Nutrient regulation in a predator, the wolf spider Pardosa prativaga

Animal Behaviour 81 (2011) 993e999

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Nutrient regulation in a predator, the wolf spider Pardosa prativaga Kim Jensen a, b, c, *, David Mayntz c, d,1, Søren Toft c,1, David Raubenheimer e, 2, Stephen James Simpson b, 3 a

Department of Zoology, University of Oxford School of Biological Sciences, University of Sydney c Department of Biological Sciences, Ecology and Genetics, University of Aarhus d Department of Genetics and Biotechnology, University of Aarhus e Institute of Natural Sciences, Massey University b

a r t i c l e i n f o Article history: Received 9 November 2010 Initial acceptance 4 January 2011 Final acceptance 28 January 2011 Available online 8 March 2011 MS. number: 10-00778 Keywords: diet geometric framework lipid:protein ratio Lycosidae nutrient balancing Pardosa prativaga performance wolf spider

Nutrient balancing is well known in herbivores and omnivores, but has only recently been demonstrated in predators. To test how a predator might regulate nutrients when the prey varies in nutrient composition, we restricted juvenile Pardosa prativaga wolf spiders to diets of one of six fruit fly, Drosophila melanogaster, prey types varying in lipid:protein composition during their second instar. We collected all fly remnants to estimate food and nutrient intake over each meal. The spiders adjusted their capture rate and nutrient extraction in response to prey mass and nutrient composition irrespective of energy intake. Intake was initially regulated to a constant lipid plus protein mass, but later spiders fed on prey with high proportions of protein increased consumption relative to spiders fed on other prey types. This pattern indicates that the spiders were prepared to overconsume vast amounts of protein to gain more lipids and energy. The spiders also regulated protein after ingestion, and ingested protein was incorporated less efficiently into body tissue when the prey was protein rich. Despite both preand postingestive nutrient regulation, the body lipid:protein compositions of the spiders were highly affected by the nutrient compositions of their prey, and growth in carapace length and lean body mass increased with increasing prey protein:lipid ratio. Our results demonstrate that prey nutrient composition affects these predators, but also that the spiders possess behavioural and physiological adaptations that lead to partial compensation for these effects. Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

In contrast to herbivores (Behmer 2009; Felton et al. 2009) and omnivores (Raubenheimer & Jones 2006; Lee et al. 2008), predators have traditionally been thought not to balance nutrient intake. This belief has partly been based on the assumption that animal tissue as a food source varies little and is nutritionally balanced (Westoby 1978; Stephens & Krebs 1986; Galef 1996). In addition, predators are generally considered energy limited, and the amount of prey a predator can catch is typically considered the limiting factor for predator performance (Kessler 1971; Riechert 1992; Wise 1993). Regarding the first assumption, chemical analysis of invertebrate prey has revealed remarkable variation in nutrient composition among species (Elser et al. 2000; Fagan et al. 2002), and even within

* Correspondence and present address: K. Jensen, Biosciences, University of Exeter, Cornwall Campus, Daphne du Maurier, Penryn, TR10 9EZ, U.K. E-mail address: [email protected] (K. Jensen). 1 D. Mayntz and S. Toft are at the Department of Biological Sciences, Ecology and Genetics, University of Aarhus, Building 1540, 8000 Århus C, Denmark. 2 D. Raubenheimer is at the Institute of Natural Sciences, Massey University, Albany, Private Bag 102 904, North Shore Mail Centre, Auckland, New Zealand. 3 S. J. Simpson is at the School of Biological Sciences, University of Sydney, Heydon-Laurence Building A08, NSW 2006, Sydney, Australia.

species, nutrient composition may vary considerably depending on food source and feeding state (Lee et al. 2002; Simpson et al. 2002; Mayntz et al. 2005; Raubenheimer et al. 2007; Salomon et al. 2008). It is probably true that predators are often energy limited, but although a predator may face shortage of prey for extended periods, it would benefit from nutrient balancing during times of plenty. Nutrient balancing would also be of benefit in cases where the qualitative nutritional requirements of the predator change during development. In such cases, regulating energy intake alone might not match nutrient demands, for example during periods of fast growth. Greenstone (1979) first suggested that predators may select food items according to their nutrient contents. This hypothesis has later been supported experimentally in fish (Oncorhynchus mykiss: Sánchez-Vázquez et al. 1999; Dicentrarchus labrax: Rubio et al. 2003), the ground beetle Anchomenus dorsalis (Mayntz et al. 2005), mink, Mustela vison (Mayntz et al. 2009) and the anteating spider Zodarion rubidum (Pékar et al. 2010). As many predators are sedentary sit-and-wait hunters, however, balancing nutrients through active prey selection may be less pronounced in these predators compared to predators that actively pursue their prey. Sit-and-wait predators will be likely to catch whatever prey

