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Embryo retrieval and kin recognition in an amphipod (Crustacea)
ANIMAL BEHAVIOUR, 2008, 76, 717e722 doi:10.1016/j.anbehav.2008.03.018
Available online at www.sciencedirect.com
Embryo retrieval and kin recognition in an amphipod (Crustacea) LYNSEY PAT TERS ON, JA IM IE T. A . DI CK & RO BERT W. ELWOOD
School of Biological Sciences, Queen’s University Belfast (Received 12 February 2008; initial acceptance 5 March 2008; final acceptance 11 March 2008; published online 16 June 2008; MS. number: 08-00081R)
Active maternal care directed towards embryos within the brood pouch has been identified in amphipod crustaceans from harsh aquatic environments. This involves ‘curl’ and ‘stretch’ components and brood flushing that alters in distinct ways in response to developmental and environmental cues. However, a cost of active brood care in crustaceans is the susceptibility to embryo loss, this being further predisposed by the structure of the amphipod brood pouch. We found embryo retrieval by females of the rock-pool amphipod Apherusa jurinei, whereby females inserted experimentally offered embryos into their brood pouches. Females early in brood development retrieved embryos to a greater degree than both nonovigerous and later stage females. In this experiment, all offered embryos were from other females, indicating a motivation to retrieve embryos that often overrides any kin recognition. In a second experiment, we found kin discrimination, with both early stage and late stage females retrieving more of their own embryos than those from other females. Recognition was not simply of embryos of similar developmental stages. There were high levels of embryo cannibalism in both experiments, but females were significantly less likely to consume their own compared to foreign embryos. We thus further show that ‘lower’ crustaceans such as amphipods engage in elaborate active maternal care including kin recognition and discrimination. Their maternal behaviour appears to balance the costs and benefits of embryo retrieval, minimizing fitness reductions due to embryo loss and adoption of foreign embryos. Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Keywords: amphipod; Apherusa jurinei; cannibalism; embryo retrieval; kin discrimination; kin recognition; maternal care
Female amphipods provide ‘passive’ care for their developing embryos by carrying them within a ventral brood pouch or marsupium. In many species, there is no obvious further ‘active’ brood care but, as with higher crustaceans, those inhabiting harsh or unpredictable environments may give such directed care (Hazlett 1983; Diesel 1989; Trumbo 1996; Dick et al. 1998, 2002; Tarutis et al. 2005; Thiel 2007). For example, the littoral rock-pool amphipod Apherusa jurinei shows active brood care by altering levels of ‘curl’ and ‘stretch’ activities towards embryos in the brood pouch in response to developmental and environmental cues (Dick et al. 2002). These activities circulate the embryos within the pouch, flush the pouch with water and may ameliorate fluctuations in the environment (Dick et al. 2002).
Correspondence: J. T. A. Dick, School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, U.K. (email: [email protected]). 0003e 3472/08/$34.00/0
Due to a number of trade-offs between embryo size and number and the structure of the brood plates or oostegites (see Steele 1991), the amphipod brood pouch is a compromise between embryo retention and exposure to the environment. Embryos may be lost from the brood pouch, a particular problem when active brood care is shown in crustaceans (Shillaker & Moore 1987; Ferna´ndez et al. 2000). We have direct and indirect evidence of embryo loss in the amphipods A. jurinei and Crangonyx pseudogracilis (Dick et al. 1998, 2002). Loss of embryos would have major fitness consequences; however, retrieval has been reported, with embryos placed back in the marsupium (Borowsky 1983; Shillaker & Moore 1987). However, not all embryos encountered may be the female’s own offspring and if a female mistakenly places a foreign embryo into her brood pouch, she may incur the cost of carrying and brooding that embryo with no genetic benefit. Costs of accepting foreign eggs/young have been examined in birds and high costs have led to increased host defence against brood parasitism (Robert & Sorci 1999). Avian
717 Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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species with altricial young have an enhanced ability to discriminate between their own eggs and those of brood parasites because of the substantial costs of caring for those parasites and potential danger to their own offspring (Davies & Brooke 1989a). In contrast, species with precocial young may suffer fewer costs when parasitized and are less likely to reject foreign eggs (Sorenson 1997). Indeed, when the costs of adopting foreign eggs are low, the high cost of potential rejection of own eggs may lead to females accepting all eggs (Davies & Brooke 1989b). There is some indication that active brood care in amphipods has some costs, as females radically reduce care activities when juveniles hatch and self-ventilate within the marsupium (Dick et al. 1998, 2002), and care is reduced in the long term (but not the short term) when brood sizes are experimentally reduced (Dick et al. 2002; Dick & Elwood 2006). Thus, it is likely that an additional embryo may not immediately increase the level of active brood care, but may do so in the longer term; thus some limited cost of mistaken retrieval is expected. This cost may be higher if the brood is relatively large because the ability to ventilate the young might decrease, while the chance of losing an embryo might increase. Nevertheless, with the high cost of rejecting their own embryos, we predict that females should be highly motivated to retrieve embryos in general. However, they might also show some recognition and discrimination in favour of kin with regard to embryo retrieval. If kin recognition occurs, it may be specific recognition of a phenotypic signature of the young or simply recognition of a developmental stage of embryos, or a ‘maternal state’ may allow retrieval only when the female might be expected to have embryos of her own (Elwood 1994). To test these predictions and possibilities we determined: (1) whether females collected from the wild with brood pouches, both empty and with embryos either early or late in development, inserted offered embryos from conspecifics into their brood pouches; (2) whether there is any selectivity between embryos of the same stage as the female’s embryos and those of different stages of development; (3) whether there is discrimination between their own embryos and those of another conspecific; (4) whether that selectivity varies with brooding stage of the female; and (5) whether females with relatively large broods are less likely than females with smaller broods to retrieve embryos. In addition, we examined cannibalism of encountered embryos and assessed whether this activity is moderated by kin recognition. We thus tested the ability of females to minimize potential fitness loss due to embryos being lost from their brood pouch or from carrying foreign embryos and whether they maximize fitness gain by using foreign embryos as food.
Donaghadee, County Down, Northern Ireland (Grid reference J477746), using fine mesh nets swept through the water and seaweed (predominantly Cladophora). Daytime water temperatures were 7e10 C. We took the amphipods to the laboratory in rock-pool water and stored them at 10 C in aerated tanks (60 cm long 30 cm wide 15 cm high) containing seaweed. After 48 h, we identified females as being nonovigerous (but with a brood pouch) or ovigerous, further subdividing the latter into: (1) early stage females, carrying recently extruded, black embryos, and (2) late stage females, carrying well-developed, orange embryos with eyespots. These two groups correspond to stages 1e3 and stages 4 and 5 of Dick et al. (2002). We established embryo numbers and volumes for early and late stage females (N ¼ 10 each) by taking the mean volume of three embryos from each brood pouch. This allowed the analysis of any effect of brood volume on embryo retrieval in the first experiment. We measured egg radius as the longest plus the smallest axis divided by 4 and the volume as 4/3 pr3. In the field, we often found scores of individuals, including females of differing embryonic developmental stages, in small pools of just a few litres of water. These pools were formed over rock substrate, where lost embryos (see the introduction) could conceivably be encountered by females as they walk around exploring their environment (as they do in laboratory aquaria). To mimic this scenario, experimental arenas were 5 cm diameter petri dishes with 9 ml seawater and a small piece of Cladophora. To obtain embryos for potential retrieval, we anaesthetized females by placing them in a petri dish with seawater and chilling them at approximately 2 C for 2e3 min until motionless. We removed embryos from the marsupium using a syringe with a fine needle, gently forcing the embryos out between the oostegites with a stream of seawater. Females recovered within minutes, engaging in walking, swimming, grooming and maternal care activities. When we presented females with embryos from other females, they were from a pool of embryos (excluding the receiving mothers’ embryos), such that offered embryos were not all from the same female. Due to the logistics of the experiment in terms of replication and manipulations, we observed the immediate behaviour of females over 15 min and then at 72 h, reasoning that if embryos were retrieved and brooded they would be observed at this time. Because embryos may have disappeared due to natural degeneration over 72 h, we tested embryo ‘robustness’ by placing individual embryos in petri dishes with rock-pool water (N ¼ 10 each for early and late) and monitoring them for 72 h.
