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Metabolic consequences of feeding and fasting on nutritionally different diets in the wolf spider Pardosa prativaga
Journal of Insect Physiology 56 (2010) 1095–1100
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Metabolic consequences of feeding and fasting on nutritionally different diets in the wolf spider Pardosa prativaga Kim Jensen a,b,*, David Mayntz c,d, Tobias Wang e, Stephen J. Simpson b, Johannes Overgaard e a
Department of Zoology, University of Oxford, South Parks Road, OX1 3PS, Oxford, UK School of Biological Sciences, University of Sydney, Heydon-Laurence Building A08, NSW 2006, Sydney, Australia c Ecology and Genetics, Department of Biological Sciences, University of Aarhus, Building 1540, 8000 A˚rhus C, Denmark d Department of Genetics and Biotechnology, University of Aarhus, Research Centre Foulum, 8830 Tjele, Denmark e Zoophysiology, Department of Biological Sciences, University of Aarhus, Building 1131, 8000 A˚rhus C, Denmark b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2009 Received in revised form 2 March 2010 Accepted 2 March 2010
We investigated whether spiders fed lipid-rich rather than protein-rich prey elevate metabolism to avoid carrying excessive lipid deposits, or whether they store ingested lipids as a buffer against possible future starvation. We fed wolf spiders (Pardosa prativaga) prey of different lipid:protein compositions and measured the metabolic rate of spiders using closed respirometry during feeding and fasting. After a 16day feeding period, spider lipid:protein composition was significantly affected by the lipid:protein composition of their prey. Feeding caused a large and fast increase in metabolism. The cost of feeding and digestion was estimated to average 21% of the ingested energy irrespective of diet. We found no difference in basal metabolic rate between dietary treatments. During starvation V˙ O2 and V˙ CO2 decreased gradually, and the larger lipid stores in spiders fed lipid-rich prey appeared to extend survival of these spiders under starvation compared to spiders fed protein-rich prey. The results show that these spiders do not adjust metabolism in order to maintain a constant body composition when prey nutrient composition varies. Instead, lipids are stored efficiently and help to prepare the spiders for the long periods of food deprivation that may occur as a consequence of their opportunistic feeding strategy. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Nutrient balancing Respiration Predator Starvation
1. Introduction All animals must balance energy intake with energy requirements, and must match the intake of protein, carbohydrates and lipid (i.e. the major macronutrients) to the requirements for metabolism, growth and reproduction. Predatory animals, such as spiders, may experience prolonged periods of food deprivation interrupted by sudden encounters with large prey items. Such animals are often endowed with physiological adaptations that enable them to maintain function during fasting and enable them to digest large meals when the opportunity arises (Wang et al., 2006). Although the infrequent encounters with appropriate prey items can limit the ability of predators to control the nutrient composition of their diet, it has been shown that predatory animals, like herbivores and omnivores (Kyriazakis and Emmans, 1991; Kyriazakis et al., 1991; Raubenheimer and Simpson, 1997;
* Corresponding author at: Department of Zoology, University of Oxford, Tinbergen Building, South Parks Road, OX1 3PS, Oxford, UK. Tel.: +44 1865 271253; fax: +44 1865 310447. E-mail addresses: [email protected], [email protected] (K. Jensen). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.03.001
Berthoud and Seeley, 2000; Raubenheimer and Jones, 2006; Simpson et al., 2006), can regulate the intake of macronutrients in their diets (Mayntz et al., 2005; Raubenheimer et al., 2007). If nutrient composition of the prey deviates from the optimal balance, animals may over-consume food to meet minimal requirements of a given macronutrient, even though the overall energy requirement is surpassed (Raubenheimer and Simpson, 1993). For example, herbivores and omnivores fed a proteindeficient diet typically increase their overall intake and, hence, overingest lipids and carbohydrates to meet their protein requirement (Simpson and Raubenheimer, 2005; Sørensen et al., 2008). Unless this surplus energy is voided, the excess energy intake is stored as body fat, which could result in obesity (Simpson and Raubenheimer, 2005; Sørensen et al., 2008). One way of voiding excess ingested energy when feeding on a low-protein diet is to increase metabolic rate to burn off excessive energy by a process known as facultative diet-induced thermogenesis, as is seen in some herbivores and omnivores (Zanotto et al., 1993, 1997; Stock, 1999; Trier and Mattson, 2003). The extent to which facultative diet-induced thermogenesis is employed might be expected to reflect the balance between the costs of fat storage and the longer term benefit of fat stores as energy reserves in times of food shortage (Warbrick-Smith et al., 2006). As for most spiders,