0003-3472/$38.00 Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2011.01.035

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K. Jensen et al. / Animal Behaviour 81 (2011) 993e999

comes within their range, and prey would be caught and killed for as long as the predator needed nutrients. Nutrient balancing would then be more likely to occur at later stages, that is, during feeding, digestion and assimilation. In accordance with this, the wolf spider Pardosa prativaga, which is an intermediately mobile sit-and-wait predator, has been shown to regulate nutrient intake by extracting more dry mass from a prey item if it contained a higher proportion of a nutrient that was deficient in the previous prey (Mayntz et al. 2005). Here we investigated a different situation where juvenile P. prativaga were provided with prey of a constant lipid to protein ratio during an entire instar of development. We then tested how prey nutrient composition would affect the spider’s body composition and performance. By measuring nutrient extraction over each meal and by comparing nutrient extraction with nutrient growth over the instar, we furthermore investigated all possible behavioural and physiological mechanisms the spiders could use to regulate nutrients and reduce nutritional imbalance when feeding on nutritionally imbalanced prey.

METHODS Diets Drosophila melanogaster fruit flies of different body lipid to protein (L:P) ratio were produced by rearing the larvae in six different media (Table 1). Cultures were held in vials, 3.4 cm in diameter, containing 5 g of dry medium plus water and a few drops of dissolved yeast. Parental flies (40
5) were allowed 5 days in the tubes for egg laying. The temperature was held at 24e26  C. Only female flies were used, to reduce the variation in prey nutrient composition within diets.

Experimental Set-up Pardosa prativaga wolf spiders carrying eggsacs were collected in a wet meadow at Brabrand, Denmark, and brought to the laboratory. Two days after hatchlings emerged they were gently removed from their mother’s back with a paint brush and distributed to individual translucent plastic vials (diameter 2.0 cm, height 6 cm), each with a 1 cm, regularly moistened plaster of Paris bottom and a foam rubber stopper. To ensure that the young spiders were strong enough to catch and kill experimental fruit flies from the start of the experiment, we fed all hatchlings one Sinella curviseta collembolan daily from a culture reared on yeast until they moulted to the second instar. Spiderlings were starved over the 5 days following the moult to ensure that they would be hungry

and willing to eat from day 1. The experiment was performed at 21e25  C. Experimental Procedure On day 1, the spiderlings from nine mother spiders were weighed to the nearest mg. The spiderlings within and between mothers were then distributed as equally as possible among seven groups, totalling 27e28 spiderlings per group. One of the groups formed a start sample where spiderlings were killed by freezing at 18  C. Spiderlings in the remaining six groups each received one of the six different fly types only (Table 1). One fresh fly of the allocated nutritional composition was provided daily to each spider. Flies that were still alive the next day were replaced. Killed flies were recorded. Their remnants were stored individually in the freezer for chemical analysis the day after they were presented to the spiders. A reference sample of 25 flies was collected daily from each growth medium to ensure reliable estimates of nutrient contents of provided flies. On a few days with low fly populations on some media, samples down to 15 reference flies were taken. Spiders were killed on the day of moulting into the next instar by freezing at 18  C, and the carapace lengths of all spiders were measured with an eye piece scale under a microscope. Nutritional Analyses and Growth At the end of the experiment, all spiders, food remnants and reference flies were dried in a vacuum oven over 4 days at 60  C and weighed. Lipids (L) in each sample were extracted in two 24 h washes of 2 ml of chloroform, and samples were again dried and weighed. Lipid masses were calculated by subtracting sample lean (lipid extracted) dry masses from sample dry masses. Nitrogen content was analysed in a combustion analyser (Na 2000, Carlo Erba, Rodano, Italy). Because of technical problems, 16 spiders across diets were lost during nitrogen analysis. Food and nutrient intake of each meal consumed in the second instar were calculated by subtracting the dry masses and nutrient contents left in the fly remnants from the average dry masses and nutrient contents of the corresponding reference flies sampled on the feeding day. Initial carapace length, dry mass and nutrient contents of the spiderlings were estimated from their initial wet masses using linear correlations from the spiderlings in the initially killed start sample. Growth in linear body size, dry mass and nutrient masses were then calculated by subtracting estimated values from the values measured at the end of the experiment. Crude protein (P) masses and food energy contents were calculated using standard estimates of 6.25 mg protein per mg nitrogen, and 17 or 37 joules per mg protein or lipid, respectively (AOAC 2006).

