- Email: [email protected]
Oviposition depth in response to egg parasitism in the water strider: high-risk experience promotes deeper oviposition
Animal Behaviour 78 (2009) 935–941
Contents lists available at ScienceDirect
Animal Behaviour journal homepage: www.elsevier.com/locate/anbehav
Oviposition depth in response to egg parasitism in the water strider: high-risk experience promotes deeper oviposition Hiroyuki Hirayama*, Eiiti Kasuya Laboratory of Ecology, Department of Biology, Faculty of Sciences, Kyushu University
a r t i c l e i n f o Article history: Received 2 April 2009 Initial acceptance 14 May 2009 Final acceptance 18 July 2009 Published online 21 August 2009 MS. number: 09-00215R Keywords: Aquarium paludum egg parasitoid wasp experienced predation risk memory oviposition site selection submerged oviposition water strider
In many species, predation risk at oviposition sites causes females to avoid high-risk sites. Previous studies, however, have focused on assessment of current risk at the site during oviposition. Whether memory of previous risk plays a role has not been considered. We examined whether memorized risk affects oviposition site selection in the water strider Aquarius paludum insularis. Aquarius paludum eggs are laid on vegetation on or in the water and are frequently parasitized by a parasitoid wasp. Eggs at deeper positions are less frequently parasitized than those near the surface. After one of three treatments (no, low or high wasp density), A. paludum females were allowed to oviposit in parasitoid-free aquaria. The depth of oviposition of females that had previously experienced high wasp density was greater than that of those that had not experienced wasps. However, the difference in oviposition depth between lowdensity and no-wasp treatments was slight although significant. This indicates that A. paludum makes the decision on oviposition depth in response to the egg parasitism risk experienced before oviposition. The effect of the memorized risk was largest just after the treatment and became weaker over time. A measurable effect persisted for 28 days. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
In egg-laying species with no further parental care, offspring survival and growth strongly depend on the quality of the oviposition site. In particular, the presence of predators or parasitoids strongly affects decision making at oviposition (hereafter, predators and parasitoids are referred to collectively as predators). Most studies on this topic show that females avoid oviposition at sites where predators or cues of predators are present (e.g. Kiflawi et al. 2003a, b; Blaustein et al. 2004; Brodin et al. 2006). However, whether animals can memorize the predation risk and use it for subsequent oviposition site selection has not yet been examined. If animals can memorize the predation risk, oviposition site selection would be affected by predation risk on a broader scale, in terms of both time and area for oviposition, than has previously been considered. Specifically, the memorized predation risk may first affect oviposition site selection over a longer timescale. For example, the risk previously experienced upon arrival at the site may be used as well as the current risk at the site. Second, the risk may also affect oviposition site selection at a larger spatial scale. For example, the previously experienced risk at places other than the oviposition site (e.g. on the way to the site) may be used. Therefore, not only
* Correspondence: H. Hirayama, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka 812-8581, Japan. E-mail address: [email protected] (H. Hirayama).
present but also past distributions of predators may affect oviposition site selection. If females can memorize the predation risk, two questions arise. One is how long and strongly the predation risk needs to be experienced before it affects the subsequent choice of the oviposition site. The second is whether a predation risk experienced in a location other than the potential/intended oviposition site may affect the choice of the subsequent site. The water strider Aquarius paludum insularis (Heteroptera: Gerridae) and its egg parasitoid wasp Tiphodytes gerriphagus (Hymenoptera: Scelionidae) are suitable subjects to help resolve these two questions. Adult females of A. paludum usually oviposit on stems or leaves of vegetation in the water. The oviposition site of A. paludum is usually close to, or actually within, the area where they forage and mate. Therefore, A. paludum is likely to encounter the egg parasitoid wasp not only when it oviposits and searches for oviposition sites but also during activities other than oviposition on the water surface (e.g. foraging, resting and mating). Thus, A. paludum is expected to maintain a memory of risk at many sites other than oviposition sites. In A. paludum, ovipositing at deep sites from the surface enables the use of oviposition sites that are safe from egg parasitism. In the wild, A. paludum eggs are frequently parasitized but the proportion of parasitized eggs depends on the depth of eggs oviposited. Eggs oviposited underwater are less frequently parasitized than those on the water surface (Spence 1986) and, in the water, eggs oviposited
0003-3472/$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2009.07.019
936
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
at deeper positions are safer from egg parasitism than those at shallower positions (Amano et al. 2008). Female A. paludum can oviposit in two ways (personal observation). They either dip only the abdomen in the water and oviposit at a very shallow position (surface oviposition), or they completely submerge themselves underwater and oviposit at deeper positions (submerged oviposition). Submerged oviposition enables A. paludum to oviposit at places safe from egg parasitism but it involves costs (e.g. energetic cost, drowning risk and predation risk for the ovipositing females). Submerged oviposition is the only time during the adult phase when A. paludum females submerge themselves in the water. When the adult A. paludum performs submerged oviposition and lays eggs deep in the water to avoid egg parasitism, the decision to submerge and the subsequent depth of eggs oviposited are expected to vary in response to the risk of egg parasitism experienced before oviposition. When the risk experienced before oviposition is low, A. paludum females are expected to oviposit near the water surface (shallow position); when the risk is high, they are expected to submerge and oviposit in a deeper position. Our aim in this study was to examine two questions. One is whether A. paludum makes the decisions on submersion and egg depth in response to the presence of the egg parasitoid before oviposition at places other than the oviposition site. The other is, for how long and how strongly does the predation risk experienced affect subsequent oviposition site selection? METHODS Materials Aquarius paludum insularis Aquarius paludum has a trivoltine life cycle in Fukuoka, Japan, and eggs of the next generation of overwintered A. paludum are frequently parasitized by T. gerriphagus. Twenty pairs (a female and a male) of overwintered adult A. paludum insularis were caught at Todoroki pond in Hisayama, Japan (33 360 2900 N, 130 290 3400 E) in July 2006. We used the next generation of these 20 pairs as the experimental individuals. They were reared in a parasitoid-free experimental room (natural light conditions or longer photoperiod, with temperature kept at 25 C). Therefore, they had no prior experience of parasitoids before the experiment. We recorded the date of emergence, the body size, sex and wing morph (short or long winged), and marked them individually with a paint mark on the thorax (opaque color, Teranishi chemical industry, Osaka, Japan). The methods here were the same as in Hirayama & Kasuya (2008). The adults that had been observed to mate within 3 weeks of emergence were used in the experiments. Throughout all the developmental stages, A. paludum were kept in aquaria (30 45 cm and 15 cm high, water filled 5 cm deep), and given frozen blow flies for food every day and fragments of foam polystyrene for the oviposition substrate and for resting sites. Water in the aquaria was exchanged at least once every two days. The adult life span was approximately 2–3 months, and females oviposited every 2–4 days in the laboratory. Oviposition behaviour in A. paludum During surface oviposition (or no submersion) females hold the oviposition substrate (e.g. a plant stem in the wild) by the forelegs, dip the abdomen in the water and usually oviposit up to 30 eggs on the substrate. The maximum depth of eggs oviposited is approximately equivalent to the body length of adult females (1.5 cm). During submerged oviposition females hold the oviposition substrate by their forelegs and turn their heads to the water surface. They enter the water by moving down the substrate head first and
oviposit on the substrate. The number of eggs oviposited in one submerged bout is usually up to 40. After oviposition, the females release the substrate and float to the surface. The maximum depth of eggs oviposited in the wild is 80 cm below the water surface (Amano et al. 2008). Both surface oviposition and submerged oviposition are performed either by solitary females or tandem pairs (with the male on the back of the female). In submerged oviposition, the submerging method (solitary females or tandem pairs) affects the duration of the subsequent submerging bout and the number of eggs oviposited in the bout (Hirayama & Kasuya 2008). Tiphodytes gerriphagus Tiphodytes gerriphagus has a multivoltine life cycle in Japan. Since T. gerriphagus is small (about 1 mm), capturing adults is difficult. So instead we collected A. paludum eggs parasitized by T. gerriphagus at Higashi Koen Pond in Fukuoka, Japan (33 360 2000 N, 130 2501200 E) from July to September 2006. Since the frequency of egg parasitism was very high in this period, we were able to get parasitized eggs by collecting eggs near the water surface. These eggs were kept in vials (40 mm in diameter, 75 mm high, water filled 40 mm deep) in the wasp room. This room (16:8 h photoperiod and temperature kept at 25 C) was different from the experimental room. Emerging adult wasps were moved to a new vial with fresh A. paludum eggs and were allowed to oviposit. We bred the wasps by repeating this procedure. Adult wasps were used for experiments within 24 h of emergence. The wasps were reared and all experimental procedures where the wasps were used were conducted in the wasp room. Adult female T. gerriphagus search for eggs of water striders on or near the water surface. They frequently submerge in the water on the vegetation (similar to the manner of A. paludum females) to find eggs of water striders. When female wasps discover the eggs, they insert their ovipositor and lay eggs (one egg per host egg, Henriquez & Spence 1993). The period from the egg to emergence of the adult is about 17 days and the longevity of adults in the laboratory is 3–4 days (personal observation). Experimental Methods Experiment 1: low-density treatment The experimental period was July to September 2006. On a given day, we randomly chose four to eight pairs (a female and a male) of water striders. Half of them (two to four pairs) were allocated to the control group and half to the low-density treatment. In the control, a pair of water strides was kept in a tightly sealed transparent plastic container (7 cm in diameter, 5 cm high, water filled 1 cm deep) for 24 h. In the low-density treatment, a pair of water striders was kept in the container with a pair (a female and a male) of T. gerriphagus for 24 h. The containers of the control were placed in an incubator (16:8 h photoperiod and temperature kept at 25 C) in the experimental room, and to prevent contamination of control groups with chemical, visual or any other cues of wasps, those of the low-density treatment were, placed in the wasp room (photoperiod and temperature were identical to the incubator). Since the container had no oviposition substrate, the water striders experienced the wasp at a place where they could not oviposit. Just after the treatments, the wasps were removed from the containers and the water striders were taken to the experimental room where control groups were housed. All procedures after the treatment were conducted in the experimental room. To exclude any effect of movement on the experiment, we also moved the control group from the experimental room to the wasp room, and back to the experimental room. Therefore, except for the wasp