Embryo Retrieval and Female/Embryo Stages METHODS
Animal Collection, Preparation and Preliminary Data We collected A. jurinei between October 2002 and March 2003 from intertidal rock pools at ‘The Commons’,
We determined the brood sizes of early and late stage females by counting the embryos in the marsupium using a binocular microscope (easy because of the small numbers of large embryos and the transparency of the female body and marsupium), enabling subsequent embryo retrievals to be determined by the increase in the number of
PATTERSON ET AL.: KIN RECOGNITION IN AN AMPHIPOD
Retrieval of Own and Foreign Embryos There were six experimental groups (N ¼ 16 each) in a 2 3 factorial design. We removed broods from females (early stage and late stage), gave them 12e16 h to recover, then presented each with either five of her own embryos or five embryos from other females of the same stage or the other stage. We carried out observations (1)e(5) and analysed the data as above.
RESULTS
Behavioural Observations and Preliminary Analyses Females presented with embryos treated them in one of three ways: cannibalizing them, placing them in their brood pouch, or leaving them lying in the dish. While walking and swimming around the dish, females encountered embryos and appeared to investigate them with their antennae. Embryos were often then ‘nudged’ across the dish, sometimes resulting in the female standing over the embryo. The female could move the embryo to the middle of her body by lifting it with her gnathopods while either lying on her side or standing over it. We observed some females cannibalizing the embryo in this situation, but did not observe embryo retrieval directly and inferred it from an increase in embryo number within the brood pouch. Embryos were robust with respect to being left isolated in petri dishes, all being intact at 72 h, although development appeared to have ceased. Thus, we recorded embryos not retrieved by females into their brood pouch or left in the dish as cannibalized (see below).
Early stage females carried significantly more embryos than late stage females (means SE ¼ 6.1 0.33 and 3.2 0.24; t98 ¼ 7.01, P < 0.0001), but early embryos were significantly smaller (0.04 0.003 mm3 versus 0.07 0.004 mm3; t18 ¼ 7.94, P < 0.0001), resulting in no significant difference in the mean overall brood volumes of early compared to late stage females (0.25 0.01 and 0.22 0.02 mm3; t98 ¼ 1.40, NS).
Embryo Retrieval and Female/Embryo Stages There was no significant difference in the mean numbers of contacts of embryos per 15 min among the three females states (F2,144 ¼ 0.65, NS) or between the two offered embryo stages (F1,144 ¼ 0.21, NS). This was also true for lifts of embryos per contact (F2,144 ¼ 0.46, NS; F1,144 ¼ 1.80, NS) and numbers of embryos cannibalized in 15 min (F2,144 ¼ 0.21, NS; F1,144 ¼ 1.03, NS). There were no interaction effects. There was a significant difference in the mean numbers of embryo retrievals at 72 h among the three female states (F2,144 ¼ 4.65, P < 0.01; Fig. 1), with early stage females retrieving significantly more embryos than either nonovigerous or late stage females (FPLSD: P < 0.01 and P < 0.02, respectively; Fig. 1), but there was no significant difference between nonovigerous and late stage females. There was no significant difference in embryo retrievals of early compared to late stage embryos (F1,144 ¼ 0.33, NS; Fig. 1) and there was no interaction effect (F2,144 ¼ 1.2, NS; Fig. 1). Significantly more early stage females retrieved one or more embryos (36%) than nonovigerous (14%) and late stage (14%) females (c22 ¼ 9.61, P < 0.008). There was no significant difference in the frequencies of females retrieving one or more early (25%) compared to late (17%) stage embryos (c21 ¼ 1.4, NS). There was no significant difference in the mean numbers of embryos cannibalized at 72 h among the three female states (F2,144 ¼ 1.72, NS; Fig. 2). However, significantly more late than early stage embryos were cannibalized (F1,144 ¼ 4.32, P < 0.04; Fig. 2), but there was no
Number of embryos retrieved by females
embryos observed in the marsupium. There were six experimental groups (N ¼ 25 each) in a 2 3 factorial design. That is, we presented each female (nonovigerous, early stage and late stage) with five embryos that were not her own (either early or late stage), lying on the arena floor, and observed her behaviour for 15 min and at 72 h. The subsequent data for analyses were: (1) number of contacts with embryos in 15 min; (2) number of embryo lifts per contact in 15 min; (3) number of embryos cannibalized (actually observed) in 15 min; (4) number of embryos placed in the brood pouch (at 72 h); and (5) number of embryos cannibalized at 72 h. Then, we took the length of the female from the base of the antennae to the base of the telson. We analysed data from (1) and (2) using two-factor ANOVA (Statview, Abacus Concepts, Inc.). Data from (3)e (5) were arcsine transformed proportions (Sokal & Rohlf 1995) and we analysed them by two-factor ANOVA. We conducted post hoc pairwise comparisons of means with Fisher’s protected least significant difference test (FPLSD). Figures show raw data for clarity. We used contingency analysis to test for associations between either female state or embryo stage and retrieval at 72 h. We used regression analysis and subsequent residuals to test if females with large broods were less likely to place encountered embryos into the pouch.
0.9 0.8
early stage embryos late stage embryos
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Non-gravid Early Status of female
Late
Figure 1. Mean (þSE) numbers of embryos of each developmental stage retrieved after 72 h by nongravid, early stage and late stage females.
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1.6
early stage embryos late stage embryos Number of embryos retrieved
5
4
3
2
1
1.4
0
Non-gravid Early Status of female
1 0.8 0.6 0.4 0.2
Late
Figure 2. Mean (þSE) numbers of embryos of each developmental stage cannibalized after 72 h by nongravid, early stage and late stage females.
interaction effect (F2,144 ¼ 1.7, NS; Fig. 2). All females cannibalized one or more embryos, thus precluding contingency analysis. Regression analysis showed a significant relationship between female length and overall brood volume for both early and late stage females (F1,48 ¼ 35.86, P < 0.001; F1,48 ¼ 20.06, P < 0.001). We generated residuals from these analyses, negative values of which indicate a relatively small brood and positive values a relatively large brood, referred to as ‘brood load’. For both early and late stage females, there was no significant difference in brood loads between females that retrieved embryos and those that did not (t48 ¼ 0.6, NS, and t48 ¼ 0.2, NS).