Table 1 Growth media and body data of the six nutritionally different Drosophila melanogaster fruit fly types used in the experiment Fly type

Growth medium Dry mass (mg) Lean dry mass (mg) Lipids (%) Crude protein (%) L:P ratio No. of samples

LP0.89

LP0.64

LP0.40

LP0.25

LP0.15

LP0.10

1:4 Sucrose:Carolina 329
7cb 222
5a 32
1.1a 37
0.8a 0.89
0.05a 17

Pure Carolina 347
7cd 253
5b 27
1.1b 42
0.8b 0.64
0.03b 20

1:9 Casein:Carolina 383
10e 306
8c 20
0.8c 50
1.2c 0.40
0.02c 18

1:4 Casein:Carolina 352
8d 303
5c 14
1.0d 58
1.8d 0.25
0.02d 20

2:3 Casein:Carolina 316
7b 286
7c 10
0.9e 68
2.2e 0.15
0.02e 18

3:2 Casein:Carolina 287
7a 264
7b 8
0.7e 78
2.2f 0.10
0.01e 17

The media were based on Carolina Instant Drosophila Medium Formula 4-24 (Burlington, NC, U.S.A.), which was used in its pure form or mixed with sucrose (Fluka, 84097, Neu-Ulm, Germany) or casein (Sigma C-5890, SigmaeAldrich, Steinheim, Germany) at varying ratios. The fly data are mean
SE from 17 to 20 samples each consisting of 25 female flies collected from each medium per feeding day, except a few days when down to 15 female flies were collected from some media owing to shortage of flies. All ratios are mass based. Different letters indicate significant differences (Student’s t test: P < 0.05).

Number of flies killed and intakes of dry mass and energy were compared across diets using analysis of variance (ANOVA) tests. Nutrient compositions remaining in discarded remnants were compared to available nutrient compositions in the reference flies using post hoc Bonferroni-corrected t tests. To analyse data on nutrient regulation in the spiders, we used the geometric framework, which is a state-space modelling approach that was designed specifically to quantify the main and interactive effects of two or more nutritional components on intake and postingestive allocation (Raubenheimer & Simpson 1993; Simpson et al. 2004). Cumulative intake from the first five killed flies and the total intake across the instar were used to plot lipid versus protein intake arrays. The shapes of such arrays reveal how the intake of one nutrient is prioritized relative to the intake of the other. To test for differences in lipid plus protein mass extracted over consecutive meals, we fitted a line with slope 1 through cumulative lipid versus protein intake for each consumed fly and compared the residuals across diets using ANOVA tests. A slope of 1 would be consistent with spiders maintaining a constant nutrient mass intake rather than a constant energy intake (where the slope would be 0.5 given that lipid is twice as energy dense as protein). The utilization efficiencies of lipids and protein were tested using analysis of covariance (ANCOVA) tests with nutrient growth as the dependent variable and linear or logarithmic nutrient intake as the covariate. Final spider body L:P ratios were compared using ANOVA after arcsine transformation (Zar 1999). Growth in body dry mass, body lean dry mass and carapace length were analysed using ANCOVA tests with spider initial wet mass as the covariate (Raubenheimer & Simpson 1992, 1994; Raubenheimer 1995). Duration of the second instar was compared across diets with a Wilcoxon test. Treatment groups were compared individually using Student’s t test. Where parametric measures were used, residuals were normal or nearly normal. All statistical analyses were performed in JMP 7.0 (SAS Institute, Raleigh, NC, U.S.A.).