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
density, the conditions of the two treatments were similar. A pair of water striders from the container was put into one section of the oviposition vessel and allowed to oviposit for 5 h. The oviposition vessels (polypropylene, 30 36 cm and 60 cm high, water depth 50 cm) were partitioned into four sections (15 18 cm, 60 cm high, water depth 50 cm) with black polypropylene boards, which prevented visual and physical interference including ripples between neighbouring sections. A wooden stake (6 mm in diameter, 53 cm in length) was set in the centre of each section as the oviposition substrate. Weights were attached at the bottom end of the stakes. A new stake was used in each trial. Since dissolved oxygen in the water affects the duration of the submerging bout and the number and depth of eggs oviposited in the bout (Hirayama & Kasuya 2008), water in the vessels was exchanged the night before the trials and was exposed to the air. One night was sufficient for saturation of dissolved oxygen. During the trials, oviposition behaviours were recorded by four video cameras (Sony handycam video CCD-TR250) connected to a digital video recorder (Tsukamoto Musen, W-DVR100). A trial consisted of a 5 h period within the vessel. We conducted the first trial just after the treatments and a second 4–7 days later. After the first trial, we counted the eggs oviposited on the substrate and measured the depth of eggs oviposited (1 cm). We recorded the manner of oviposition (surface or submerged oviposition) from the video records. We also recorded the submerging method (single female or tandem pair) and the duration submerged (1 s; methods were identical to Hirayama & Kasuya 2008). After the first trial, the water striders were moved to aquaria (30 45 cm and 15 cm, high, water filled 5 cm deep) without an oviposition substrate. After 3–6 days in the aquaria, the water striders in one aquarium were randomly paired and used for the second trial. All pairs from one aquarium were tested on the same day. The second trial was conducted in the same manner as the first, but the water striders did not experience the wasps between the first and second trials (only before the first trial). We conducted third, fourth and fifth trials by repeating this procedure. When females or/and males died in one aquarium, we did not add new individuals, so there was sometimes a shortage of males to pair with females. In these cases, only females were used for the trials. Trials where only females were used were excluded from subsequent analysis. Experiment 2: high-density treatment The experimental period was August to November 2006. On a given day, we randomly chose four to eight pairs (one female and one male) of water striders. Half of them (two to four pairs) were allocated to the control and half to the high-density treatment. The control was conducted in the same manner as in experiment 1. In the high-density treatment, a pair of water striders was kept in the container with six wasps for 24 h and a container held both sexes of the wasps. Other conditions and procedures were identical to those in experiment 1. Statistical analysis We analysed the effects of the treatments (low density in experiment 1; high density in experiment 2) and the order of the trial on the oviposition decision (to oviposit or not), on the submersion decision (to submerge or not), on the depth of eggs oviposited, on the number of eggs oviposited and on the duration of submersion by using generalized liner models (GLMs; McCullagh & Nelder 1989). For the oviposition decision the response variable was binary: females oviposited or not. We considered that oviposition had occurred when at least one egg was seen on the substrate. We did
937
not distinguish between surface oviposition and submerged oviposition in this analysis. The proportion of ovipositing pairs (the number of ovipositing pairs/the number of pairs used) in each trial was used as the response variable. For the submersion decision, the response variable was also binary: females submerged or not. We considered oviposition to be submerged when we observed it at least once in a trial. In this analysis, submerged oviposition includes cases where females performed both surface and submerged oviposition in a trial. Females that did not oviposit in a trial were excluded. For egg depth, the response variable was the mean depth of eggs oviposited by any given female in a trial. Females that had not oviposited in a trial were excluded from this analysis. Since females sometimes submerged more than once in a trial (five females in experiment 1; seven females in experiment 2), the duration of submersion was analysed based on two measures: total duration of submersion (sum of the duration of all the bouts of submerged oviposition of a female in a trial) and the mean duration of submersion (total duration of submersion divided by the number of bouts of submerged oviposition of a single female in a trial). However, we give only the result for mean duration submersion because both measures gave similar results. Females that did not perform submerged oviposition were excluded from this analysis. Ten females performed submerged oviposition both solitarily and in tandem pairs in a single trial (three cases of all the 14 submerged ovipositions in experiment 1; seven cases of all the 40 submerged ovipositions in experiment 2). Since the submerging method (solitary females or tandem pairs) did not affect the results of the analyses, we pooled submerged oviposition by solitary females and that by tandem pairs in the analysis. The treatment, the order of the trials and the interaction between these variables were used as the explanatory variables in all the analyses. If the interaction was not significant, it was excluded from the model. The variable density had a binary distribution (0 denotes the control and 1 the low-density treatment in experiment 1; 0 denotes the control and 1 the high-density treatment in experiment 2). We assigned scores to trials according to their order (e.g. the first trial as 1, the second trial as 2). The binominal family and the logit link were used when the oviposition decision or submersion decision was the response variable. The data on the depth of eggs oviposited, the number of eggs oviposited and duration of submersion showed unequal variances. Therefore, we used GLMs with the quasilikelihoood (for details, see Hirayama & Kasuya 2008). In all the cases where the quasilikelihood was used, we adopted the variance proportional to the mean and the identity link. We used a likelihood ratio test (McCullagh & Nelder 1989) to analyse the effect of the wasp density on the depth of eggs oviposited by submerging females, because the data showed unequal variances. The null hypothesis was that the effect of the low-density treatment on the depth was the same as that of the high-density treatment (the difference in depth between the control and the wasp treatment in experiment 1 was same as that in experiment 2). The alternative hypothesis was that the effect of the low-density treatment in depth was different from that of the high-density treatment (the difference of the depth between the control and the wasp treatment was different in the two experiments). Log likelihoods of models corresponding to the null and alternative hypotheses for the same data set were compared. As the test statistic, we used the log likelihood ratio statistic, which is 2 (the difference between the log likelihood of the two models), where one model represents the null hypothesis and the other represents the alternative one. The variances were allowed to differ between the combinations of treatments and experiments and
938
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
were estimated from the data. Since the model that represents the alternative hypothesis has one more parameter than that for the null hypothesis, the likelihood ratio statistic was compared with the chi-square distribution with one degree of freedom. The individual identity of A. paludum, the body size (female: mean SD ¼ 14.22 0.53 mm, N ¼ 62; male: 11.67 0.46 mm, N ¼ 64) and the wing morph (short-winged female: 40; longwinged female: 22; short-winged male: 34; long-winged male: 30) did not affect the results of the analysis. In the high-density treatment of experiment 2, the sex ratio of the wasp (male proportion: mean SD ¼ 0.43 0.19, N ¼ 16) did not affect the results of the analysis. All the tests were two tailed and the level of significance was 0.05. All the statistical procedures were performed using the software package R 2.6.0 (R Development Core Team 2008). RESULTS Sixteen pairs of A. paludum were used in each treatment. For the low-density treatment of experiment 1 and in the control of experiment 2, in each case one female failed to oviposit throughout all the trials. These two females were excluded from the analysis. In the high-density treatment (experiment 2), in four cases we were not able to confirm the duration of submerged oviposition owing to insufficient video-recording imagery. These four cases were also excluded from the analysis of the duration of submersion. Experiment 1: Low-density Treatment The mean depth of eggs oviposited in the low-density treatment was slightly, but significantly, greater than that of the control through all the trials (Table 1, Fig. 1d). The order of the trials had no significant effect (Table 1). For the number of eggs oviposited, only the interaction was significant (Table 1). This shows that the difference in the number of eggs oviposited between the control and the low-density treatment became larger in later trials (Fig. 1c). Neither experience with the parasitoid nor the order of trials had an effect on the proportion of ovipositing pairs, the proportion of pairs that submerged and the mean duration of submersion in experiment 1 (Table 1, Fig. 1a, b, e). Experiment 2: High-density Treatment The proportion of submerging pairs was larger in the highdensity treatment than in the control in eight of the nine trials (the exception being the ninth trial; Fig. 1g), and the effect of the treatment was significant (Table 2). The order of trials did not have a significant affect (Table 2).