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wolf spiders are frequently exposed to periods of starvation interspersed with periods of plenty (Riechert and Harp, 1987; Wise, 1993). It is interesting, therefore, to study whether these spiders possess regulatory adaptations to handle nutrient imbalance and/or starvation. It has previously been shown that prey low in protein and rich in lipids reduce growth, survival and fecundity in wolf spiders (Mayntz and Toft, 2001). It is unclear, however, whether wolf spiders increase metabolic rate when exposed to protein-poor, lipid-rich diets. Such a response would aid the spiders in achieving a better nutritional balance, but could also potentially render the animals more vulnerable to depletion of energy stores during food deprivation. It has been shown that wolf spiders can reduce metabolism in response to starvation (Tanaka and Itoˆ, 1982; Tanaka et al., 1985; Nakamura, 1987), but the metabolic response to changes in body and prey nutrient composition, as well as its interaction with food deprivation, is unknown. To gain further insight into this question, the present study investigated how prey of variable macronutrient composition (lipids vs. protein) affects body composition, basal metabolic rate (BMR), metabolic response to feeding and digestion (SDA), and metabolism during fasting in the wolf spider Pardosa prativaga. 2. Materials and methods
Fig. 1. Lipid vs. crude protein content (mean SE) of Pardosa prativaga wolf spiders immediately after winter hibernation and after 16 days of ad libitum feeding on lipidrich, intermediate, or protein-rich Drosophila melanogaster fruit flies. The slopes of the inserted lines, indicated by the number at the end of each line, represent the lipid: crude protein ratio of the three D. melanogaster fly types. Crude protein content is calculated by multiplying the nitrogen content with a constant factor of 6.25 (AOAC, 2000). Data for fly body compositions are from similarly produced D. melanogaster (Jensen, 2010).
2.1. Experimental diets Fruit flies (Drosophila melanogaster) used as food for the spiders were raised on three different media based on mixtures (g:g) of a basic medium (Carolina Instant Drosophila Medium Formula 4-24, Burlington, NC, USA), casein (Sigma C-5890, Sigma–Aldrich, Steinheim, Germany) and sucrose (Fluka, 84097, Neu-Ulm, Germany). Lipid-rich flies were raised in a medium with a 1:4 ratio of sucrose and basic medium; intermediate flies were raised on a 1:9 ratio of casein and basic medium; and protein-rich flies were raised on a 3:2 ratio of casein and basic medium. All flies were reared in 3.4 cm Ø vials containing 2.5 g dry medium mixed with 15–22 ml water and a few drops of satiated yeast solution covering the surface. Cultures were inoculated with 40–50 adult D. melanogaster of mixed sexes for 3 days and the resulting larvae were raised at 24–26 8C until emergence. The different rearing media produce fruit flies with markedly different protein and lipid contents (Fig. 1). 2.2. Experimental animals Juvenile P. prativaga wolf spiders (Lycosidae) were collected in wet meadows around Aarhus (Jutland, Denmark) in February 2008 as they were hibernating under vegetation. They were kept in individual transparent plastic vials (2 cm Ø, 6 cm height) with moist Plaster-of-Paris bottoms and foam rubber stoppers at 5 8C in darkness without food for 1–2 months before experiments started. This mimicked the conditions in nature during winter when the spiders were caught, and experiments started in middle spring. Five days before initiation of feeding, the spiders were transferred to an incubator at 25 8C and a 16:8 h light:dark photoperiod. Each spider (N = 268) was weighed to the nearest 1 mg and 16 randomly selected spiders were frozen at 18 8C to determine initial body composition. The remaining spiders were distributed randomly between three dietary treatments: lipid-rich flies, intermediate flies, or protein-rich flies for 16 days. During the feeding period, the spiders were allowed to feed to satiety every second day. 2.3. Experimental protocol Basal metabolic rate (BMR) of 44 randomly selected spiders from each feeding regime was measured just before the feeding
period, and measurements of mass and BMR were repeated after the 16 days on the respective diets. Ten other spiders from each feeding regime were frozen to assess how the diets had influenced body composition. The remaining spiders were then fasted for 7 days, weighed, and BMR measurements were repeated. To measure the metabolic cost of feeding, often termed the specific dynamic action of a meal (SDA), we fed the spiders a single preweighed fly from their respective diet directly in the respiration chamber. This measure therefore includes the metabolic cost of handling, ingesting, digesting, and assimilating a prey item (McCue, 2006). When the fly was taken, the respiration chamber was closed immediately and gas exchange was measured over two consecutive 3-h periods. All spiders completed meal extraction within the first 3-h period. Gas exchange was measured again over the following 14 h after which respiration levels had returned to resting rates. The spiders were then returned to their vials and fed flies ad libitum for 24 h to ensure that they were fully fed before exposure to long-term starvation. During the starvation period, mass and BMR were measured after 5, 11, 17, and 35 days. 2.4. Measurements of metabolism Gas exchange was measured by closed respirometry, in which the spiders were enclosed in individual 10-ml glass syringes sealed with vacuum grease. Except during SDA measurements, enclosure ˙ periods were 24 h, after which the decrease in O2 volume VO2 and increase in CO2 volume V˙ CO2 were measured. The syringes contained a small drop of water to keep the air saturated, and were submerged in a water bath at 25 8C to stabilize temperature. Measurements of V˙ O2 and V˙ CO2 were performed by injecting 8 ml gas from the respiratory chambers into a CO2 and O2 analyser (Applied Electrochemistry, Sunnyvale, CA, USA). This gas sample was then drawn through the analyser at a known and constant rate so that the O2 decrease and CO2 increase could be calculated relative to injections of standard gas samples of known O2 and CO2 contents. V˙ O2 and V˙ CO2 were corrected to standard temperature and pressure under dry conditions (STPD), and mass specific metabolic rate was calculated using the latest measure of body mass prior to the measurement. In most cases, CO2 of the respiratory chamber did not exceed 2% and O2 did not decline below 18%. For comparison, standard concentrations of CO2 and O2 in the air were