995

1 (a) 0.8 0.6 LP0.89 LP0.64 LP0.40 LP0.25 LP0.15 LP0.10

0.4 0.2 0 1

2

3

4 Day

5

6

7

0.25 Lipid + protein extracted (mg)

Statistical Analyses

Proportion of spiders killing the fly

K. Jensen et al. / Animal Behaviour 81 (2011) 993e999

(b) 0.2

0.15 LP0.89 LP0.64 LP0.40 LP0.25 LP0.15 LP0.10

0.1

0.05

1

2

3 4 Consecutive fly killed

5

Figure 1. (a) Proportion of spiders killing their fly from the first feeding day on different diets and (b) nutrients (lipid and protein) extracted from the first five killed flies per spider (mean
SE). Sample sizes in both figures are 27e28 per group for the first feeding and drop during the period as spiders that had moulted are not included. A period is included only if the sample size per group was nine or above.

RESULTS Diets The female flies produced on the six different media differed markedly in dry mass and body composition (Table 1). Flies raised on sugar-rich media contained more lipids and less protein compared to flies raised on more protein-rich media. Fly dry masses were highest on the intermediate media and smaller when flies were reared on media containing high amounts of protein or lipids (Table 1). Dry Mass and Energy Intake The daily probability that a spider would kill the provided fly (Fig. 1a) decreased during the instar for all prey types. Also, the nutrient mass extracted from successive killed flies decreased on all diets (Fig. 1b). The number of flies killed by spiders in the second instar differed across dietary treatments (ANOVA: F5,162 ¼ 3.26, P ¼ 0.008), with flies from media producing smaller dry body masses being killed in higher numbers than larger flies (Fig. 2a, Table 1). As a result, total dry mass consumption over the entire instar did not differ significantly across dietary treatments (ANOVA: F5,161 ¼ 0.10, P ¼ 0.99; Fig. 2b). Total energy intakes (ingested lipid plus protein energy) were highest on the most lipid-rich diets and lowest on the intermediary diets, with significant overall differences (ANOVA: F5,162 ¼ 2.89, P ¼ 0.016; Fig. 2c).

Nutrient Extraction In almost all cases, the nutrient contents of the discarded fly remnants did not differ significantly from the contents of the live flies (Bonferroni, t tests: P > 0.05; Fig. 3a), which indicates that the spiders extracted nutrients in proportions that were not different from the proportions in the prey (Fig. 3a). After the first meal, though, remnants of the LP0.89 flies contained significantly lower L:P ratios than the corresponding reference flies (Bonferroni, t test: P ¼ 0.001; Fig. 3b), indicating that the spiders feeding on the most lipid-rich flies extracted lipids in a higher proportion than if extracting at random during their first meal. Spiders feeding on the LP0.40 flies apparently extracted a higher proportion of protein than the proportion in the flies over the experiment (Bonferroni, t test: P ¼ 0.027; Fig. 3a), but since no clear pattern was found in the other diet treatments this might be a type 1 error. When protein intake was plotted against lipid intake, we found a distinct pattern of macronutrient regulation (Fig. 4). Over the first meals, intake points aligned along a slope that was not significantly different from 1 (ANOVA: P > 0.05), indicating that, at this point, the spiders’ mass intake of the two combined nutrients was nearly constant or regulated so that mass-based excesses and deficits were equal. During the last meals before moulting, though, protein was progressively ingested on the two most protein-rich diets, which by the fifth meal bent the intake array outwards about a pivot point at L:P ¼ 0.25, and the array from this meal no longer followed a line

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K. Jensen et al. / Animal Behaviour 81 (2011) 993e999

6 (a)

P = 0.008

1

(a)

Flies Remnants

0.8

a 5

0.6 ab

4.5

bc

bc

4 3.5

0.2

c 0

0.2

0.4

0.6

0.8

(b) Dry mass intake (mg)

0.4

bc

1

P = 0.99

0

L:P content

No. of flies killed

5.5

1

a

0.95

LP0.25

LP0.15

LP0.10

(b)

a

a

0.6

a 0.4 0.2

0.9 0

0.2

0.4

0.6

0.8

1 0

22 (c)

Flies Remnants First killed LP0.89 flies

P = 0.016

Figure 3. Nutrient compositions for the reference flies and the discarded remnants (a) for all fly types over the experiment (mean
SE) and (b) for the most lipid-rich fly type (LP0.89) from the first spider meal in the second instar (mean
SE).