Retrieval of Own and Foreign Embryos There was no significant difference in the mean numbers of contacts of embryos per 15 min between early and late stage females (F1,90 ¼ 0.21, NS) or among the three embryo origins (F2,90 ¼ 0.28, NS). Early stage females lifted more embryos per contact (F1,90 ¼ 4.5, P < 0.05), but there was no effect of embryo origin (F2,90 ¼ 0.32, NS). There was no significant difference in the numbers of embryos cannibalized in 15 min between early and late stage females or among embryo origins (F1,90 ¼ 0.81, NS, and F2,90 ¼ 0.68, NS). There were no interaction effects. There was no significant difference in the mean numbers of embryos retrieved by early and late stage females (F1,90 ¼ 0.008, NS; Fig. 3). However, females retrieved significantly more of their own compared to foreign embryos (F2,90 ¼ 11.17, P < 0.001; Fig. 3). The interaction was not significant (F2,90 ¼ 2.7, NS; Fig. 3). There was no significant difference in the numbers of early females (48%) and late females (50%) retrieving one or more embryos (c21 ¼ 0.03, NS). Significantly more females retrieved one or more of their own embryos (63%) than same stage foreign embryos (9%) or other stage foreign embryos (25%) (c22 ¼ 21.82, P < 0.0001).
own embryos other, same stage other, different stage
1.2
0
Early Late Status of female
Figure 3. Mean (þSE) numbers of own and foreign embryos retrieved by females after 72 h.
There was no significant difference in the mean numbers of embryos cannibalized at 72 h by early and late stage females (F1,90 ¼ 1.6, NS; Fig. 4); however, females cannibalized significantly fewer of their own than foreign embryos (F2,90 ¼ 7.7, P < 0.001; Fig. 4). The interaction was not significant (F2,90 ¼ 0.6, NS; Fig. 4). There was no significant difference in the numbers of early females (89%) and late females (95%) cannibalizing one or more embryos (c21 ¼ 0.03, NS). Significantly fewer females cannibalized one or more of their own embryos (81%) than same stage foreign embryos (97%; c21 ¼ 4.01, P < 0.05), but this comparison was not significant with other stage foreign embryos (91%; c21 ¼ 1.2, NS). DISCUSSION The general activities and initial behaviour towards experimentally presented embryos early and late in development were similar among nongravid, early stage
5 Number of embryos cannibalized
Number of embryos cannibalized
720
own embryos other, same stage other, different stage
4
3
2
1
0
Early
Late
Status of female Figure 4. Mean (þSE) numbers of own and foreign embryos cannibalized by females after 72 h.
PATTERSON ET AL.: KIN RECOGNITION IN AN AMPHIPOD
and late stage female A. jurinei. While we did not directly observe embryo retrieval, we observed increased numbers of embryos in female brood pouches. These could not have been extruded from the oviducts, as all eggs are extruded simultaneously in amphipods and indeed numbers of embryos generally decline as development proceeds (see above and Dick et al. 1998, 2002). Thus, we are confident that retrievals occurred. We are similarly confident about counts of cannibalism, because isolated embryos were robust over the durations used in the experiments and embryos that disappeared must have been consumed. Females in all groups in experiment 1 retrieved conspecific embryos that were not their own and placed them in their brood pouches. However, females with early stage broods retrieved more of the offered embryos than did nonovigerous or late stage females. Furthermore, more early stage females retrieved than did the females of the other two groups. Thus, reproductive stage of the female influences her response to encountered embryos. This is consistent with our previous finding that females enter a maternal state manifested by frequent curls and stretches, which subsequently declines as embryos develop (Dick et al. 1998, 2002). Because the stretch activity expands the brood plates, embryos may be lost at this point, particularly if the stretching is vigorous and the embryos are small, as with early stage females. Thus, if early stage females are more likely to lose embryos, it is not surprising that they are more motivated to retrieve embryos. In the amphipod Gammarus insensibilis, however, egg collection was observed to be done only by females with embryos near to hatching, as this was a critical time for accidental embryo spillage in this species (Sheader 1996). This seems to be an example of temporal-based recognition (Elwood 1994); other examples are known in isopods (Linsenmair 1987, 2007), starlings (Pinxten et al. 1991) and rodents (Elwood 1977). Despite the finding that females show some selectivity in retrieval based upon the female’s maternal state, there was no selectivity in retrieval based upon embryo stage. We had expected that females would reject embryos that could not have been their own because they differed in developmental stage to those of their own. Remarkably, following brood removal, we have also observed female A. jurinei attempting to place other objects into their brood pouches, such as small snails, and one early stage female placed all five foreign late stage embryos in her brood pouch in experiment 2 (see effect in Fig. 3). Furthermore, females with relatively large broods were equally likely to retrieve embryos as those with relatively small broods. Perhaps there is little effect of existing brood load on the cost of mistaken retrieval in these animals. We had expected the pattern of cannibalism of embryos to be the reciprocal of that for retrieving, but this was not the case. Indeed, the results for embryo consumption seemed to contradict those of retrieval, in that female stage had no effect on cannibalism, but stage of embryo did. Females consumed more of the embryos if they were late stage and the reasons for this are not immediately explained in fitness terms. We also note that the effects seen here, showing discrimination between female stages for retrieval and between embryo stages for cannibalism,
although significant, are nevertheless small because the majority of embryos were cannibalized (Fig. 2). This consumption, however, may have been due to embryos dying over time, as although they remained intact, development appeared to cease. Indeed, the mean number cannibalized after 15 min was only 0.6, rising to 4.6 at 72 h, indicating cannibalism may have occurred at high rates due to declines in embryonic cues as they died. Indeed, we would not expect amphipod embryos to survive long outside the controlled environment of the brood pouch (see also Morritt & Spicer 1996), and this requires further work with regard to embryonic cues and their decay over time outside of the marsupium. In experiment 2, early and late stage female A. jurinei that had their broods removed were offered either their own embryos or those from different females, either of the same stage or of a stage different from that of the female’s original brood. Thus, we examined possible recognition cues that were specific to the embryos that might have been learned by the mother during interaction with them. This recognition seems to occur, because females retrieved significantly more of their own embryos compared to other females’ embryos of the same or a different developmental stage. Furthermore, the data on cannibalism in this experiment are congruent with those on retrieval, because fewer own embryos were cannibalized compared to those of different females irrespective of the embryo stage. However, in contrast to the first experiment, there was no indication in this experiment that early stage females were generally more maternal than late stage females. This may be because of the overriding effect of accepting own embryos in experiment 2. Thus, there is a clear finding that females specifically recognize phenotypic cues associated with their own embryos and treat them preferentially and this is consistent with findings from a variety of taxa (Beecher 1991). Examples include sticklebacks that discriminate between own and foreign embryos by embryo cues alone and totally