The mean depth of eggs oviposited was greater in the highdensity treatment than in the control in eight of the nine trials (with the exception of the ninth; Fig. 1i). The treatment and the interaction were significant, and the order of trials was not significant (Table 2). This probably shows that the depth in the first trial was greater than that in the other eight trials in the high-density treatment, while the depth in the control group varied slightly between trials (Fig. 1i). In the high-density treatment, the depth in the first trial was significantly greater than that in the second to ninth trials (mean SD ¼ 1.97 2.51, N ¼ 53; GLM, quasifamily and identity link: b SE ¼ 8.2 2.4, df ¼ 60, Z ¼ 3.35, P < 0.01; Fig. 1i). This is consistent with our explanation that the depth in the first trial was greater than that in the other eight trials. Neither experience with the parasitoid nor the order of trials had an effect on the proportion of ovipositing pairs, the number of eggs oviposited and the mean duration of submersion in experiment 2 (Table 2, Fig. 1f, h, j). The results of experiments 1 and 2 indicate that, when A. paludum had experienced a high density of wasps just before a trial (as in the first trial of the high-density treatment), they then submerged more frequently and oviposited at a greater depth. In contrast, when they had experienced a low wasp density just before (as in the first trial of the low-density treatment), they oviposited at a slightly greater depth but did not increase their frequency of submersion. The oviposition decision, the number of eggs oviposited and the duration of submersion were not affected by experience of the wasps in experiments 1 and 2. The effect of experience in the high-density treatment on the depth of eggs oviposited was strongest just after the treatment (the first trial) and persisted, although weakening, in subsequent trials. Effect of Wasp Density on Depth of Eggs The difference in the mean depth of eggs oviposited by submerging females between the control and the wasp treatment was significantly greater in experiment 2 (control: mean SD 1.86 1.15 cm, N ¼ 12; high-density treatment: 5.58 8.24 cm, N ¼ 28) than in experiment 1 (control: 0.79 0.60 cm, N ¼ 6; highdensity treatment: 1.59 0.98 cm, N ¼ 8; likelihood ratio statistic ¼ 4.16, P ¼ 0.041). This indicates that the females that performed submerged oviposition in the high-density treatment oviposited deeper than those in the low-density treatment. In this analysis, we pooled all the trials because the order of trials did not influence the results. DISCUSSION Previous studies on oviposition site selection have not examined the effect of experienced risk on subsequent oviposition are
Table 1 Results of the GLM analysis in experiment 1 Response variables
df
Explanatory variables Treatment
Oviposition decision Submersion decision Mean depth of eggs No. of eggs Duration of submersion
98 69 69 69 13
Order of trials
Interaction
bSE
Z
P
bSE
Z
P
bSE
Z
P
0.310.45 0.440.60 0.320.15 6.527.45 54.1141.4
0.70 0.73 2.09 0.87 0.38
0.48 0.46 0.04 0.38 0.70
0.280.24 0.080.23 0.0790.057 2.791.57 3.275.0
1.16 0.37 1.37 1.77 0.043
0.24 0.70 0.17 0.08 0.96
d d d 6.102.71 d
d d d 2.24 d
d d d 0.03 d
Oviposition decision: females oviposited or not; submersion decision females performed submerged oviposition at least once in the trial or performed only surface oviposition (did not submerge). The binomial family and logit link were used in the analysis of oviposition decision and submersion decision. The quasilikelihood with the variance proportional to the mean and the identity link were used in the analysis of mean depth of eggs, the number of eggs and the duration of submersion. For further explanation, see text.
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
Experiment 1
Proportion of ovipositing pairs
1
(a) 15 0.75
14 15
0.5
16
0.25
8
10
Experiment 2 4
(f)
8
4
15
Proportion of submerging pairs
15
16
Control Low density
0 1
9 15
10
939
(b)
(g)
60 (c)
(h)
14
14
14 15 14 Control High density
14
14
5
5
5
5
13 9
0.75 0.5 0.25
No. of eggs
0
40 20 0
Submerging duration (s)
Depth of eggs (cm)
25
(d)
(i)
20 15 10 5 0 1200
(e)
(j)
800 400 0
1st
2nd
3rd
4th
5th
Order of trials
1st 2nd 3rd 4th 5th 6th 7th 8th 9th Order of trials
Figure 1. Summary of experiments 1 (a–e) and 2 (f–j). (a), (f) Number of ovipositing pairs divided by number of experimental pairs. The number of pairs used in the trials is shown. (b), (g) Number of submerging pairs divided by number of ovipositing pairs. (c), (h) Number of eggs oviposited. (d), (i) Mean depth of eggs oviposited. (e), (j) Mean duration of submersion. In (c), (d), (h) and (i), pairs that did not oviposit were excluded. Similarly, in (e) and (j), pairs that did not perform submerged oviposition were excluded. Mean þ SDs are shown in (c), (d), (e), (h), (i) and (j).
Table 2 Results of the GLM analysis in experiment 2 Response variables
df
Explanatory variables Treatment
Oviposition decision Submersion decision Mean depth of eggs No. of eggs Duration of submersion
210 119 119 119 35
Order of trials
Interaction
bSE
Z
P
bSE
Z
P
bSE
Z
P
0.0560.28 1.200.41 4.591.28 20.115.8 84.2101.6
0.20 2.91 3.58 1.27 0.82
0.84 <0.01 <0.001 0.20 0.41
0.110.061 0.0120 083 0.0300.133 2.233.09 8.0816.9
1.91 0.14 0.22 0.72 0.47
0.06 0.88 0.82 0.47 0.63
d d 0.520.22 d d
d d 2.28 d d
d d 0.02 d d
For further explanation, see text. The terms in the table are the same as in Table 1.
