surface (Fig. 4). In contrast, when an empty plastic bag was presented in the same manner in the control, the young did not restrict their movement, and there were significant differences in the movement distances of the fish between the experimental and control treatments (GLMM: F ¼ 144.95, P ¼ 0.001; Fig. 4). At Kasenga and Isanga, R. horei did not have white stripes. The white-striped morph occurred more frequently at Katoto and Wonzye than at Kasenga and Isanga, but less so than at Mutondwe (Fig. 5a, Appendix Table A1). At Kasenga and Isanga, black/white

79

100

50

0

Experiment

Control

Figure 4. Difference in the distance moved by young Neolamprologus furcifer when experimentally presented with a predator and the control. The box plots show the median and 25th and 75th percentiles; the whiskers indicate the values within 1.5 times the interquartile range and the circles are outliers.

colour patches were observed only in small fry, which were coloured similarly to those at Wonzye (see Fig. 1b). However, the white coloration of young N. furcifer had mostly disappeared in slightly larger fish (trunk length < 10 mm; Fig. 5b, Appendix Table A2), and they lacked the white stripes. In contrast, at three other localities, the colour of the fry transitioned to white stripes (Appendix Table A2), and white-striped model snails were plentiful at these locations. An interaction between fish size and the frequency of white-striped R. horei affected the coloration of young N. furcifer (Table 1). The number of R. horei with white stripes inside territories also affected the colour pattern of young N. furcifer, but territory conditions did not (Table 1).

9

DISCUSSION

8

Most of our results support the hypothesis that young N. furcifer with white stripes on a dark brown trunk masquerade as the model snail R. horei in nest territories. The coloration, shape and size range of the fish trunk were similar to those of the model snail R. horei in the nest territories at the Wonzye study site (Fig. 1). Young fish ceased movements on rock surfaces in the presence of a predatory fish, which may represent a general antipredator behaviour. In the shaded rock surface breeding territories of N. furcifer, the black/ white coloration of fish would be more noticeable to predatory fish than a dark coloration (conspicuous for human observers at least) and was unlikely to serve as crypsis, camouflage or disruptive coloration (Skelhorn, 2015; Skelhorn, Rowland, & Ruxton, 2010; Skelhorn, Rowland, Speed, & Ruxton, 2010). Therefore, if this coloration is advantageous for avoiding predation risk, it should be referred to as a masquerade. After experimental removal of the model snails, we found that parental females attacked predators twice as frequently as they did before removal, and the control showed that this increase was not due to the experimental manipulation. The frequent attacks by females imply that the young would be at greater risk from predators in the absence of model snails. The increased attack frequency

7 6 5 4 3 2 1 0

Before

After

Figure 3. Difference in the number of attacks against predators per 10 min by female Neolamprologus furcifer parents before and after the removal of the snail Reymondia horei from nests (black dot) and in the control (white).

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S. Satoh et al. / Animal Behaviour 124 (2017) 75e82

(a)

Lake Tanganyika Burundi b

c c

Tanzania

Isanga

a

Mutondwe

Congo

Kasenga Wonzye

Zambia a

N

Katoto

5 km

(b) Katoto

a

a

Locality

Wonzye

Mutondwe

a

b

Kasenga

Isanga

b

3

6

9

12

15

18

21

24

Trunk length (mm) Figure 5. Relationship between young Neolamprologus furcifer and model Reymondia horei colour morph types at five localities (Katoto, Wonzye, Mutondwe, Kasenga and Isanga) along the shore of Lake Tanganyika. (a) The frequency of snails with and without white stripes (white and black, respectively, in the pie chart). (b) Because all fry (<8 mm trunk length) of N. furcifer had white/black patches, the probability (%) of the disappearance of white coloration as a function of size is shown: 5% (black dot), 50% (grey) and 95% (white) (for the methods used see the Statistical analyses section). The white patch and white stripes were difficult to distinguish and were therefore combined. Different letters indicate statistical differences between localities.