cannibalize clutches mixed with foreign embryos (Frommen et al. 2007) and birds that learn features of their own eggs and subsequently reject eggs with a differing appearance (Lyon 2007). The few studies that examine offspring recognition in lower crustaceans, however, generally report the ready adoption of foreign juveniles by mothers (Thiel 2007), but even less is known regarding recognition of embryos. Some mysids adopt larvae and appear to discriminate among own and foreign larvae; however, this appears to be based on simple discrimination against larvae of ages different from those of the adopting female (Wittmann 1978). Furthermore, Borowsky (1983) found no discrimination between own and foreign conspecific embryos in two Gammarus species, although a degree of interspecific embryo recognition was observed. We speculate that, because Gammarus species do not engage in the distinct brood pouch expansion and flushing seen in A. jurinei, they may suffer less embryo loss and hence reduced selection for recognition of kin. Female Corophium bonnellii, which were observed to lose embryos, frequently placed embryos derived from other C. bonnellii females into their brood pouches, but refused embryos from other species. In contrast, female Lembos websteri never retrieved
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other females’ embryos due to the lower incidence of embryo loss predisposed by the more rigid structure of their brood pouch (Shillaker & Moore 1987). Embryo consumption may also be interpreted within a kin-recognition paradigm. Borowsky (1983) showed that the more distantly related embryos were to the female, the greater the proportion that would be consumed. Similarly, female L. websteri show a strong preference for ingesting all embryos encountered even if the individuals have a full gut (Shillaker & Moore 1987). As L. websteri are less likely to lose embryos, they are more prone to eating any embryos they encounter (Shillaker & Moore 1987). Thus, in the amphipods, embryo loss appears to have selected for embryo retrieval and cannibalism avoidance with differing degrees of discrimination. The observation of specific recognition of own offspring in this study has not been described in other amphipods. Furthermore, we have additionally shown temporal-based kin recognition, because in the first experiment early stage females were more likely to retrieve than were females at other reproductive stages. In this species there appear to be different mechanisms that ensure maximum care of own young while minimizing misdirected care to unrelated offspring and these are similar to those seen in various vertebrates (Elwood 1977, 1994; Beecher 1991; Pinxten et al. 1991). Acknowledgment L.P. was supported by a DARDNI studentship. References Beecher, M. D. 1991. Successes and failures of parenteoffspring recognition in animals. In: Kin Recognition (Ed. by P. G. Hepper), pp. 94e124. Cambridge, U.K.: Cambridge University Press. Borowsky, B. 1983. Placement of eggs in their brood pouches by females of the amphipod Crustacea Gammarus palustris and Gammarus mucronatus. Marine Behaviour and Physiology, 9, 319e325. Davies, N. B. & Brooke, M. L. 1989a. An experimental study of coevolution between the cuckoo, Cuculus canorus, and its hosts. I. Host egg discrimination. Journal of Animal Ecology, 58, 207e224. Davies, N. B. & Brooke, M. L. 1989b. An experimental study of coevolution between the cuckoo, Cuculus canorus, and its hosts. II. Host egg markings, chick discrimination and general discussion. Journal of Animal Ecology, 58, 225e236. Dick, J. T. A. & Elwood, R. W. 2006. Parenteoffspring conflict and motivational control of brooding in an amphipod (Crustacea). Biology Letters, 2, 501e504. Dick, J. T. A., Faloon, S. E. & Elwood, R. W. 1998. Active brood care in an amphipod: influences of embryonic development, temperature and oxygen. Animal Behaviour, 56, 663e672. Dick, J. T. A., Bailey, R. J. E. & Elwood, R. W. 2002. Maternal care in the rockpool amphipod Apherusa jurinei: development and environmental cues. Animal Behaviour, 63, 707e713. Diesel, R. 1989. Parental care in an unusual environment: Metopaulias depressus (Decapoda: Grapsidae), a crab that lives in epiphytic bromeliads. Animal Behaviour, 38, 561e575. Elwood, R. W. 1977. Changes in the responsiveness of male and female gerbils (Meriones unguicutaus) towards test pups during the pregnancy of the female. Animal Behaviour, 25, 46e51.
Elwood, R. W. 1994. Temporal based kinship recognition: a switch in time saves mine. Behavioural Processes, 33, 15e24. ¨ rtner, H.-O. 2000. The cost of being Ferna´ndez, M., Bock, C. & Po a caring mother: the ignored factor in the reproduction of marine invertebrates. Ecology Letters, 3, 487e494. Frommen, J. G., Brendler, C. & Bakker, T. C. M. 2007. The tale of the bad stepfather: male three spined sticklebacks Gasterosteus aculeatus L. recognize foreign eggs in their manipulated nest by egg cues alone. Journal of Fish Biology, 70, 1295e1301. Hazlett, B. A. 1983. Parental behaviour in decapod Crustacea. In: Studies in Adaptation: the Behaviour of Higher Crustacea (Ed. by S. Rebach & D. W. Dunham), pp. 171e193. New York: J. Wiley. Linsenmair, K. E. 1987. Kin recognition in subsocial arthropods; in particular in the desert isopod Hemilepistus reamuri. In: Kin Recognition in Animals (Ed. by D. J. C. Fletcher & C. D. Michener), pp. 121e208. New York: J. Wiley. Linsenmair, K. E. 2007. Sociobiology of terrestrial isopods. In: Evolutionary Ecology of Social and Sexual Systems: Crustaceans as Model Organisms (Ed. by J. E. Duffy & M. Thiel), pp. 339e364. Oxford: Oxford University Press. Lyon, B. 2007. Mechanism of egg recognition in defenses against conspecific brood parasitism: American coots (Fulica americana) know their own eggs. Behavioral Ecology and Sociobiology, 61, 455e463. Morritt, D. & Spicer, J. I. 1996. Developmental ecophysiology of the beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda). 1. Female control of the embryonic environment. Journal of Experimental Marine Biology and Ecology, 207, 191e203. Pinxten, R., Eens, M. & Verheyen, R. F. 1991. Responses of male starlings to experimental brood parasitism. Animal Behaviour, 35, 236e246. Robert, M. & Sorci, G. 1999. Rapid increase of host defence against brood parasites in a recently parasitized area: the case of village weavers in Hispaniola. Proceedings of the Royal Society of London, Series B, 266, 941e946. Sheader, M. 1996. Factors influencing egg size in the gammarid amphipod Gammarus insensibilis. Marine Biology, 124, 519e526. Shillaker, R. O. & Moore, P. G. 1987. The biology of brooding in the amphipods Lembos websteri Bate and Corophium bonnellii Milne Edwards. Journal of Experimental Marine Biology and Ecology, 110, 113e132. Sokal, R. R. & Rohlf, F. J. 1995. Biometry: the Principles and Practice of Statistics in Biological Research. New York: W. H. Freeman. Sorenson, M. D. 1997. Effects of intra- and interspecific brood parasitism on a precocial host, the canvasback, Aythya valisineria. Behavioral Ecology, 8, 153e161. Steele, D. H. 1991. Is the oostegite structure of amphipods determined by their phylogeny or is it an adaptation to their environment? Hydrobiologia, 223, 27e34. Tarutis, J., Lewis, S. & Dyke, M. 2005. Active parental care in a freshwater amphipod (Crustacea: Gammarus pseudolimnaeus): effects of environmental factors. American Midland Naturalist, 153, 276e283. Thiel, M. 2007. Social behavior of parenteoffspring groups in crustaceans. In: Evolutionary Ecology of Social and Sexual Systems: Crustaceans as Model Organisms (Ed. by J. E. Duffy & M. Thiel), pp. 294e318. Oxford: Oxford University Press. Trumbo, S. T. 1996. Parental care in invertebrates. In: Parental Care: Evolution, Mechanisms, and Adaptive Significance (Ed. by J. S. Rosenblatt & C. T. Snowdon), pp. 3e51. San Diego: Academic Press. Wittmann, K. J. 1978. Adoption, replacement and identification of young in marine Mysidacea (Crustacea). Journal of Experimental Marine Biology and Ecology, 32, 259e274.