940
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
detected. We gave a number of A. paludum pairs experience of the egg parasitoid wasp T. gerriphagus at sites without any oviposition substrate (i.e. sites that were not potential oviposition sites). In both experiments 1 and 2, females that had experienced the wasps oviposited at a greater depth than those that had not (Fig. 1 d, i). This shows that A. paludum takes into account the risk of egg parasitism from the presence of wasps at sites other than oviposition sites when choosing a place to oviposit. The finding that A. paludum oviposited at deeper positions even though at the oviposition site itself no wasps or cues of them were present suggests females had to use the memorized risk when choosing oviposition sites. These results suggest that the assessment of predation risk occurs and affects oviposition site selection over larger spatial and time scales than previous studies have demonstrated. In both the low- and high-density treatments A. paludum oviposited at deeper places than the controls. In experiment 1, where the water striders had experienced the low density of wasps, the proportion of females submerging did not increase and the difference in the depth of eggs oviposited from the control was small (Fig. 1 b, d). However, in experiment 2, where the water striders had experienced a high density of wasps, the proportion of females submerging increased and the difference in the depth of eggs oviposited from the control was large, particularly in the first trial (immediately after experience of the wasp; Fig. 1g, i). These results indicate that A. paludum evaluates the risk of egg parasitism from the density of wasps and the perceived risk is used to make decisions on submersion and the depth of eggs oviposited. Why did the females in the low-density treatment not increase the frequency of submerged oviposition? This was probably because the benefits of submerged oviposition did not exceed the costs in this case. Possible benefits of submerged oviposition are the avoidance of egg parasitism (Amano et al. 2008), the avoidance of harassment by males and protection of eggs from desiccation (Fincke 1986; Corbet 1999). On the other hand, possible costs of submerged oviposition are the risk of predation and drowning for parents (Fincke 1986; Corbet 1999), low hatching success at deeper sites (unpublished data) and the extra energy costs. The decision on submersion is expected to be affected by a combination of these costs and benefits. If the risk of egg parasitism is low (e.g. the control or the low-density treatment), the benefit of avoiding egg parasitism probably does not exceed the costs of submersion. In experiment 2, the depth of eggs oviposited in the highdensity treatment was greater than that of the control from the first to the eighth trial. The difference in the depth between the treatments was largest in the first trial (just after the treatments; Fig. 1i). These results show that the evaluated risk affects the decision on depth of oviposition not only immediately after the experience, although the effect became weaker with time. The oviposition depth in the high-density treatment was greater than that in the control from the first to the eighth trial (the minimum interval between trials was 3 days). The effect, therefore, of experience of a high density of wasps persists for at least 28 days. Why is the memory of the risk kept for so long? One possibility is that the experience of high egg parasitism risk means a high future risk for females. Since the wasp density may be similar on the day of the experience and on subsequent days, females may avoid egg parasitism from wasps of the same generation after a few days to a week from the experience. And since the wasp ecloses about 17 days after oviposition (in our laboratory), females may avoid egg parasitism from wasps of the next generation after around 20 days from the experience. Therefore, A. paludum keeps the memory of risk for a long period. However, the expected risk becomes less reliable with time. Therefore, from the second trial, the difference in the depth between the
treatments became smaller than in the first trial. In the wild, A. paludum probably experiences the wasp continuously. Therefore, it may use both current risk and memorized risk when deciding on the depth of oviposition. However, we need a further study to explore this. Owing to the small size of T. gerriphagus, it is difficult to estimate the density of wasps in the wild and thus to compare the wasp density in the experiments with those in the wild. However, a very high proportion of parasitism in the wild suggests a fairly high density of wasps. For example, in August, almost all A. paludum eggs oviposited at or near the water surface had been parasitized by the wasps (personal observation). Although we do not have adequate data on the adult density in the wild, it might be comparable to that in our experiments. Animals generally do not oviposit at the site where predators or their cues are present (e.g. Blaustein 1998). Few studies (e.g. Montserrat et al. 2007) have shown that females retain eggs in the presence of predators. The perceived risk of predation is expected to affect whether or not to oviposit, or the number of eggs oviposited afterwards, particularly just after experience of the predator. However A. paludum neither refrained from oviposition nor oviposited fewer eggs. This is probably because females are able to oviposit at places safe from egg parasitism. For A. paludum and T. gerriphagus, the response of the host to the risk of egg parasitism can be measured by the depth of A. paludum eggs oviposited (a continuous measure). This is a very useful feature for investigating oviposition site selection. Previous studies on oviposition site selection mainly focused on the oviposition decision (to oviposit or not) at one site. If the degree of oviposition avoidance at the site is small or the sample size is modest, the effect on oviposition site selection is difficult to detect by such a binary measure. Such a difficulty is removed by using A. paludum and T. gerriphagus. Previous studies on oviposition site selection examined whether the presence of the predator or its cues at potential oviposition sites affects the oviposition decision (Blaustein 1998; Stav et al. 1999; Eitam et al. 2002; Spencer et al. 2002; Kiflawi et al. 2003a, b; Blaustein et al. 2004, 2005; Brodin et al. 2006). The present study shows that the predation risk evaluated at other than potential oviposition sites affected the subsequent oviposition. Therefore, to examine the effect of predation risk on oviposition site selection, it is not sufficient only to examine the effect of the risk at potential oviposition sites and at the time of oviposition. The risk that females experience prior to the oviposition decision should also be considered, even if it is experienced at places other than potential oviposition sites. The effect of predation risk at one site on the population dynamics and the community structure through oviposition site selection is probably larger than previous studies have indicated. Acknowledgments We express our gratitude to all our colleagues at our laboratory, for their help and encouragement. In particular, we thank Takashi Kuriwada and Gen Sakurai for their assistance and advice. We also thank Chris Wood for correcting our paper and Dr Mike Speed and two anonymous referees for their helpful comments. This study was partly supported by JPSS Research Fellowships for Young Scientists. References Amano, H., Hayashi, K. & Kasuya, E. 2008. Avoidance of egg parasitism through submerged oviposition by tandem pairs in the water strider, Aquarius paludum insularis (Heteroptera: Gerridae). Ecological Entomology, 33, 560–563. Blaustein, L. 1998. Influence of the predatory backswimmer, Notonecta maculata, on invertebrate community structure. Ecological Entomology, 23, 246–252. Blaustein, L., Kiflawi, M., Eitam, A., Mangel, M. & Cohen, J. 2004. Oviposition habitat selection in response to risk of predation in temporary pools: mode
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941 of detection and consistency across experimental venue. Oecologia, 138, 300–305. Blaustein, L., Blaustein, J. & Chase, J. 2005. Chemical detection of the predator Notonecta irrorata by ovipositing Culex mosquitoes. Journal of Vector Ecology, 30, 299–301. Brodin, T., Johansson, F. & Bergsten, J. 2006. Predator related oviposition site selection of aquatic beetles (Hydroporus spp.) and effects on offspring lifehistory. Freshwater Biology, 51, 1277–1285. Corbet, P. S. 1999. Dragonflies: Behaviour and Ecology of Odonata. Colchester: Harley Books. Eitam, A., Blaustein, L. & Mangel, M. 2002. Effects of Anisops sardea (Hemiptera: Notonectidae) on oviposition habitat selection by mosquitoes and other dipterans and on community structure in artificial pools. Hydrobiologia, 485, 183–189. Fincke, O. M. 1986. Underwater oviposition in a damselfly (Odonata, Coenagrionidae) favors male vigilance and multiple mating by females. Behavioral Ecology and Sociobiology, 18, 405–412. Henriquez, N. P. & Spence, J. R. 1993. Host location by the gerrid egg parasitoid Tiphodytes gerriphagus (Marchal) (Hymenoptera, Scelionidae). Journal of Insect Behavior, 6, 455–466. Hirayama, H. & Kasuya, E. 2008. Factors affecting submerged oviposition in a water strider: level of dissolved oxygen and male presence. Animal Behaviour, 76, 1919–1926.
941
Kiflawi, M., Blaustein, L. & Mangel, M. 2003a. Predation-dependent oviposition habitat selection by the mosquito Culiseta longiareolata: a test of competing hypotheses. Ecology Letters, 6, 35–40. Kiflawi, M., Blaustein, L. & Mangel, M. 2003b. Oviposition habitat selection by the mosquito Culiseta longiareolata in response to risk of predation and conspecific larval density. Ecological Entomology, 28, 168–173. McCullagh, P. & Nelder, J. A. 1989. Generalized Linear Models, 2nd edn. London and New York: Chapman & Hall. Montserrat, M., Bas, C., Magalha˜es, S., Sabelis, M., Roos, M. & Janssen, A. 2007. Predators induce egg retention in prey. Oecologia, 150, 699–705. R Development Core Team 2008. R: a Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. Spence, J. R. 1986. Interactions between the scelionid egg parasitoid Tiphodytes gerriphagus (Hymenoptera) and its gerrid hosts (Heteroptera). Canadian Journal of Zoology, 64, 2728–2738. Spencer, M., Blaustein, L. & Cohen, J. E. 2002. Oviposition habitat selection by mosquitoes (Culiseta longiareolata) and consequences for population size. Ecology, 83, 669–679. Stav, G., Blaustein, L. & Margalith, J. 1999. Experimental evidence for predation risk sensitive oviposition by a mosquito, Culiseta longiareolata. Ecological Entomology, 24, 202–207.