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20.95% and 0.03%, respectively. The respiratory gas exchange ratio (RE) was calculated as V˙ CO2 =V˙ O2 . 2.5. Calculation of SDA The specific dynamic action (SDA) was calculated as the energy consumption during feeding and digestion that exceeded the BMR immediately prior to feeding (Overgaard et al., 2002). Thus, the increase in V˙ O2 caused by feeding and digestion was calculated by subtracting the resting levels measured before feeding from the levels integrated over the 20 h period following prey capture. This V˙ O2 was then converted to Joules using a conversion factor of 19.67 J/ ml O2 (Gessaman and Nagy, 1988). Fly lipid and protein contents were calculated using known values of protein and lipid compositions of similarly raised dry flies (Jensen, 2010). Dry masses of the provided flies were estimated using average fly mass/dry mass ratios from 20 reference flies collected from each diet. Using standard energetic contents of lipid and protein (AOAC, 2000) and fly body masses, the energetic content of the lipid-rich, intermediate, and protein-rich flies was estimated to 5.5, 4.7 and 4.6 J/mg wet mass, respectively. Finally, the SDA coefficient was calculated as energy expended/energy ingested (Jobling and Davies, 1980). 2.6. Starvation tolerance Spiders (N = 30) from each of the feeding regimes were used to assess the longevity of fasting spiders. These spiders had been allowed to feed on the three diets ad libitum for 16 days after which they were provided water but no food. Deaths were recorded every morning and evening. 2.7. Body measurements
Fig. 2. Relative body mass growth of Pardosa prativaga wolf spiders after feeding and fasting (mean SE). Spiders were fed one of three experimental diets that was either protein-rich (solid), intermediate (dashed) or lipid-rich (dotted). After a 16-day ad libitum feeding period, the spiders were fasted for 1 week before being fed ad libitum for 1 day. After this the spiders were fasted. Only the first 17 fasting days are included in the figure, and only data of spiders surviving this period are shown.
ANCOVA tests using initial mass as the covariate. Diet effects on the number of molts during the feeding period were analysed using Nominal Logistic Regression. Survival during starvation was compared using Wilcoxon Chi Square test. Statistical analyses were made in JMP 7.0 (SAS Institute 2007). 3. Results 3.1. Spider nutrient composition, mass, size, and developmental stage Body lipid vs. crude protein compositions of the spiders before and after feeding for 16 days on the three diets are shown in Fig. 1.
At the end of the experiment the spiders sampled before and after the 16-day feeding period were dried over 4 days at 60 8C and weighed to nearest 1 mg after measuring carapace lengths with a microscope eyepiece scale. Lipids were extracted over two 24-h washes in 2 ml petroleum ether, and the lean corpses were dried and weighed. The lipid mass of each spider was calculated by subtracting the lean dry body mass from the total dry body mass. Last, corpses were analysed for proportion of nitrogen relative to dry mass in a combustion analyser (Na 2000, Carlo Erba, Italy). Absolute nitrogen masses were calculated by multiplying nitrogen proportions with the respective dry masses, and crude protein masses were calculated using a protein:nitrogen factor of 6.25 (AOAC, 2000). Spider lipid energy stores were calculated by multiplying spider lipid masses with standard lipid energy content (AOAC, 2000). To get an estimate of predicted survivorship within each feeding treatment, we divided the calculated energy from lipid storages with the basal energy expenditure measured during the first 17 days of the last fasting period (Fig. 2), assuming constant BMR after the 17th fasting day. As metabolism is known to decline during fasting in wolf spiders (Tanaka and Itoˆ, 1982; Nakamura, 1987), this estimate expresses survivorship if no further adjustments to starvation were made after 17 days of starvation. 2.8. Statistical analysis Differences in nutritional composition and SDA coefficient across treatment groups were tested using ANOVA. ANOVA was also used to test for differences in V˙ O2 , V˙ CO2 , and RE across diet treatments at individual time points. Fractions and percentages were arcsine transformed before analysis. Tukey-Kramer tests were used to identify differences between individual dietary groups. Effects of diet on spider mass and size were compared with
Fig. 3. (a) Body mass and (b) carapace length of Pardosa prativaga wolf spiders (mean SE) after being fed 16 days ad libitum on lipid-rich, intermediate, or proteinrich fruit flies.