Table 1 Factors affecting the probability of young Neolamprologus furcifer having a masquerade coloration Explanatory terms

Estimate±SE

df

c2

P

Intercept Trunk length (TL) TL)Frequencya Numberb Frequencya Territory Overhanging Vertical

12.22±2.79 1.40±0.32 5.03±4.95 1.22±0.50 0.10±0.03 e Reference 0.41±0.96

e 1 1 1 1 1 e e

e 85.88 7.11 8.13 6.92 0.18 e e

e <0.001 0.004 0.008 0.310 0.674 e e

Significant terms are highlighted in bold. Territory site identity and locality are included as random factors. a Number of R. horei with white stripes divided by the total number of R. horei with and without stripes in the territory site. b Number of R. horei with white stripes in the territory site.

probably had two causes: (1) females became more sensitive to predators and/or (2) predatory fish approached nest territories more frequently than before the snail removal. Increased sensitivity of females to predators would certainly be indicative of a masquerade. A reduction in prey misidentification following the snail removal is likely to increase predator visit rates due to improved prey recognition, which would likewise indicate that the coloration of the young cichlids serves as a masquerade (Skelhorn & Ruxton, 2010). However, the increased visit rate of predators has alternative explanations, such as increased conspicuousness of young fish in the absence of the model snails (Skelhorn & Ruxton, 2014). These results suggest that the resemblance of young fish to model snails reduces predation risk and serves as a masquerade, but camouflage among abundant model snails cannot be excluded (Skelhorn, Rowland, Speed, & Roxton, 2010).

S. Satoh et al. / Animal Behaviour 124 (2017) 75e82

Females probably increase the frequency of their attacks following snail removal to compensate for the reduced efficacy of the masquerade. This explanation is consistent with our observation that the mortality rates of young fish were not different between experimental and control nests. More frequent attacks against predators require females to spend more time attending the nests, thereby reducing the time available for other activities such as foraging (Alonso-Alvarez & Velando, 2012; Clutton-Brock, 1991). The increased effort required for parental care will reduce female body condition during the 2.5-month care period and impose significant costs on current and future breeding activities (CluttonBrock, 1991). This suggests that caregiving females may benefit from the masquerade of young fish. In general, the protective value of a masquerade is higher in microhabitats in which model objects are abundant, and mobile masquerading animals select these microhabitats (Skelhorn & Ruxton, 2010, 2011). Therefore, for masquerading animals such as moth caterpillars (Wagner, 2005), mothers may broaden their ovipositing site choices (e.g. among plant species) if they can improve the efficacy of larval masquerades. Inside the territory of our study species N. furcifer, the model snails were much more abundant than outside the territories, whereas the density of other snails showed the opposite trend. The selective removal of nonmodel snails by females (1.1 snails/h) may be more important than site selection for maintaining this snail distribution. Indeed, there were no sites with high model snail densities outside nest territories. Typical territories of female N. furcifer contained about 250 R. horei individuals and 10e20 young fish of 20e30 mm SL. The large number of model animals may efficiently increase the protective value of a masquerade (Skelhorn, Rowland, Delf, Speed, & Ruxton, 2011; Skelhorn, Rowland, Speed, & Ruxton, 2010; Tinbergen, 1960). Why, then, did so many model snails occur inside nest territories (Fig. 2c)? Parental N. furcifer females also attacked nonpredatory fishes such as algal feeders at the nest site (Satoh & Kohda, 2016) as reported in other nest-brooding cichlids (e.g. Nakano & Nagoshi, 1990). These attacks may result in higher amounts of microalgae and fine organic silt, which are food sources for snails, as observed in other nest-brooding cichlids (Karino, 1998; Kohda & Takemon, 1996). This effect could explain the high snail abundance inside N. furcifer territories. The local colour variations of young N. furcifer and the model snails also supports our hypothesis (Fig. 5). Although the fry (<7 mm SL) assumed a black/white coloration at all sites, this coloration was different from the white stripes of slightly larger fish. This coloration in cichlid fry is regarded as disruptive coloration (Maan & Sefc, 2007; Stevens & Cuthill, 2006). At the three localities with white-striped model snails, large young N. furcifer had white stripes like those of the model snails, whereas at the two study sites with strictly brown R. horei, the large young lacked white stripes and instead were entirely brown. At all five localities, N. furcifer breed in similar shady rock surface microhabitats, and the presence of the model snails is a crucial factor affecting the occurrence of masquerading young (Table 1). The white stripes in large young may have evolved to resemble the appearance of model snails as a masquerade, but were probably not associated with other factors, such as a signal for intraspecific communication, in their specific habitats. When model snails were absent, parental females compensated for the reduced masquerade efficacy by investing more in nest guarding, with no apparent decrease in young fish mortality rates. Therefore, in this masquerading system, the females rather than the masquerading young appear to benefit from the masquerade. The