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Embryo retrieval and kin recognition in an amphipod (Crustacea) LYNSEY PAT TERS ON, JA IM IE T. A . DI CK & RO BERT W. ELWOOD
School of Biological Sciences, Queen’s University Belfast (Received 12 February 2008; initial acceptance 5 March 2008; final acceptance 11 March 2008; published online 16 June 2008; MS. number: 08-00081R)
Active maternal care directed towards embryos within the brood pouch has been identified in amphipod crustaceans from harsh aquatic environments. This involves ‘curl’ and ‘stretch’ components and brood flushing that alters in distinct ways in response to developmental and environmental cues. However, a cost of active brood care in crustaceans is the susceptibility to embryo loss, this being further predisposed by the structure of the amphipod brood pouch. We found embryo retrieval by females of the rock-pool amphipod Apherusa jurinei, whereby females inserted experimentally offered embryos into their brood pouches. Females early in brood development retrieved embryos to a greater degree than both nonovigerous and later stage females. In this experiment, all offered embryos were from other females, indicating a motivation to retrieve embryos that often overrides any kin recognition. In a second experiment, we found kin discrimination, with both early stage and late stage females retrieving more of their own embryos than those from other females. Recognition was not simply of embryos of similar developmental stages. There were high levels of embryo cannibalism in both experiments, but females were significantly less likely to consume their own compared to foreign embryos. We thus further show that ‘lower’ crustaceans such as amphipods engage in elaborate active maternal care including kin recognition and discrimination. Their maternal behaviour appears to balance the costs and benefits of embryo retrieval, minimizing fitness reductions due to embryo loss and adoption of foreign embryos. Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Keywords: amphipod; Apherusa jurinei; cannibalism; embryo retrieval; kin discrimination; kin recognition; maternal care
Female amphipods provide ‘passive’ care for their developing embryos by carrying them within a ventral brood pouch or marsupium. In many species, there is no obvious further ‘active’ brood care but, as with higher crustaceans, those inhabiting harsh or unpredictable environments may give such directed care (Hazlett 1983; Diesel 1989; Trumbo 1996; Dick et al. 1998, 2002; Tarutis et al. 2005; Thiel 2007). For example, the littoral rock-pool amphipod Apherusa jurinei shows active brood care by altering levels of ‘curl’ and ‘stretch’ activities towards embryos in the brood pouch in response to developmental and environmental cues (Dick et al. 2002). These activities circulate the embryos within the pouch, flush the pouch with water and may ameliorate fluctuations in the environment (Dick et al. 2002).
Correspondence: J. T. A. Dick, School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, U.K. (email: [email protected]). 0003e 3472/08/$34.00/0
Due to a number of trade-offs between embryo size and number and the structure of the brood plates or oostegites (see Steele 1991), the amphipod brood pouch is a compromise between embryo retention and exposure to the environment. Embryos may be lost from the brood pouch, a particular problem when active brood care is shown in crustaceans (Shillaker & Moore 1987; Ferna´ndez et al. 2000). We have direct and indirect evidence of embryo loss in the amphipods A. jurinei and Crangonyx pseudogracilis (Dick et al. 1998, 2002). Loss of embryos would have major fitness consequences; however, retrieval has been reported, with embryos placed back in the marsupium (Borowsky 1983; Shillaker & Moore 1987). However, not all embryos encountered may be the female’s own offspring and if a female mistakenly places a foreign embryo into her brood pouch, she may incur the cost of carrying and brooding that embryo with no genetic benefit. Costs of accepting foreign eggs/young have been examined in birds and high costs have led to increased host defence against brood parasitism (Robert & Sorci 1999). Avian
717 Ó 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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species with altricial young have an enhanced ability to discriminate between their own eggs and those of brood parasites because of the substantial costs of caring for those parasites and potential danger to their own offspring (Davies & Brooke 1989a). In contrast, species with precocial young may suffer fewer costs when parasitized and are less likely to reject foreign eggs (Sorenson 1997). Indeed, when the costs of adopting foreign eggs are low, the high cost of potential rejection of own eggs may lead to females accepting all eggs (Davies & Brooke 1989b). There is some indication that active brood care in amphipods has some costs, as females radically reduce care activities when juveniles hatch and self-ventilate within the marsupium (Dick et al. 1998, 2002), and care is reduced in the long term (but not the short term) when brood sizes are experimentally reduced (Dick et al. 2002; Dick & Elwood 2006). Thus, it is likely that an additional embryo may not immediately increase the level of active brood care, but may do so in the longer term; thus some limited cost of mistaken retrieval is expected. This cost may be higher if the brood is relatively large because the ability to ventilate the young might decrease, while the chance of losing an embryo might increase. Nevertheless, with the high cost of rejecting their own embryos, we predict that females should be highly motivated to retrieve embryos in general. However, they might also show some recognition and discrimination in favour of kin with regard to embryo retrieval. If kin recognition occurs, it may be specific recognition of a phenotypic signature of the young or simply recognition of a developmental stage of embryos, or a ‘maternal state’ may allow retrieval only when the female might be expected to have embryos of her own (Elwood 1994). To test these predictions and possibilities we determined: (1) whether females collected from the wild with brood pouches, both empty and with embryos either early or late in development, inserted offered embryos from conspecifics into their brood pouches; (2) whether there is any selectivity between embryos of the same stage as the female’s embryos and those of different stages of development; (3) whether there is discrimination between their own embryos and those of another conspecific; (4) whether that selectivity varies with brooding stage of the female; and (5) whether females with relatively large broods are less likely than females with smaller broods to retrieve embryos. In addition, we examined cannibalism of encountered embryos and assessed whether this activity is moderated by kin recognition. We thus tested the ability of females to minimize potential fitness loss due to embryos being lost from their brood pouch or from carrying foreign embryos and whether they maximize fitness gain by using foreign embryos as food.
Donaghadee, County Down, Northern Ireland (Grid reference J477746), using fine mesh nets swept through the water and seaweed (predominantly Cladophora). Daytime water temperatures were 7e10 C. We took the amphipods to the laboratory in rock-pool water and stored them at 10 C in aerated tanks (60 cm long 30 cm wide 15 cm high) containing seaweed. After 48 h, we identified females as being nonovigerous (but with a brood pouch) or ovigerous, further subdividing the latter into: (1) early stage females, carrying recently extruded, black embryos, and (2) late stage females, carrying well-developed, orange embryos with eyespots. These two groups correspond to stages 1e3 and stages 4 and 5 of Dick et al. (2002). We established embryo numbers and volumes for early and late stage females (N ¼ 10 each) by taking the mean volume of three embryos from each brood pouch. This allowed the analysis of any effect of brood volume on embryo retrieval in the first experiment. We measured egg radius as the longest plus the smallest axis divided by 4 and the volume as 4/3 pr3. In the field, we often found scores of individuals, including females of differing embryonic developmental stages, in small pools of just a few litres of water. These pools were formed over rock substrate, where lost embryos (see the introduction) could conceivably be encountered by females as they walk around exploring their environment (as they do in laboratory aquaria). To mimic this scenario, experimental arenas were 5 cm diameter petri dishes with 9 ml seawater and a small piece of Cladophora. To obtain embryos for potential retrieval, we anaesthetized females by placing them in a petri dish with seawater and chilling them at approximately 2 C for 2e3 min until motionless. We removed embryos from the marsupium using a syringe with a fine needle, gently forcing the embryos out between the oostegites with a stream of seawater. Females recovered within minutes, engaging in walking, swimming, grooming and maternal care activities. When we presented females with embryos from other females, they were from a pool of embryos (excluding the receiving mothers’ embryos), such that offered embryos were not all from the same female. Due to the logistics of the experiment in terms of replication and manipulations, we observed the immediate behaviour of females over 15 min and then at 72 h, reasoning that if embryos were retrieved and brooded they would be observed at this time. Because embryos may have disappeared due to natural degeneration over 72 h, we tested embryo ‘robustness’ by placing individual embryos in petri dishes with rock-pool water (N ¼ 10 each for early and late) and monitoring them for 72 h.