20 a 18

16

ab

abc bc

bc c

14 0

0.2

0.4

0.6

0.8

1

Fly L:P content Protein-rich prey

Lipid-rich prey

Figure 2. (a) Total number of flies killed (mean
SE), (b) dry mass extracted (mean
SE) and (c) lipid þ protein energy extracted (mean
SE) by P. prativaga spiders during the second instar. The P values are from ANOVA tests across dietary treatments. Different letters indicate significant differences between dietary treatments (Student’s t test: P < 0.05).

with a slope of 1 (ANOVA: P < 0.01; Fig. 4), which indicates increased food intake by spiders feeding on protein-rich flies relative to spiders on the other diets.

Nutrient Utilization The lipid utilization plot (Fig. 5a) shows a close to linear relationship between lipids consumed and lipids accumulated in spider bodies (linear regression: R2 ¼ 0.60, F1,161 ¼ 240.71, P < 0.0001). An ANCOVA on lipid growth with lipid consumption as covariate revealed no significant covariate*diet interaction (ANCOVA:

F5,161 ¼ 1.17, P > 0.05). Furthermore, the diet treatment factor remained nonsignificant after we removed the interaction term from the statistical model (ANCOVA: F5,161 ¼ 1.99, P > 0.05). This indicates that ingested lipids were stored with nearly equal efficiency in spiders across dietary treatments, and the relation between lipid intake and lipid body growth could be described as Lgrowth ¼ 0.00 þ 0.48  Lintake. The utilization plot for protein (Fig. 5b) decelerated at higher protein intakes and was thus described better by a logarithmic

0.4 Lipid intake (mg)

Energy intake (J)

LP0.40

0.8

a a

LP0.64

Fly type

1.05

1

LP0.89

Total* LP0.89 Fly 5* Fly 4 LP0.64 Fly 3 LP0.40 0.2 Fly 2 LP0.25 LP0.15 Fly 1

0

0.2

0.4 0.6 Crude protein intake (mg)

0.8

LP0.10

1

Figure 4. Cumulative protein and lipid consumption from successive flies killed by P. prativaga spiders during the second instar (mean
SE). As spiders killed unequal numbers of flies, only consumption from the first five killed flies plus total consumption over the instar is included in the figure. *Arrays that are significantly different from a line with slope 1.

K. Jensen et al. / Animal Behaviour 81 (2011) 993e999

0.2

(a)

LP0.89

f

LP0.40 LP0.25

0.1

P < 0.0001

0.6

LP0.64 L:P content

Lipid growth (mg)

(a)

997

LP0.15 LP0.10

e

0.4 d c

0.2 0.1

0

0.2

0.3

0.4

a 0

(b) 0.4 LP0.25 LP0.15 LP0.40

LP0.64 LP0.89

0.2

0

LP0.10

0.2

0.4

0.6

0.8

1

Lean dry mass growth (mg)

Crude protein growth (mg)

Lipid intake (mg)

Crude protein intake (mg)

Body Composition and Growth The ratio of lipid to protein (L:P ratio) in spider bodies at the end of the instar was highly affected by diet (ANOVA: F5,139 ¼ 108.81, P < 0.0001). Spider bodies reflected the fly nutrient composition (Table 1), and the L:P ratio of the spiders increased stepwise about fourfold from the most protein-rich fly type to the most lipid-rich fly type (Fig. 6a). Spider survival was very high and in no treatment did more than one spider die during the experiment. The duration of the second instar lasted 13.1
0.11 (mean
SE) days and did not differ significantly across dietary treatments (Wilcoxon: c5,1622 ¼ 2.77, P ¼ 0.74). The total dry mass growth of spiders likewise did not vary significantly across dietary treatments (ANCOVA: F5,162 ¼ 0.38, P ¼ 0.86). However, spiders fed on more protein-rich flies had significantly more growth in lean body mass (ANCOVA: F5,162 ¼ 11.20, P < 0.0001; Fig. 6b) and carapace length (ANCOVA: F5,161 ¼ 2.44, P ¼ 0.037; Fig. 6c) compared to spiders fed more lipidrich flies. The increase in carapace length spanned from 21
3% (mean
SE) in spiders fed the most lipid-rich (LP0.89) flies to 33
3% (mean
SE) in spiders fed the protein-rich LP0.15 flies.