Contents lists available at ScienceDirect
Animal Behaviour journal homepage: www.elsevier.com/locate/anbehav
Oviposition depth in response to egg parasitism in the water strider: high-risk experience promotes deeper oviposition Hiroyuki Hirayama*, Eiiti Kasuya Laboratory of Ecology, Department of Biology, Faculty of Sciences, Kyushu University
a r t i c l e i n f o Article history: Received 2 April 2009 Initial acceptance 14 May 2009 Final acceptance 18 July 2009 Published online 21 August 2009 MS. number: 09-00215R Keywords: Aquarium paludum egg parasitoid wasp experienced predation risk memory oviposition site selection submerged oviposition water strider
In many species, predation risk at oviposition sites causes females to avoid high-risk sites. Previous studies, however, have focused on assessment of current risk at the site during oviposition. Whether memory of previous risk plays a role has not been considered. We examined whether memorized risk affects oviposition site selection in the water strider Aquarius paludum insularis. Aquarius paludum eggs are laid on vegetation on or in the water and are frequently parasitized by a parasitoid wasp. Eggs at deeper positions are less frequently parasitized than those near the surface. After one of three treatments (no, low or high wasp density), A. paludum females were allowed to oviposit in parasitoid-free aquaria. The depth of oviposition of females that had previously experienced high wasp density was greater than that of those that had not experienced wasps. However, the difference in oviposition depth between lowdensity and no-wasp treatments was slight although significant. This indicates that A. paludum makes the decision on oviposition depth in response to the egg parasitism risk experienced before oviposition. The effect of the memorized risk was largest just after the treatment and became weaker over time. A measurable effect persisted for 28 days. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
In egg-laying species with no further parental care, offspring survival and growth strongly depend on the quality of the oviposition site. In particular, the presence of predators or parasitoids strongly affects decision making at oviposition (hereafter, predators and parasitoids are referred to collectively as predators). Most studies on this topic show that females avoid oviposition at sites where predators or cues of predators are present (e.g. Kiflawi et al. 2003a, b; Blaustein et al. 2004; Brodin et al. 2006). However, whether animals can memorize the predation risk and use it for subsequent oviposition site selection has not yet been examined. If animals can memorize the predation risk, oviposition site selection would be affected by predation risk on a broader scale, in terms of both time and area for oviposition, than has previously been considered. Specifically, the memorized predation risk may first affect oviposition site selection over a longer timescale. For example, the risk previously experienced upon arrival at the site may be used as well as the current risk at the site. Second, the risk may also affect oviposition site selection at a larger spatial scale. For example, the previously experienced risk at places other than the oviposition site (e.g. on the way to the site) may be used. Therefore, not only
* Correspondence: H. Hirayama, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka 812-8581, Japan. E-mail address: [email protected] (H. Hirayama).
present but also past distributions of predators may affect oviposition site selection. If females can memorize the predation risk, two questions arise. One is how long and strongly the predation risk needs to be experienced before it affects the subsequent choice of the oviposition site. The second is whether a predation risk experienced in a location other than the potential/intended oviposition site may affect the choice of the subsequent site. The water strider Aquarius paludum insularis (Heteroptera: Gerridae) and its egg parasitoid wasp Tiphodytes gerriphagus (Hymenoptera: Scelionidae) are suitable subjects to help resolve these two questions. Adult females of A. paludum usually oviposit on stems or leaves of vegetation in the water. The oviposition site of A. paludum is usually close to, or actually within, the area where they forage and mate. Therefore, A. paludum is likely to encounter the egg parasitoid wasp not only when it oviposits and searches for oviposition sites but also during activities other than oviposition on the water surface (e.g. foraging, resting and mating). Thus, A. paludum is expected to maintain a memory of risk at many sites other than oviposition sites. In A. paludum, ovipositing at deep sites from the surface enables the use of oviposition sites that are safe from egg parasitism. In the wild, A. paludum eggs are frequently parasitized but the proportion of parasitized eggs depends on the depth of eggs oviposited. Eggs oviposited underwater are less frequently parasitized than those on the water surface (Spence 1986) and, in the water, eggs oviposited
0003-3472/$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2009.07.019
936
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
at deeper positions are safer from egg parasitism than those at shallower positions (Amano et al. 2008). Female A. paludum can oviposit in two ways (personal observation). They either dip only the abdomen in the water and oviposit at a very shallow position (surface oviposition), or they completely submerge themselves underwater and oviposit at deeper positions (submerged oviposition). Submerged oviposition enables A. paludum to oviposit at places safe from egg parasitism but it involves costs (e.g. energetic cost, drowning risk and predation risk for the ovipositing females). Submerged oviposition is the only time during the adult phase when A. paludum females submerge themselves in the water. When the adult A. paludum performs submerged oviposition and lays eggs deep in the water to avoid egg parasitism, the decision to submerge and the subsequent depth of eggs oviposited are expected to vary in response to the risk of egg parasitism experienced before oviposition. When the risk experienced before oviposition is low, A. paludum females are expected to oviposit near the water surface (shallow position); when the risk is high, they are expected to submerge and oviposit in a deeper position. Our aim in this study was to examine two questions. One is whether A. paludum makes the decisions on submersion and egg depth in response to the presence of the egg parasitoid before oviposition at places other than the oviposition site. The other is, for how long and how strongly does the predation risk experienced affect subsequent oviposition site selection? METHODS Materials Aquarius paludum insularis Aquarius paludum has a trivoltine life cycle in Fukuoka, Japan, and eggs of the next generation of overwintered A. paludum are frequently parasitized by T. gerriphagus. Twenty pairs (a female and a male) of overwintered adult A. paludum insularis were caught at Todoroki pond in Hisayama, Japan (33 360 2900 N, 130 290 3400 E) in July 2006. We used the next generation of these 20 pairs as the experimental individuals. They were reared in a parasitoid-free experimental room (natural light conditions or longer photoperiod, with temperature kept at 25 C). Therefore, they had no prior experience of parasitoids before the experiment. We recorded the date of emergence, the body size, sex and wing morph (short or long winged), and marked them individually with a paint mark on the thorax (opaque color, Teranishi chemical industry, Osaka, Japan). The methods here were the same as in Hirayama & Kasuya (2008). The adults that had been observed to mate within 3 weeks of emergence were used in the experiments. Throughout all the developmental stages, A. paludum were kept in aquaria (30 45 cm and 15 cm high, water filled 5 cm deep), and given frozen blow flies for food every day and fragments of foam polystyrene for the oviposition substrate and for resting sites. Water in the aquaria was exchanged at least once every two days. The adult life span was approximately 2–3 months, and females oviposited every 2–4 days in the laboratory. Oviposition behaviour in A. paludum During surface oviposition (or no submersion) females hold the oviposition substrate (e.g. a plant stem in the wild) by the forelegs, dip the abdomen in the water and usually oviposit up to 30 eggs on the substrate. The maximum depth of eggs oviposited is approximately equivalent to the body length of adult females (1.5 cm). During submerged oviposition females hold the oviposition substrate by their forelegs and turn their heads to the water surface. They enter the water by moving down the substrate head first and
oviposit on the substrate. The number of eggs oviposited in one submerged bout is usually up to 40. After oviposition, the females release the substrate and float to the surface. The maximum depth of eggs oviposited in the wild is 80 cm below the water surface (Amano et al. 2008). Both surface oviposition and submerged oviposition are performed either by solitary females or tandem pairs (with the male on the back of the female). In submerged oviposition, the submerging method (solitary females or tandem pairs) affects the duration of the subsequent submerging bout and the number of eggs oviposited in the bout (Hirayama & Kasuya 2008). Tiphodytes gerriphagus Tiphodytes gerriphagus has a multivoltine life cycle in Japan. Since T. gerriphagus is small (about 1 mm), capturing adults is difficult. So instead we collected A. paludum eggs parasitized by T. gerriphagus at Higashi Koen Pond in Fukuoka, Japan (33 360 2000 N, 130 2501200 E) from July to September 2006. Since the frequency of egg parasitism was very high in this period, we were able to get parasitized eggs by collecting eggs near the water surface. These eggs were kept in vials (40 mm in diameter, 75 mm high, water filled 40 mm deep) in the wasp room. This room (16:8 h photoperiod and temperature kept at 25 C) was different from the experimental room. Emerging adult wasps were moved to a new vial with fresh A. paludum eggs and were allowed to oviposit. We bred the wasps by repeating this procedure. Adult wasps were used for experiments within 24 h of emergence. The wasps were reared and all experimental procedures where the wasps were used were conducted in the wasp room. Adult female T. gerriphagus search for eggs of water striders on or near the water surface. They frequently submerge in the water on the vegetation (similar to the manner of A. paludum females) to find eggs of water striders. When female wasps discover the eggs, they insert their ovipositor and lay eggs (one egg per host egg, Henriquez & Spence 1993). The period from the egg to emergence of the adult is about 17 days and the longevity of adults in the laboratory is 3–4 days (personal observation). Experimental Methods Experiment 1: low-density treatment The experimental period was July to September 2006. On a given day, we randomly chose four to eight pairs (a female and a male) of water striders. Half of them (two to four pairs) were allocated to the control group and half to the low-density treatment. In the control, a pair of water strides was kept in a tightly sealed transparent plastic container (7 cm in diameter, 5 cm high, water filled 1 cm deep) for 24 h. In the low-density treatment, a pair of water striders was kept in the container with a pair (a female and a male) of T. gerriphagus for 24 h. The containers of the control were placed in an incubator (16:8 h photoperiod and temperature kept at 25 C) in the experimental room, and to prevent contamination of control groups with chemical, visual or any other cues of wasps, those of the low-density treatment were, placed in the wasp room (photoperiod and temperature were identical to the incubator). Since the container had no oviposition substrate, the water striders experienced the wasp at a place where they could not oviposit. Just after the treatments, the wasps were removed from the containers and the water striders were taken to the experimental room where control groups were housed. All procedures after the treatment were conducted in the experimental room. To exclude any effect of movement on the experiment, we also moved the control group from the experimental room to the wasp room, and back to the experimental room. Therefore, except for the wasp