Embryo Retrieval and Female/Embryo Stages METHODS
Animal Collection, Preparation and Preliminary Data We collected A. jurinei between October 2002 and March 2003 from intertidal rock pools at ‘The Commons’,
We determined the brood sizes of early and late stage females by counting the embryos in the marsupium using a binocular microscope (easy because of the small numbers of large embryos and the transparency of the female body and marsupium), enabling subsequent embryo retrievals to be determined by the increase in the number of
PATTERSON ET AL.: KIN RECOGNITION IN AN AMPHIPOD
Retrieval of Own and Foreign Embryos There were six experimental groups (N ¼ 16 each) in a 2 3 factorial design. We removed broods from females (early stage and late stage), gave them 12e16 h to recover, then presented each with either five of her own embryos or five embryos from other females of the same stage or the other stage. We carried out observations (1)e(5) and analysed the data as above.
RESULTS
Behavioural Observations and Preliminary Analyses Females presented with embryos treated them in one of three ways: cannibalizing them, placing them in their brood pouch, or leaving them lying in the dish. While walking and swimming around the dish, females encountered embryos and appeared to investigate them with their antennae. Embryos were often then ‘nudged’ across the dish, sometimes resulting in the female standing over the embryo. The female could move the embryo to the middle of her body by lifting it with her gnathopods while either lying on her side or standing over it. We observed some females cannibalizing the embryo in this situation, but did not observe embryo retrieval directly and inferred it from an increase in embryo number within the brood pouch. Embryos were robust with respect to being left isolated in petri dishes, all being intact at 72 h, although development appeared to have ceased. Thus, we recorded embryos not retrieved by females into their brood pouch or left in the dish as cannibalized (see below).
Early stage females carried significantly more embryos than late stage females (means SE ¼ 6.1 0.33 and 3.2 0.24; t98 ¼ 7.01, P < 0.0001), but early embryos were significantly smaller (0.04 0.003 mm3 versus 0.07 0.004 mm3; t18 ¼ 7.94, P < 0.0001), resulting in no significant difference in the mean overall brood volumes of early compared to late stage females (0.25 0.01 and 0.22 0.02 mm3; t98 ¼ 1.40, NS).
Embryo Retrieval and Female/Embryo Stages There was no significant difference in the mean numbers of contacts of embryos per 15 min among the three females states (F2,144 ¼ 0.65, NS) or between the two offered embryo stages (F1,144 ¼ 0.21, NS). This was also true for lifts of embryos per contact (F2,144 ¼ 0.46, NS; F1,144 ¼ 1.80, NS) and numbers of embryos cannibalized in 15 min (F2,144 ¼ 0.21, NS; F1,144 ¼ 1.03, NS). There were no interaction effects. There was a significant difference in the mean numbers of embryo retrievals at 72 h among the three female states (F2,144 ¼ 4.65, P < 0.01; Fig. 1), with early stage females retrieving significantly more embryos than either nonovigerous or late stage females (FPLSD: P < 0.01 and P < 0.02, respectively; Fig. 1), but there was no significant difference between nonovigerous and late stage females. There was no significant difference in embryo retrievals of early compared to late stage embryos (F1,144 ¼ 0.33, NS; Fig. 1) and there was no interaction effect (F2,144 ¼ 1.2, NS; Fig. 1). Significantly more early stage females retrieved one or more embryos (36%) than nonovigerous (14%) and late stage (14%) females (c22 ¼ 9.61, P < 0.008). There was no significant difference in the frequencies of females retrieving one or more early (25%) compared to late (17%) stage embryos (c21 ¼ 1.4, NS). There was no significant difference in the mean numbers of embryos cannibalized at 72 h among the three female states (F2,144 ¼ 1.72, NS; Fig. 2). However, significantly more late than early stage embryos were cannibalized (F1,144 ¼ 4.32, P < 0.04; Fig. 2), but there was no
Number of embryos retrieved by females
embryos observed in the marsupium. There were six experimental groups (N ¼ 25 each) in a 2 3 factorial design. That is, we presented each female (nonovigerous, early stage and late stage) with five embryos that were not her own (either early or late stage), lying on the arena floor, and observed her behaviour for 15 min and at 72 h. The subsequent data for analyses were: (1) number of contacts with embryos in 15 min; (2) number of embryo lifts per contact in 15 min; (3) number of embryos cannibalized (actually observed) in 15 min; (4) number of embryos placed in the brood pouch (at 72 h); and (5) number of embryos cannibalized at 72 h. Then, we took the length of the female from the base of the antennae to the base of the telson. We analysed data from (1) and (2) using two-factor ANOVA (Statview, Abacus Concepts, Inc.). Data from (3)e (5) were arcsine transformed proportions (Sokal & Rohlf 1995) and we analysed them by two-factor ANOVA. We conducted post hoc pairwise comparisons of means with Fisher’s protected least significant difference test (FPLSD). Figures show raw data for clarity. We used contingency analysis to test for associations between either female state or embryo stage and retrieval at 72 h. We used regression analysis and subsequent residuals to test if females with large broods were less likely to place encountered embryos into the pouch.
0.9 0.8
early stage embryos late stage embryos
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Non-gravid Early Status of female
Late
Figure 1. Mean (þSE) numbers of embryos of each developmental stage retrieved after 72 h by nongravid, early stage and late stage females.
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1.6
early stage embryos late stage embryos Number of embryos retrieved
5
4
3
2
1
1.4
0
Non-gravid Early Status of female
1 0.8 0.6 0.4 0.2
Late
Figure 2. Mean (þSE) numbers of embryos of each developmental stage cannibalized after 72 h by nongravid, early stage and late stage females.
interaction effect (F2,144 ¼ 1.7, NS; Fig. 2). All females cannibalized one or more embryos, thus precluding contingency analysis. Regression analysis showed a significant relationship between female length and overall brood volume for both early and late stage females (F1,48 ¼ 35.86, P < 0.001; F1,48 ¼ 20.06, P < 0.001). We generated residuals from these analyses, negative values of which indicate a relatively small brood and positive values a relatively large brood, referred to as ‘brood load’. For both early and late stage females, there was no significant difference in brood loads between females that retrieved embryos and those that did not (t48 ¼ 0.6, NS, and t48 ¼ 0.2, NS).