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females invest in improving the protective value of the masquerade by increasing the density of model snails. The costs of removing nonmodel snails about once per hour may be lower than the costs associated with increased nest defence. This represents a novel type of antipredator strategy among nest-brooding cichlids in which the young develop a variety of coloration. This is be also the first example of masquerade whereby a third party increases, and benefits from, the protective value of the masquerade. The present study suggests masquerade systems will be more diversified than documented previously, and future research on this subject will be interesting and fruitful. Acknowledgments We thank the members of the Laboratory of Animal Sociology, Osaka City University, for their fruitful discussions regarding this work. This study was financially supported by KAKENHI (Nos. 25304017, 26540070, 26118511 and 16H05773) to M.K. References Alonso-Alvarez, C., & Velando, A. (2012). Benefits and costs of parental care. In N. J. Royle, P. T. Smiseth, & M. Kolliker (Eds.), The evolution of parental care (pp. 133e149). Oxford, U.K.: Oxford University Press. Bandel, K. (2006). Families of the Cerithioidea and related superfamilies (PalaeoCaenogastropoda; Mollusca) from the Triassic to the recent characterized by protoconch morphology e Including the description of new taxa. Freiberger Forschungshefte C, 511, 59e138. Brichard, P. (1979). Unusual behaviors in Lake Tanganyika cichlids. Buntbarsche Bulletin, 74, 10e12. Caro, T. (2005). Antipredator defences in birds and mammals. Chicago, IL: University of Chicago Press. Clutton-Brock, T. H. (1991). The evolution of parental care. Princeton, NJ: Princeton University Press. Cuthill, I. C., Stevens, M., Sheppard, J., Maddocks, T., Parraga, C. A., & Troscianko, T. S. (2005). Disruptive coloration and background pattern matching. Nature, 434, 72e74. Edmunds, M. (1981). On defining ‘mimicry’. Biological Journal of the Linnean Society, 16, 9e10. Endler, J. A. (1981). An overview of the relationships between mimicry and crypsis. Biological Journal of the Linnean Society, 16, 25e31. Hori, M., Yamaoka, K., & Takamura, K. (1983). Abundance and micro-distribution of cichlid fishes on a rocky shore of Lake Tanganyika. African Study Monographs, 3, 25e38. Karino, K. (1998). Depth-related differences in territory size and defense in the herbivorous cichlid, Neolamprologus moorii, in Lake Tanganyika. Ichthyological Research, 45, 89e94. Kohda, M., & Takemon, H. (1996). Group foraging by the herbivorous cichlid fish, Petrochromis fasciolatus, in Lake Tanganyika. Ichthyological Research, 43, 55e63. Konings, A. (1998). Tanganyika cichlids in their natural habitat. Miami, FL: Cichlid Press. Kuwamura, T. (1997). The evolution of parental care and mating systems among Tanganyikan cichlids. In T. Kawanabe, M. Hori, & M. Nagoshi (Eds.), Fish communities in Lake Tanganyika (pp. 57e86). Kyoto, Japan: Kyoto University Press. Liu, M. H., Blamires, S. J., Liao, C. P., & Tso, I. M. (2014). Evidence of bird dropping masquerading by a spider to avoid predators. Scientific Reports, 4, 5058. Maan, M. E., & Sefc, K. M. (2007). Colour variation in cichlid fish: Developmental mechanisms, selective pressures and evolutionary consequences. Seminars in Cell & Developmental Biology, 24, 516e528. Nakano, S., & Nagoshi, M. (1990). Brood defence and parental roles in a biparental cichlid fish Lamprologus toae in Lake Tanganyika. Japanese Journal of Ichthyology, 36, 468e476. R Core Team. (2014). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. http://www.r-project.org. Ruxton, G., Sherratt, T., & Speed, M. (2004). Avoiding attack: The evolutionary ecology of crypsis, warning signals and mimicry. Oxford, U.K.: Oxford University Press. Satoh, S., & Kohda, M. (2016). Brood and territorial defence in substrate-brooding cichlid Neolamprologus furcifer endemic to Lake Tanganyika. Manuscript in preparation. Skelhorn, J. (2015). Masquerade. Current Biology, 25, 643e644. Skelhorn, J., Rowland, H. M., Delf, J., Speed, M. P., & Ruxton, G. D. (2011). Densitydependent predation influences the evolution and behavior of masquerading prey. Proceedings of the National Academy of Sciences of the United States of America, 108, 6532e6536.