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
density, the conditions of the two treatments were similar. A pair of water striders from the container was put into one section of the oviposition vessel and allowed to oviposit for 5 h. The oviposition vessels (polypropylene, 30 36 cm and 60 cm high, water depth 50 cm) were partitioned into four sections (15 18 cm, 60 cm high, water depth 50 cm) with black polypropylene boards, which prevented visual and physical interference including ripples between neighbouring sections. A wooden stake (6 mm in diameter, 53 cm in length) was set in the centre of each section as the oviposition substrate. Weights were attached at the bottom end of the stakes. A new stake was used in each trial. Since dissolved oxygen in the water affects the duration of the submerging bout and the number and depth of eggs oviposited in the bout (Hirayama & Kasuya 2008), water in the vessels was exchanged the night before the trials and was exposed to the air. One night was sufficient for saturation of dissolved oxygen. During the trials, oviposition behaviours were recorded by four video cameras (Sony handycam video CCD-TR250) connected to a digital video recorder (Tsukamoto Musen, W-DVR100). A trial consisted of a 5 h period within the vessel. We conducted the first trial just after the treatments and a second 4–7 days later. After the first trial, we counted the eggs oviposited on the substrate and measured the depth of eggs oviposited (1 cm). We recorded the manner of oviposition (surface or submerged oviposition) from the video records. We also recorded the submerging method (single female or tandem pair) and the duration submerged (1 s; methods were identical to Hirayama & Kasuya 2008). After the first trial, the water striders were moved to aquaria (30 45 cm and 15 cm, high, water filled 5 cm deep) without an oviposition substrate. After 3–6 days in the aquaria, the water striders in one aquarium were randomly paired and used for the second trial. All pairs from one aquarium were tested on the same day. The second trial was conducted in the same manner as the first, but the water striders did not experience the wasps between the first and second trials (only before the first trial). We conducted third, fourth and fifth trials by repeating this procedure. When females or/and males died in one aquarium, we did not add new individuals, so there was sometimes a shortage of males to pair with females. In these cases, only females were used for the trials. Trials where only females were used were excluded from subsequent analysis. Experiment 2: high-density treatment The experimental period was August to November 2006. On a given day, we randomly chose four to eight pairs (one female and one male) of water striders. Half of them (two to four pairs) were allocated to the control and half to the high-density treatment. The control was conducted in the same manner as in experiment 1. In the high-density treatment, a pair of water striders was kept in the container with six wasps for 24 h and a container held both sexes of the wasps. Other conditions and procedures were identical to those in experiment 1. Statistical analysis We analysed the effects of the treatments (low density in experiment 1; high density in experiment 2) and the order of the trial on the oviposition decision (to oviposit or not), on the submersion decision (to submerge or not), on the depth of eggs oviposited, on the number of eggs oviposited and on the duration of submersion by using generalized liner models (GLMs; McCullagh & Nelder 1989). For the oviposition decision the response variable was binary: females oviposited or not. We considered that oviposition had occurred when at least one egg was seen on the substrate. We did
937
not distinguish between surface oviposition and submerged oviposition in this analysis. The proportion of ovipositing pairs (the number of ovipositing pairs/the number of pairs used) in each trial was used as the response variable. For the submersion decision, the response variable was also binary: females submerged or not. We considered oviposition to be submerged when we observed it at least once in a trial. In this analysis, submerged oviposition includes cases where females performed both surface and submerged oviposition in a trial. Females that did not oviposit in a trial were excluded. For egg depth, the response variable was the mean depth of eggs oviposited by any given female in a trial. Females that had not oviposited in a trial were excluded from this analysis. Since females sometimes submerged more than once in a trial (five females in experiment 1; seven females in experiment 2), the duration of submersion was analysed based on two measures: total duration of submersion (sum of the duration of all the bouts of submerged oviposition of a female in a trial) and the mean duration of submersion (total duration of submersion divided by the number of bouts of submerged oviposition of a single female in a trial). However, we give only the result for mean duration submersion because both measures gave similar results. Females that did not perform submerged oviposition were excluded from this analysis. Ten females performed submerged oviposition both solitarily and in tandem pairs in a single trial (three cases of all the 14 submerged ovipositions in experiment 1; seven cases of all the 40 submerged ovipositions in experiment 2). Since the submerging method (solitary females or tandem pairs) did not affect the results of the analyses, we pooled submerged oviposition by solitary females and that by tandem pairs in the analysis. The treatment, the order of the trials and the interaction between these variables were used as the explanatory variables in all the analyses. If the interaction was not significant, it was excluded from the model. The variable density had a binary distribution (0 denotes the control and 1 the low-density treatment in experiment 1; 0 denotes the control and 1 the high-density treatment in experiment 2). We assigned scores to trials according to their order (e.g. the first trial as 1, the second trial as 2). The binominal family and the logit link were used when the oviposition decision or submersion decision was the response variable. The data on the depth of eggs oviposited, the number of eggs oviposited and duration of submersion showed unequal variances. Therefore, we used GLMs with the quasilikelihoood (for details, see Hirayama & Kasuya 2008). In all the cases where the quasilikelihood was used, we adopted the variance proportional to the mean and the identity link. We used a likelihood ratio test (McCullagh & Nelder 1989) to analyse the effect of the wasp density on the depth of eggs oviposited by submerging females, because the data showed unequal variances. The null hypothesis was that the effect of the low-density treatment on the depth was the same as that of the high-density treatment (the difference in depth between the control and the wasp treatment in experiment 1 was same as that in experiment 2). The alternative hypothesis was that the effect of the low-density treatment in depth was different from that of the high-density treatment (the difference of the depth between the control and the wasp treatment was different in the two experiments). Log likelihoods of models corresponding to the null and alternative hypotheses for the same data set were compared. As the test statistic, we used the log likelihood ratio statistic, which is 2 (the difference between the log likelihood of the two models), where one model represents the null hypothesis and the other represents the alternative one. The variances were allowed to differ between the combinations of treatments and experiments and
938
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
were estimated from the data. Since the model that represents the alternative hypothesis has one more parameter than that for the null hypothesis, the likelihood ratio statistic was compared with the chi-square distribution with one degree of freedom. The individual identity of A. paludum, the body size (female: mean SD ¼ 14.22 0.53 mm, N ¼ 62; male: 11.67 0.46 mm, N ¼ 64) and the wing morph (short-winged female: 40; longwinged female: 22; short-winged male: 34; long-winged male: 30) did not affect the results of the analysis. In the high-density treatment of experiment 2, the sex ratio of the wasp (male proportion: mean SD ¼ 0.43 0.19, N ¼ 16) did not affect the results of the analysis. All the tests were two tailed and the level of significance was 0.05. All the statistical procedures were performed using the software package R 2.6.0 (R Development Core Team 2008). RESULTS Sixteen pairs of A. paludum were used in each treatment. For the low-density treatment of experiment 1 and in the control of experiment 2, in each case one female failed to oviposit throughout all the trials. These two females were excluded from the analysis. In the high-density treatment (experiment 2), in four cases we were not able to confirm the duration of submerged oviposition owing to insufficient video-recording imagery. These four cases were also excluded from the analysis of the duration of submersion. Experiment 1: Low-density Treatment The mean depth of eggs oviposited in the low-density treatment was slightly, but significantly, greater than that of the control through all the trials (Table 1, Fig. 1d). The order of the trials had no significant effect (Table 1). For the number of eggs oviposited, only the interaction was significant (Table 1). This shows that the difference in the number of eggs oviposited between the control and the low-density treatment became larger in later trials (Fig. 1c). Neither experience with the parasitoid nor the order of trials had an effect on the proportion of ovipositing pairs, the proportion of pairs that submerged and the mean duration of submersion in experiment 1 (Table 1, Fig. 1a, b, e). Experiment 2: High-density Treatment The proportion of submerging pairs was larger in the highdensity treatment than in the control in eight of the nine trials (the exception being the ninth trial; Fig. 1g), and the effect of the treatment was significant (Table 2). The order of trials did not have a significant affect (Table 2).