Retrieval of Own and Foreign Embryos There was no significant difference in the mean numbers of contacts of embryos per 15 min between early and late stage females (F1,90 ¼ 0.21, NS) or among the three embryo origins (F2,90 ¼ 0.28, NS). Early stage females lifted more embryos per contact (F1,90 ¼ 4.5, P < 0.05), but there was no effect of embryo origin (F2,90 ¼ 0.32, NS). There was no significant difference in the numbers of embryos cannibalized in 15 min between early and late stage females or among embryo origins (F1,90 ¼ 0.81, NS, and F2,90 ¼ 0.68, NS). There were no interaction effects. There was no significant difference in the mean numbers of embryos retrieved by early and late stage females (F1,90 ¼ 0.008, NS; Fig. 3). However, females retrieved significantly more of their own compared to foreign embryos (F2,90 ¼ 11.17, P < 0.001; Fig. 3). The interaction was not significant (F2,90 ¼ 2.7, NS; Fig. 3). There was no significant difference in the numbers of early females (48%) and late females (50%) retrieving one or more embryos (c21 ¼ 0.03, NS). Significantly more females retrieved one or more of their own embryos (63%) than same stage foreign embryos (9%) or other stage foreign embryos (25%) (c22 ¼ 21.82, P < 0.0001).
own embryos other, same stage other, different stage
1.2
0
Early Late Status of female
Figure 3. Mean (þSE) numbers of own and foreign embryos retrieved by females after 72 h.
There was no significant difference in the mean numbers of embryos cannibalized at 72 h by early and late stage females (F1,90 ¼ 1.6, NS; Fig. 4); however, females cannibalized significantly fewer of their own than foreign embryos (F2,90 ¼ 7.7, P < 0.001; Fig. 4). The interaction was not significant (F2,90 ¼ 0.6, NS; Fig. 4). There was no significant difference in the numbers of early females (89%) and late females (95%) cannibalizing one or more embryos (c21 ¼ 0.03, NS). Significantly fewer females cannibalized one or more of their own embryos (81%) than same stage foreign embryos (97%; c21 ¼ 4.01, P < 0.05), but this comparison was not significant with other stage foreign embryos (91%; c21 ¼ 1.2, NS). DISCUSSION The general activities and initial behaviour towards experimentally presented embryos early and late in development were similar among nongravid, early stage
5 Number of embryos cannibalized
Number of embryos cannibalized
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own embryos other, same stage other, different stage
4
3
2
1
0
Early
Late
Status of female Figure 4. Mean (þSE) numbers of own and foreign embryos cannibalized by females after 72 h.
PATTERSON ET AL.: KIN RECOGNITION IN AN AMPHIPOD
and late stage female A. jurinei. While we did not directly observe embryo retrieval, we observed increased numbers of embryos in female brood pouches. These could not have been extruded from the oviducts, as all eggs are extruded simultaneously in amphipods and indeed numbers of embryos generally decline as development proceeds (see above and Dick et al. 1998, 2002). Thus, we are confident that retrievals occurred. We are similarly confident about counts of cannibalism, because isolated embryos were robust over the durations used in the experiments and embryos that disappeared must have been consumed. Females in all groups in experiment 1 retrieved conspecific embryos that were not their own and placed them in their brood pouches. However, females with early stage broods retrieved more of the offered embryos than did nonovigerous or late stage females. Furthermore, more early stage females retrieved than did the females of the other two groups. Thus, reproductive stage of the female influences her response to encountered embryos. This is consistent with our previous finding that females enter a maternal state manifested by frequent curls and stretches, which subsequently declines as embryos develop (Dick et al. 1998, 2002). Because the stretch activity expands the brood plates, embryos may be lost at this point, particularly if the stretching is vigorous and the embryos are small, as with early stage females. Thus, if early stage females are more likely to lose embryos, it is not surprising that they are more motivated to retrieve embryos. In the amphipod Gammarus insensibilis, however, egg collection was observed to be done only by females with embryos near to hatching, as this was a critical time for accidental embryo spillage in this species (Sheader 1996). This seems to be an example of temporal-based recognition (Elwood 1994); other examples are known in isopods (Linsenmair 1987, 2007), starlings (Pinxten et al. 1991) and rodents (Elwood 1977). Despite the finding that females show some selectivity in retrieval based upon the female’s maternal state, there was no selectivity in retrieval based upon embryo stage. We had expected that females would reject embryos that could not have been their own because they differed in developmental stage to those of their own. Remarkably, following brood removal, we have also observed female A. jurinei attempting to place other objects into their brood pouches, such as small snails, and one early stage female placed all five foreign late stage embryos in her brood pouch in experiment 2 (see effect in Fig. 3). Furthermore, females with relatively large broods were equally likely to retrieve embryos as those with relatively small broods. Perhaps there is little effect of existing brood load on the cost of mistaken retrieval in these animals. We had expected the pattern of cannibalism of embryos to be the reciprocal of that for retrieving, but this was not the case. Indeed, the results for embryo consumption seemed to contradict those of retrieval, in that female stage had no effect on cannibalism, but stage of embryo did. Females consumed more of the embryos if they were late stage and the reasons for this are not immediately explained in fitness terms. We also note that the effects seen here, showing discrimination between female stages for retrieval and between embryo stages for cannibalism,
although significant, are nevertheless small because the majority of embryos were cannibalized (Fig. 2). This consumption, however, may have been due to embryos dying over time, as although they remained intact, development appeared to cease. Indeed, the mean number cannibalized after 15 min was only 0.6, rising to 4.6 at 72 h, indicating cannibalism may have occurred at high rates due to declines in embryonic cues as they died. Indeed, we would not expect amphipod embryos to survive long outside the controlled environment of the brood pouch (see also Morritt & Spicer 1996), and this requires further work with regard to embryonic cues and their decay over time outside of the marsupium. In experiment 2, early and late stage female A. jurinei that had their broods removed were offered either their own embryos or those from different females, either of the same stage or of a stage different from that of the female’s original brood. Thus, we examined possible recognition cues that were specific to the embryos that might have been learned by the mother during interaction with them. This recognition seems to occur, because females retrieved significantly more of their own embryos compared to other females’ embryos of the same or a different developmental stage. Furthermore, the data on cannibalism in this experiment are congruent with those on retrieval, because fewer own embryos were cannibalized compared to those of different females irrespective of the embryo stage. However, in contrast to the first experiment, there was no indication in this experiment that early stage females were generally more maternal than late stage females. This may be because of the overriding effect of accepting own embryos in experiment 2. Thus, there is a clear finding that females specifically recognize phenotypic cues associated with their own embryos and treat them preferentially and this is consistent with findings from a variety of taxa (Beecher 1991). Examples include sticklebacks that discriminate between own and foreign embryos by embryo