Carapace length growth (mm)

model between protein intake and protein growth than by a linear relationship: Pgrowth ¼ 0.11 þ 0.030  lnPintake (R2 ¼ 0.60). When an ANCOVA on protein growth was performed with protein intake as the covariate, we found a significant covariate*diet interaction (F5,147 ¼ 3.56, P ¼ 0.005) indicating that protein utilization varied across diets, with lower protein utilization on the most protein-rich diets (Fig. 5b).

0.2

0.4

0.6

0.4 (b)

0.8

1

P < 0.0001

a abc 0.3

ab bc cd

0.2

d

0

Figure 5. Utilization plots of growth versus consumption over the second instar by P. prativaga spiders on the six diets (mean
SE). (a) Lipid growth versus lipid consumption. (b) Protein growth versus protein consumption.

b

0.35

0.2

0.4

0.6

(c)

0.8

1

P = 0.037

a

0.3 ab

ab

ab

0.25 b b

0.2 0

0.2

Protein-rich prey

0.4 0.6 Fly L:P content

0.8

1

Lipid-rich prey

Figure 6. (a) Body L:P composition (mean
SE) of P. prativaga spiders after the moult to the third instar. (b) Lean body mass growth (mean
SE) and (c) carapace length growth (mean
SE) of P. prativaga spiders from the start of the second instar to the start of the third instar. The P values are from (a) ANOVA or (b,c) ANCOVA tests across dietary treatments. Different letters indicate significant differences between dietary treatments (Student’s t test: P < 0.05).

nutrients potentially available in killed prey, nutrients actually extracted from the killed prey and nutrients allocated to growth during the instar. Spiders were provided with more flies than they killed and did not completely consume all nutrients from killed prey. However, the nutrient compositions available in the killed flies generally did not differ from the nutrients actually extracted.

Summary of Nutrient Acquisition and Allocation Responses

DISCUSSION

Figure 7 summarizes the relationship between nutrients available to the spiders (i.e. the nutritional value of flies provided),

Our study showed clear effects on body composition and growth in juvenile P. prativaga during one instar of development after we

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K. Jensen et al. / Animal Behaviour 81 (2011) 993e999

0.8

Flies provided LP0.89

Lipids (mg)

LP0.64 LP0.40

Flies killed 0.4

Extracted

LP0.25 LP0.15

Grown LP0.10 0

0.4

0.8

1.2

1.6

Crude protein (mg) Figure 7. Arrays summarizing nutrient acquisition and allocation by P. prativaga spiders over the second instar. All values are mean
SE. The outermost array shows the lipid and protein masses available in the provided flies. The next array shows the nutrient masses available in the flies that were killed by the spiders. The third array shows the nutrient masses that were extracted from the flies. Finally, the innermost array shows lipid and protein growth by the spiders. Reference fly means from the actual feeding days are used in all calculations.

added pure protein (casein) or sugar to the diet of their prey (Fig. 6). Earlier studies have shown that enriching the growth medium of D. melanogaster prey with dog food also increases performance of their wolf spider predators (Mayntz & Toft 2001; Wilder & Rypstra 2008). High rates of growth are important performance estimates for several reasons, including escaping similar-sized predators (Borre et al. 2006; Mayntz & Toft 2006), being able to reproduce earlier or having higher reproductive potential (Petersen 1950; Kessler 1971; Vollrath 1987; Honek 1993) and being favoured in courtship (Maklakov et al. 2004; Lomborg & Toft 2009). The increased growth on low L:P ratios indicates that wolf spiders, like many other predators, are adapted to a diet rich in protein (White 1978). However, our experiment also showed evidence that the spiders possess both behavioural and physiological mechanisms for nutrient regulation, which partially compensate for the costs of feeding on nutritionally imbalanced prey. Probability of Killing Prey Optimal foraging theory assumes that the feeding behaviour of predators has evolved under a strong selection pressure to maximize capture rates and energy intake (Stephens & Krebs 1986), and according to this it would be plausible to predict that predators would have evolved to kill all prey they could catch. This was not the case. Spiders did not always kill flies and did not exhibit superfluous killing as has sometimes been reported for other predators (Maupin & Riechert 2001; Fantinou et al. 2008). The spiders killed at a high rate in the early days of the experiment (Fig. 1a) when their need for nutrients was highest. Later, declining nutrient masses were extracted from the flies (Fig. 1b), and prey was more often left alive (Fig. 1a), even though the experimental setting allowed easy capture. The different number of prey items killed over the instar depending on fly type (Fig. 2a) suggests that the spiders varied the number of prey they killed depending on their need for food mass (Fig. 2b) and nutrients (Fig. 4). Total Intake and Nutrient Intake Arrays As a result of differences in the number of flies killed, the total mass of macronutrients consumed did not differ across dietary treatments, whereas total energy gain varied widely (Fig. 2c). Hence, we cannot reject the simple hypothesis that regulation of dry mass intake alone determined food intake. The intake arrays for protein and lipid at the beginning of the instar reflect this