The mean depth of eggs oviposited was greater in the highdensity treatment than in the control in eight of the nine trials (with the exception of the ninth; Fig. 1i). The treatment and the interaction were significant, and the order of trials was not significant (Table 2). This probably shows that the depth in the first trial was greater than that in the other eight trials in the high-density treatment, while the depth in the control group varied slightly between trials (Fig. 1i). In the high-density treatment, the depth in the first trial was significantly greater than that in the second to ninth trials (mean SD ¼ 1.97 2.51, N ¼ 53; GLM, quasifamily and identity link: b SE ¼ 8.2 2.4, df ¼ 60, Z ¼ 3.35, P < 0.01; Fig. 1i). This is consistent with our explanation that the depth in the first trial was greater than that in the other eight trials. Neither experience with the parasitoid nor the order of trials had an effect on the proportion of ovipositing pairs, the number of eggs oviposited and the mean duration of submersion in experiment 2 (Table 2, Fig. 1f, h, j). The results of experiments 1 and 2 indicate that, when A. paludum had experienced a high density of wasps just before a trial (as in the first trial of the high-density treatment), they then submerged more frequently and oviposited at a greater depth. In contrast, when they had experienced a low wasp density just before (as in the first trial of the low-density treatment), they oviposited at a slightly greater depth but did not increase their frequency of submersion. The oviposition decision, the number of eggs oviposited and the duration of submersion were not affected by experience of the wasps in experiments 1 and 2. The effect of experience in the high-density treatment on the depth of eggs oviposited was strongest just after the treatment (the first trial) and persisted, although weakening, in subsequent trials. Effect of Wasp Density on Depth of Eggs The difference in the mean depth of eggs oviposited by submerging females between the control and the wasp treatment was significantly greater in experiment 2 (control: mean SD 1.86 1.15 cm, N ¼ 12; high-density treatment: 5.58 8.24 cm, N ¼ 28) than in experiment 1 (control: 0.79 0.60 cm, N ¼ 6; highdensity treatment: 1.59 0.98 cm, N ¼ 8; likelihood ratio statistic ¼ 4.16, P ¼ 0.041). This indicates that the females that performed submerged oviposition in the high-density treatment oviposited deeper than those in the low-density treatment. In this analysis, we pooled all the trials because the order of trials did not influence the results. DISCUSSION Previous studies on oviposition site selection have not examined the effect of experienced risk on subsequent oviposition are
Table 1 Results of the GLM analysis in experiment 1 Response variables
df
Explanatory variables Treatment
Oviposition decision Submersion decision Mean depth of eggs No. of eggs Duration of submersion
98 69 69 69 13
Order of trials
Interaction
bSE
Z
P
bSE
Z
P
bSE
Z
P
0.310.45 0.440.60 0.320.15 6.527.45 54.1141.4
0.70 0.73 2.09 0.87 0.38
0.48 0.46 0.04 0.38 0.70
0.280.24 0.080.23 0.0790.057 2.791.57 3.275.0
1.16 0.37 1.37 1.77 0.043
0.24 0.70 0.17 0.08 0.96
d d d 6.102.71 d
d d d 2.24 d
d d d 0.03 d
Oviposition decision: females oviposited or not; submersion decision females performed submerged oviposition at least once in the trial or performed only surface oviposition (did not submerge). The binomial family and logit link were used in the analysis of oviposition decision and submersion decision. The quasilikelihood with the variance proportional to the mean and the identity link were used in the analysis of mean depth of eggs, the number of eggs and the duration of submersion. For further explanation, see text.
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
Experiment 1
Proportion of ovipositing pairs
1
(a) 15 0.75
14 15
0.5
16
0.25
8
10
Experiment 2 4
(f)
8
4
15
Proportion of submerging pairs
15
16
Control Low density
0 1
9 15
10
939
(b)
(g)
60 (c)
(h)
14
14
14 15 14 Control High density
14
14
5
5
5
5
13 9
0.75 0.5 0.25
No. of eggs
0
40 20 0
Submerging duration (s)
Depth of eggs (cm)
25
(d)
(i)
20 15 10 5 0 1200
(e)
(j)
800 400 0
1st
2nd
3rd
4th
5th
Order of trials
1st 2nd 3rd 4th 5th 6th 7th 8th 9th Order of trials
Figure 1. Summary of experiments 1 (a–e) and 2 (f–j). (a), (f) Number of ovipositing pairs divided by number of experimental pairs. The number of pairs used in the trials is shown. (b), (g) Number of submerging pairs divided by number of ovipositing pairs. (c), (h) Number of eggs oviposited. (d), (i) Mean depth of eggs oviposited. (e), (j) Mean duration of submersion. In (c), (d), (h) and (i), pairs that did not oviposit were excluded. Similarly, in (e) and (j), pairs that did not perform submerged oviposition were excluded. Mean þ SDs are shown in (c), (d), (e), (h), (i) and (j).
Table 2 Results of the GLM analysis in experiment 2 Response variables
df
Explanatory variables Treatment
Oviposition decision Submersion decision Mean depth of eggs No. of eggs Duration of submersion
210 119 119 119 35
Order of trials
Interaction
bSE
Z
P
bSE
Z
P
bSE
Z
P
0.0560.28 1.200.41 4.591.28 20.115.8 84.2101.6
0.20 2.91 3.58 1.27 0.82
0.84 <0.01 <0.001 0.20 0.41
0.110.061 0.0120 083 0.0300.133 2.233.09 8.0816.9
1.91 0.14 0.22 0.72 0.47
0.06 0.88 0.82 0.47 0.63
d d 0.520.22 d d
d d 2.28 d d
d d 0.02 d d
For further explanation, see text. The terms in the table are the same as in Table 1.