cues alone and totally cannibalize clutches mixed with foreign embryos (Frommen et al. 2007) and birds that learn features of their own eggs and subsequently reject eggs with a differing appearance (Lyon 2007). The few studies that examine offspring recognition in lower crustaceans, however, generally report the ready adoption of foreign juveniles by mothers (Thiel 2007), but even less is known regarding recognition of embryos. Some mysids adopt larvae and appear to discriminate among own and foreign larvae; however, this appears to be based on simple discrimination against larvae of ages different from those of the adopting female (Wittmann 1978). Furthermore, Borowsky (1983) found no discrimination between own and foreign conspecific embryos in two Gammarus species, although a degree of interspecific embryo recognition was observed. We speculate that, because Gammarus species do not engage in the distinct brood pouch expansion and flushing seen in A. jurinei, they may suffer less embryo loss and hence reduced selection for recognition of kin. Female Corophium bonnellii, which were observed to lose embryos, frequently placed embryos derived from other C. bonnellii females into their brood pouches, but refused embryos from other species. In contrast, female Lembos websteri never retrieved
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other females’ embryos due to the lower incidence of embryo loss predisposed by the more rigid structure of their brood pouch (Shillaker & Moore 1987). Embryo consumption may also be interpreted within a kin-recognition paradigm. Borowsky (1983) showed that the more distantly related embryos were to the female, the greater the proportion that would be consumed. Similarly, female L. websteri show a strong preference for ingesting all embryos encountered even if the individuals have a full gut (Shillaker & Moore 1987). As L. websteri are less likely to lose embryos, they are more prone to eating any embryos they encounter (Shillaker & Moore 1987). Thus, in the amphipods, embryo loss appears to have selected for embryo retrieval and cannibalism avoidance with differing degrees of discrimination. The observation of specific recognition of own offspring in this study has not been described in other amphipods. Furthermore, we have additionally shown temporal-based kin recognition, because in the first experiment early stage females were more likely to retrieve than were females at other reproductive stages. In this species there appear to be different mechanisms that ensure maximum care of own young while minimizing misdirected care to unrelated offspring and these are similar to those seen in various vertebrates (Elwood 1977, 1994; Beecher 1991; Pinxten et al. 1991). Acknowledgment L.P. was supported by a DARDNI studentship. References Beecher, M. D. 1991. Successes and failures of parenteoffspring recognition in animals. In: Kin Recognition (Ed. by P. G. Hepper), pp. 94e124. Cambridge, U.K.: Cambridge University Press. Borowsky, B. 1983. Placement of eggs in their brood pouches by females of the amphipod Crustacea Gammarus palustris and Gammarus mucronatus. Marine Behaviour and Physiology, 9, 319e325. Davies, N. B. & Brooke, M. L. 1989a. An experimental study of coevolution between the cuckoo, Cuculus canorus, and its hosts. I. Host egg discrimination. Journal of Animal Ecology, 58, 207e224. Davies, N. B. & Brooke, M. L. 1989b. An experimental study of coevolution between the cuckoo, Cuculus canorus, and its hosts. II. Host egg markings, chick discrimination and general discussion. Journal of Animal Ecology, 58, 225e236. Dick, J. T. A. & Elwood, R. W. 2006. Parenteoffspring conflict and motivational control of brooding in an amphipod (Crustacea). Biology Letters, 2, 501e504. Dick, J. T. A., Faloon, S. E. & Elwood, R. W. 1998. Active brood care in an amphipod: influences of embryonic development, temperature and oxygen. Animal Behaviour, 56, 663e672. Dick, J. T. A., Bailey, R. J. E. & Elwood, R. W. 2002. Maternal care in the rockpool amphipod Apherusa jurinei: development and environmental cues. Animal Behaviour, 63, 707e713. Diesel, R. 1989. Parental care in an unusual environment: Metopaulias depressus (Decapoda: Grapsidae), a crab that lives in epiphytic bromeliads. Animal Behaviour, 38, 561e575. Elwood, R. W. 1977. Changes in the responsiveness of male and female gerbils (Meriones unguicutaus) towards test pups during the pregnancy of the female. Animal Behaviour, 25, 46e51.
Elwood, R. W. 1994. Temporal based kinship recognition: a switch in time saves mine. Behavioural Processes, 33, 15e24. ¨ rtner, H.-O. 2000. The cost of being Ferna´ndez, M., Bock, C. & Po a caring mother: the ignored factor in the reproduction of marine invertebrates. Ecology Letters, 3, 487e494. Frommen, J. G., Brendler, C. & Bakker, T. C. M. 2007. The tale of the bad stepfather: male three spined sticklebacks Gasterosteus aculeatus L. recognize foreign eggs in their manipulated nest by egg cues alone. Journal of Fish Biology, 70, 1295e1301. Hazlett, B. A. 1983. Parental behaviour in decapod Crustacea. In: Studies in Adaptation: the Behaviour of Higher Crustacea (Ed. by S. Rebach & D. W. Dunham), pp. 171e193. New York: J. Wiley. Linsenmair, K. E. 1987. Kin recognition in subsocial arthropods; in particular in the desert isopod Hemilepistus reamuri. In: Kin Recognition in Animals (Ed. by D. J. C. Fletcher & C. D. Michener), pp. 121e208. New York: J. Wiley. Linsenmair, K. E. 2007. Sociobiology of terrestrial isopods. In: Evolutionary Ecology of Social and Sexual Systems: Crustaceans as Model Organisms (Ed. by J. E. Duffy & M. Thiel), pp. 339e364. Oxford: Oxford University Press. Lyon, B. 2007. Mechanism of egg recognition in defenses against conspecific brood parasitism: American coots (Fulica americana) know their own eggs. Behavioral Ecology and Sociobiology, 61, 455e463. Morritt, D. & Spicer, J. I. 1996. Developmental ecophysiology of the beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda). 1. Female control of the embryonic environment. Journal of Experimental Marine Biology and Ecology, 207, 191e203. Pinxten, R., Eens, M. & Verheyen, R. F. 1991. Responses of male starlings to experimental brood parasitism. Animal Behaviour, 35, 236e246. Robert, M. & Sorci, G. 1999. Rapid increase of host defence against brood parasites in a recently parasitized area: the case of village weavers in Hispaniola. Proceedings of the Royal Society of London, Series B, 266, 941e946. Sheader, M. 1996. Factors influencing egg size in the gammarid amphipod Gammarus insensibilis. Marine Biology, 124, 519e526. Shillaker, R. O. & Moore, P. G. 1987. The biology of brooding in the amphipods Lembos websteri Bate and Corophium bonnellii Milne Edwards. Journal of Experimental Marine Biology and Ecology, 110, 113e132. Sokal, R. R. & Rohlf, F. J. 1995. Biometry: the Principles and Practice of Statistics in Biological Research. New York: W. H. Freeman. Sorenson, M. D. 1997. Effects of intra- and interspecific brood parasitism on a precocial host, the canvasback, Aythya valisineria. Behavioral Ecology, 8, 153e161. Steele, D. H. 1991. Is the oostegite structure of amphipods determined by their phylogeny or is it an adaptation to their environment? Hydrobiologia, 223, 27e34. Tarutis, J., Lewis, S. & Dyke, M. 2005. Active parental care in a freshwater amphipod (Crustacea: Gammarus pseudolimnaeus): effects of environmental factors. American Midland Naturalist, 153, 276e283. Thiel, M. 2007. Social behavior of parenteoffspring groups in crustaceans. In: Evolutionary Ecology of Social and Sexual Systems: Crustaceans as Model Organisms (Ed. by J. E. Duffy & M. Thiel), pp. 294e318. Oxford: Oxford University Press. Trumbo, S. T. 1996. Parental care in invertebrates. In: Parental Care: Evolution, Mechanisms, and Adaptive Significance (Ed. by J. S. Rosenblatt & C. T. Snowdon), pp. 3e51. San Diego: Academic Press. Wittmann, K. J. 1978. Adoption, replacement and identification of young in marine Mysidacea (Crustacea). Journal of Experimental Marine Biology and Ecology, 32, 259e274.