possibility. The concave array that developed towards the end of the instar, however, indicates that nutrient-specific intake regulation became apparent at this point. Probably, the different diet nutrient compositions began to have pronounced physiological effects at this point. Increasing intake on nutritionally extreme diets resulted in spiders gaining more of the limiting nutrient in the diet, in this case lipids, at the cost of ingesting a greater amount of the nutrient in excess, in this case protein. The ground beetle A. dorsalis showed a similar intake pattern when restricted to nutritionally fixed foods after winter hibernation. They also formed an initial intake array with a slope of 1 and later developed an outwards curvilinear intake array, as beetles provided with proteinrich food increased intake relative to beetles on the other diets (Raubenheimer et al. 2007). The starvation period before feeding in our experiment and the hibernation period might explain the similar results. The pivot point about which the nutrient intake array opens out over time (Figs 4, 7) coincides with the LP0.25 diet. This diet also corresponded to the point of deflection in the protein utilization plot (Fig. 5b), beyond which excess ingested protein was converted to tissue growth at a lower rate. These two findings possibly reflect that the LP0.25 diet contains a nutrient composition that balances the two nutrients, so both are most efficiently utilized by the spiders (Simpson & Raubenheimer 1995). Nutrient Extraction During the first meal, the spiders provided with the most lipidrich flies (LP0.89) extracted a higher proportion of lipids than the proportion in the flies (Fig. 3b). Lipids in these flies were very abundant which might have enabled selective lipid extraction, and the spiders possibly extracted lipids selectively to restore energy stores that were depleted during the previous moult and days of starvation. This pattern corresponds to the pattern found in A. dorsalis after winter hibernation: beetles given the chance to choose their diet first selected lipid-rich food to fill up lipid stores before foraging selectively for protein (Raubenheimer et al. 2007). Whereas the sessile spider Stegodyphus lineatus was shown to redress nutritional imbalance by extracting nutrients selectively from the prey (Mayntz et al. 2005), P. prativaga was instead shown to consume higher masses from nutritionally complementary prey than from nutritionally similar prey (Mayntz et al. 2005). This is in accordance with our general finding that P. prativaga does not show extensive selective nutrient extraction but rather regulates mass intake to balance the combined intake of lipids and protein. Postingestive Nutrient Regulation Our results show that lipids were incorporated into body tissues with nearly similar efficiency regardless of prey nutrient composition (Fig. 5a), and body lipid growth was therefore closely linked to lipid intake. This is in accordance with the finding that metabolism is not raised when these spiders feed on lipid-rich prey (Jensen et al. 2010). Protein utilization, however, varied across diets with lower utilization efficiency above a certain protein intake (Fig. 5b). Postingestive regulation of protein has been shown before in cats, Felis catus (Russell et al. 2002, 2003) and in some herbivores (Simpson et al. 2002; Lee et al. 2004, 2006) and probably reflects a pronounced ability to use amino acids as a source of metabolic energy while voiding the amino groups (Zanotto et al.1993). Perhaps the ability to overingest protein as a source of energy is common in predators as an adaptation to feeding on protein-rich foods. This is in contrast to herbivores and omnivores, which are not willing to overingest protein (Raubenheimer & Simpson 1997; Raubenheimer & Jones 2006). As a consequence of the lower rate of protein utilization for growth on high-protein diets, the growth array