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Skelhorn, J., Rowland, H. M., & Ruxton, G. D. (2010). The evolution and ecology of masquerade. Biological Journal of the Linnean Society, 99, 1e8. Skelhorn, J., Rowland, H. M., Speed, M. P., & Ruxton, G. D. (2010). Masquerade: Camouflage without crypsis. Science, 327, 51. Skelhorn, J., Rowland, H. M., Speed, M. P., Wert, L. D. W., Quinn, L., Delf, J., et al. (2010). Size-dependent misclassification of masquerading prey. Behavioral Ecology, 21, 1344e1348. Skelhorn, J., & Ruxton, G. D. (2013). Size-dependent microhabitat selection by masquerading prey. Behavioral Ecology, 24, 89e97. Skelhorn, J., & Ruxton, G. D. (2010). Predators are less likely to misclassify masquerading prey when their models are present. Biology Letters, 6, 597e599. Skelhorn, J., & Ruxton, G. D. (2011). Context-dependent misclassification of masquerading prey. Evolutionary Ecology, 25, 751e761. Skelhorn, J., & Ruxton, G. D. (2014). Viewing distance affects how the presence of inedible models influence the benefit of masquerade. Evolutionary Ecology, 28, 441e455. Stevens, M., & Cuthill, I. C. (2006). Disruptive coloration, crypsis and edge detection in early visual processing. Proceedings of the Royal Society B: Biological Sciences, 273, 2141e2147. Stevens, M., & Merilaita, S. (2009). Animal camouflage: Current issues and new perspectives. Philosophical Transactions of the Royal Society B: Biological Sciences, 364, 423e427. Suzuki, T. N., & Sakurai, R. (2015). Bent posture improves the protective value of bird dropping masquerading by caterpillars. Animal Behaviour, 105, 79e84. Tinbergen, L. (1960). The natural control of insect in pinewoods 1. Factors influencing the intensity of predation by a song bird. Archives Neerlandaises de Zoologie, 13, 265e343. €ki, M., Kuusinen, E., Paloranta, M., & Mappes, J. Valkonen, J. K., Nokelainen, O., Jokima (2014). From deception to frankness: Benefits of ontogenetic shift in the antipredator strategy of alder moth Acronicta alni larvae. Current Zoology, 60, 114e122. Vane-Wright, R. I. (1980). On the definition of mimicry. Biological Journal of the Linnean Society, 13, 1e6. Wagner, D. L. (2005). Caterpillars of Eastern North America: A guide to identification and natural history. Princeton, NJ: Princeton University Press. Yanagisawa, Y. (1987). Social organization of a polygynous cichlid Lamprologus furcifer in Lake Tanganyika. Ichthyological Research, 34, 82e90. Yuma, M. (1994). Food habits and foraging behaviour of benthivorous cichlid fishes in Lake Tanganyika. Environmental Biology of Fishes, 39, 173e182.

Appendix

Table A1 Interpopulation comparison of the coloration of the model snail R. horei Explanatory terms

Estimate±SE

df

c2

P

Intercept Locality Katoto Wonzye Mutondwe Kasenga Isanga

0.19±0.19 e Reference 0.19±0.22 1.33±0.31 5.06±0.82 3.83±1.32

e 4 e e e e e

e 367.29 e e e e e

e <0.001 e 0.367 <0.001 <0.001 0.004

Significant terms are highlighted in bold.

Table A2 Factors affecting the probability of young Neolamprologus furcifer having a masquerade coloration at the five study localities Explanatory terms

Estimate±SE

df

c2

P

Intercept Trunk length Locality Katoto Wonzye Mutondwe Kasenga Isanga

12.07±2.19 1.40±0.32 e Reference 1.61 ±1.16 2.93±1.63 2.67±1.32 4.89±1.34

e 1 4 e e e e e

e 85.88 23.77 e e e e e

 <0.001 <0.001 e 0.167 0.072 0.043 <0.001

Significant terms are highlighted in bold. Territory site identity and locality are included as random factors.