940
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941
detected. We gave a number of A. paludum pairs experience of the egg parasitoid wasp T. gerriphagus at sites without any oviposition substrate (i.e. sites that were not potential oviposition sites). In both experiments 1 and 2, females that had experienced the wasps oviposited at a greater depth than those that had not (Fig. 1 d, i). This shows that A. paludum takes into account the risk of egg parasitism from the presence of wasps at sites other than oviposition sites when choosing a place to oviposit. The finding that A. paludum oviposited at deeper positions even though at the oviposition site itself no wasps or cues of them were present suggests females had to use the memorized risk when choosing oviposition sites. These results suggest that the assessment of predation risk occurs and affects oviposition site selection over larger spatial and time scales than previous studies have demonstrated. In both the low- and high-density treatments A. paludum oviposited at deeper places than the controls. In experiment 1, where the water striders had experienced the low density of wasps, the proportion of females submerging did not increase and the difference in the depth of eggs oviposited from the control was small (Fig. 1 b, d). However, in experiment 2, where the water striders had experienced a high density of wasps, the proportion of females submerging increased and the difference in the depth of eggs oviposited from the control was large, particularly in the first trial (immediately after experience of the wasp; Fig. 1g, i). These results indicate that A. paludum evaluates the risk of egg parasitism from the density of wasps and the perceived risk is used to make decisions on submersion and the depth of eggs oviposited. Why did the females in the low-density treatment not increase the frequency of submerged oviposition? This was probably because the benefits of submerged oviposition did not exceed the costs in this case. Possible benefits of submerged oviposition are the avoidance of egg parasitism (Amano et al. 2008), the avoidance of harassment by males and protection of eggs from desiccation (Fincke 1986; Corbet 1999). On the other hand, possible costs of submerged oviposition are the risk of predation and drowning for parents (Fincke 1986; Corbet 1999), low hatching success at deeper sites (unpublished data) and the extra energy costs. The decision on submersion is expected to be affected by a combination of these costs and benefits. If the risk of egg parasitism is low (e.g. the control or the low-density treatment), the benefit of avoiding egg parasitism probably does not exceed the costs of submersion. In experiment 2, the depth of eggs oviposited in the highdensity treatment was greater than that of the control from the first to the eighth trial. The difference in the depth between the treatments was largest in the first trial (just after the treatments; Fig. 1i). These results show that the evaluated risk affects the decision on depth of oviposition not only immediately after the experience, although the effect became weaker with time. The oviposition depth in the high-density treatment was greater than that in the control from the first to the eighth trial (the minimum interval between trials was 3 days). The effect, therefore, of experience of a high density of wasps persists for at least 28 days. Why is the memory of the risk kept for so long? One possibility is that the experience of high egg parasitism risk means a high future risk for females. Since the wasp density may be similar on the day of the experience and on subsequent days, females may avoid egg parasitism from wasps of the same generation after a few days to a week from the experience. And since the wasp ecloses about 17 days after oviposition (in our laboratory), females may avoid egg parasitism from wasps of the next generation after around 20 days from the experience. Therefore, A. paludum keeps the memory of risk for a long period. However, the expected risk becomes less reliable with time. Therefore, from the second trial, the difference in the depth between the
treatments became smaller than in the first trial. In the wild, A. paludum probably experiences the wasp continuously. Therefore, it may use both current risk and memorized risk when deciding on the depth of oviposition. However, we need a further study to explore this. Owing to the small size of T. gerriphagus, it is difficult to estimate the density of wasps in the wild and thus to compare the wasp density in the experiments with those in the wild. However, a very high proportion of parasitism in the wild suggests a fairly high density of wasps. For example, in August, almost all A. paludum eggs oviposited at or near the water surface had been parasitized by the wasps (personal observation). Although we do not have adequate data on the adult density in the wild, it might be comparable to that in our experiments. Animals generally do not oviposit at the site where predators or their cues are present (e.g. Blaustein 1998). Few studies (e.g. Montserrat et al. 2007) have shown that females retain eggs in the presence of predators. The perceived risk of predation is expected to affect whether or not to oviposit, or the number of eggs oviposited afterwards, particularly just after experience of the predator. However A. paludum neither refrained from oviposition nor oviposited fewer eggs. This is probably because females are able to oviposit at places safe from egg parasitism. For A. paludum and T. gerriphagus, the response of the host to the risk of egg parasitism can be measured by the depth of A. paludum eggs oviposited (a continuous measure). This is a very useful feature for investigating oviposition site selection. Previous studies on oviposition site selection mainly focused on the oviposition decision (to oviposit or not) at one site. If the degree of oviposition avoidance at the site is small or the sample size is modest, the effect on oviposition site selection is difficult to detect by such a binary measure. Such a difficulty is removed by using A. paludum and T. gerriphagus. Previous studies on oviposition site selection examined whether the presence of the predator or its cues at potential oviposition sites affects the oviposition decision (Blaustein 1998; Stav et al. 1999; Eitam et al. 2002; Spencer et al. 2002; Kiflawi et al. 2003a, b; Blaustein et al. 2004, 2005; Brodin et al. 2006). The present study shows that the predation risk evaluated at other than potential oviposition sites affected the subsequent oviposition. Therefore, to examine the effect of predation risk on oviposition site selection, it is not sufficient only to examine the effect of the risk at potential oviposition sites and at the time of oviposition. The risk that females experience prior to the oviposition decision should also be considered, even if it is experienced at places other than potential oviposition sites. The effect of predation risk at one site on the population dynamics and the community structure through oviposition site selection is probably larger than previous studies have indicated. Acknowledgments We express our gratitude to all our colleagues at our laboratory, for their help and encouragement. In particular, we thank Takashi Kuriwada and Gen Sakurai for their assistance and advice. We also thank Chris Wood for correcting our paper and Dr Mike Speed and two anonymous referees for their helpful comments. This study was partly supported by JPSS Research Fellowships for Young Scientists. References Amano, H., Hayashi, K. & Kasuya, E. 2008. Avoidance of egg parasitism through submerged oviposition by tandem pairs in the water strider, Aquarius paludum insularis (Heteroptera: Gerridae). Ecological Entomology, 33, 560–563. Blaustein, L. 1998. Influence of the predatory backswimmer, Notonecta maculata, on invertebrate community structure. Ecological Entomology, 23, 246–252. Blaustein, L., Kiflawi, M., Eitam, A., Mangel, M. & Cohen, J. 2004. Oviposition habitat selection in response to risk of predation in temporary pools: mode
H. Hirayama, E. Kasuya / Animal Behaviour 78 (2009) 935–941 of detection and consistency across experimental venue. Oecologia, 138, 300–305. Blaustein, L., Blaustein, J. & Chase, J. 2005. Chemical detection of the predator Notonecta irrorata by ovipositing Culex mosquitoes. Journal of Vector Ecology, 30, 299–301. Brodin, T., Johansson, F. & Bergsten, J. 2006. Predator related oviposition site selection of aquatic beetles (Hydroporus spp.) and effects on offspring lifehistory. Freshwater Biology, 51, 1277–1285. Corbet, P. S. 1999. Dragonflies: Behaviour and Ecology of Odonata. Colchester: Harley Books. Eitam, A., Blaustein, L. & Mangel, M. 2002. Effects of Anisops sardea (Hemiptera: Notonectidae) on oviposition habitat selection by mosquitoes and other dipterans and on community structure in artificial pools. Hydrobiologia, 485, 183–189. Fincke, O. M. 1986. Underwater oviposition in a damselfly (Odonata, Coenagrionidae) favors male vigilance and multiple mating by females. Behavioral Ecology and Sociobiology, 18, 405–412. Henriquez, N. P. & Spence, J. R. 1993. Host location by the gerrid egg parasitoid Tiphodytes gerriphagus (Marchal) (Hymenoptera, Scelionidae). Journal of Insect Behavior, 6, 455–466. Hirayama, H. & Kasuya, E. 2008. Factors affecting submerged oviposition in a water strider: level of dissolved oxygen and male presence. Animal Behaviour, 76, 1919–1926.
941
Kiflawi, M., Blaustein, L. & Mangel, M. 2003a. Predation-dependent oviposition habitat selection by the mosquito Culiseta longiareolata: a test of competing hypotheses. Ecology Letters, 6, 35–40. Kiflawi, M., Blaustein, L. & Mangel, M. 2003b. Oviposition habitat selection by the mosquito Culiseta longiareolata in response to risk of predation and conspecific larval density. Ecological Entomology, 28, 168–173. McCullagh, P. & Nelder, J. A. 1989. Generalized Linear Models, 2nd edn. London and New York: Chapman & Hall. Montserrat, M., Bas, C., Magalha˜es, S., Sabelis, M., Roos, M. & Janssen, A. 2007. Predators induce egg retention in prey. Oecologia, 150, 699–705. R Development Core Team 2008. R: a Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. Spence, J. R. 1986. Interactions between the scelionid egg parasitoid Tiphodytes gerriphagus (Hymenoptera) and its gerrid hosts (Heteroptera). Canadian Journal of Zoology, 64, 2728–2738. Spencer, M., Blaustein, L. & Cohen, J. E. 2002. Oviposition habitat selection by mosquitoes (Culiseta longiareolata) and consequences for population size. Ecology, 83, 669–679. Stav, G., Blaustein, L. & Margalith, J. 1999. Experimental evidence for predation risk sensitive oviposition by a mosquito, Culiseta longiareolata. Ecological Entomology, 24, 202–207.