K. Jensen et al. / Animal Behaviour 81 (2011) 993e999

approaches a vertical slope with protein growth being more strongly conserved across diets than lipid growth (Fig. 7). The spiders did not show postingestive regulation where nutrient growth points converge around a common growth target as seen in herbivores and omnivores (Simpson & Raubenheimer 2000, 2001). As spiders are adapted to periods of starvation (Wise 1993), they are likely to have adapted to utilizing the nutrients they ingest and waste as little as possible to avoid the risk of lacking these nutrients in the future. Conclusions In summary, our experiment showed that P. prativaga wolf spiders regulated nutrients at several levels of food handling. Despite this regulation, however, spider body composition was highly affected by the nutrient composition of the prey. This incomplete regulation may be explained as an opportunistic feeding strategy that has evolved as an adaptation to unpredictable prey availability. Spiders may thus have adapted to acquiring and maintaining nutrients when these are available in order to survive and thrive in periods of prey shortage. Acknowledgments This study was supported by a grant from the UK Biotechnology and Biological Sciences Research Council. D.M. was in receipt of a grant from the Danish Research Council. D.R. is part-funded by the National Research Centre for Growth and Development, New Zealand. S.J.S. was in receipt of a Federation Fellowship and currently a Laureate Fellowship from the Australian Research Council. References AOAC. 2006. Official Methods of Analysis. 18th edn. Washington, DC: Association of Official Analytical Chemists International. Behmer, S. T. 2009. Insect herbivore nutrient regulation. Annual Review of Entomology, 54, 165e187. Borre, J. V., Bonte, D. & Maelfait, J.-P. 2006. Interdemic variation of cannibalism in a wolf spider (Pardosa monticola) inhabiting different habitat types. Ecological Entomology, 31, 99e105. Elser, J. J., Fagan, W. F., Denno, R. F., Dobberfuhl, D. R., Folarin, A., Huberty, A. F., Interlandi, S., Kilham, S. S., McCauley, E. & Schulz, K. L., et al. 2000. Nutritional constraints in terrestrial and freshwater foodwebs. Nature, 408, 578e580. Fagan, W. F., Siemann, E., Mitter, C., Denno, R. F., Huberty, A. F., Woods, H. A. & Elser, J. J. 2002. Nitrogen in insects: implications for trophic complexity and species diversification. American Naturalist, 160, 784e802. Fantinou, A. A., Perdikis, D. C., Maselou, D. A. & Lambropoulos, P. D. 2008. Prey killing without consumption: does Macrolophus pygmaeus show adaptive foraging behaviour? Biological Control, 47, 187e193. Felton, A. M., Felton, A., Wood, J. T., Foley, W. J., Raubenheimer, Wallis, I. R. & Lindenmayer, D. B. 2009. Nutritional ecology of Ateles chamek in lowland Bolivia: how macronutrient balancing influences food choices. International Journal of Primatology, 30, 675e696. Galef, B. G. Jr. 1996. Food selection: problems in understanding how we choose foods to eat. Neuroscience and Biobehavioral Reviews, 20, 67e73. Greenstone, M. H. 1979. Spider feeding behaviour optimises dietary essential amino acid composition. Nature, 282, 501e503. Honek, A. 1993. Intraspecific variation in body size and fecundity in insects: a general relationship. Oikos, 66, 483e492. Jensen, K., Mayntz, D., Wang, T., Simpson, S. J. & Overgaard, J. 2010. Metabolic consequences of feeding and fasting on nutritionally different diets in the wolf spider Pardosa prativaga. Journal of Insect Physiology, 56, 1095e1100. Kessler, A. 1971. Relation between egg production and food consumption in species of the genus Pardosa (Lycosidae, Araneae) under experimental conditions of food-abundance and food-shortage. Oecologia, 8, 93e109. Lee, K. P., Behmer, S. T., Simpson, S. J. & Raubenheimer, D. 2002. A geometric analysis of nutrient regulation in the generalist caterpillar Spodoptera littoralis (Boisduval). Journal of Insect Physiology, 48, 655e665. Lee, K. P., Simpson, S. J. & Raubenheimer, D. 2004. A comparison of nutrient regulation between solitarious and gregarious phases of the specialist caterpillar, Spodoptera exempta (Walker). Journal of Insect Physiology, 50, 1171e1180. Lee, K. P., Behmer, S. T. & Simpson, S. J. 2006. Nutrient regulation in relation to diet breadth: a comparison of Heliothis sister species and a hybrid. Journal of Experimental Biology, 209, 2076e2084.

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