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The spiders had a relative lipid composition of 8.9 1.1% (mean SE) at the onset of experimental feeding. However, after 16 days of feeding on the different prey types, the relative lipid contents of the spiders were significantly affected by the nutrient composition of their diet (ANOVA, F3,46 = 23.35, P < 0.0001). A post hoc test revealed that the relative lipid composition of the spiders differed significantly between all three diets after the 16-day feeding period (TukeyKramer, P < 0.05). The relative protein composition was also significantly affected by feeding on different diets (ANOVA, F3,46 = 14.69, P < 0.0001). Post hoc analysis showed that spiders on the protein-rich diet had significantly higher relative crude protein content than spiders on the two other diets (Tukey-Kramer, P < 0.05), but crude protein content did not differ significantly between the intermediate and the lipid-rich diet (Tukey-Kramer, P > 0.05). Spiders on all diets in most cases increased body mass by at least 100% during the 16-day feeding period, and gradually lost body mass during the subsequent fasting period (Fig. 2). After the 16-day feeding period, we found a significant dietary treatment effect on both body mass (ANCOVA, F2,27 = 7.93, P = 0.002) and carapace length (ANCOVA, F2,27 = 5.32, P = 0.013). The general tendency was higher body mass (Fig. 3a) and longer carapace (Fig. 3b) in spiders fed more protein-rich prey. Spiders went through either none, one, two or three molts during the
feeding period, but there was no significant effect of diet on number of molts (Nominal Logistic Regression, F2,224 = 12.50, P > 0.05). Most spiders were subadult after one molt and adult after two molts. 3.2. Basal metabolic rate and the metabolic response to feeding and digestion Diet composition did not affect V˙ O2 or V˙ CO2 before or after the 16-day feeding period (Fig. 4). Furthermore, the maximal metabolic response during feeding and digestion did not differ among diets. The metabolic response to feeding and digestion caused a large increase in gas exchange in all three experimental groups and was associated with an increase in the respiratory exchange ratio (RE) from a fasting value of 0.79 0.005 (mean SE) to a value of 0.85 0.005 (mean SE) during feeding and digestion (Fig. 4). Following ingestion of meals corresponding to an average of 8.5% of spider mass, V˙ O2 and V˙ CO2 increased to more than 4-fold above fasting values within the 3-h period after prey capture. This cumulative SDA response did not differ among diets (ANOVA, F2,49 = 0.28, P = 0.76, Fig. 5a). Similarly, we found no difference between dietary treatments when the SDA response was calculated relative to the amount of energy ingested (SDA coefficients). The SDA coefficient was approximately 21% in all three groups (ANOVA, F2,49 = 0.08, P = 0.92, Fig. 5b). Thus, 21% of the ingested energy was respired as a consequence of feeding and digestion in all groups. 3.3. Survival during fasting Survival during fasting differed significantly across the three diets (P = 0.001, x290 ¼ 13:3, Wilcoxon). The fasting duration causing 50% mortality was 34 days for spiders fed a protein-rich diet, 40 days for intermediate spiders, and 46 days for lipid-rich spiders (Fig. 6a). When calculated solely from lipid storage and BMR, spiders from the three diet types were estimated to survive approximately 18, 23 and 32 days (Fig. 6b). Survival time thus correlated well with lipid storage.
Fig. 4. Gas exchange of digesting and fasting Pardosa prativaga wolf spiders. (a) Oxygen consumption (V˙ O2 ), (b) carbon dioxide production (V˙ CO2 ), and (c) respiratory exchange ratio (RE). The spiders were maintained at 25 8C. In order to visualize the short period associated with consumption and digestion of a single fly (1 fly), the x-axis is extended during this period. Data are presented as mean SE.
Fig. 5. Metabolic increase (SDA response) of Pardosa prativaga wolf spiders during feeding and digestion of a single D. melanogaster varying in lipid:protein content (mean SE). (a) The SDA response presented as the total oxygen consumption in excess of the basal metabolism. (b) The SDA response presented as the amount of energy used for feeding and digestion relative to the ingested energy (SDA coefficient).
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Fig. 6. (a) Survival of Pardosa prativaga wolf spiders during starvation. The spiders were fed either protein-rich (N = 30), intermediate (N = 30) or lipid-rich (N = 30) fruit flies ad libitum for 16 days immediately after winter hibernation, after which they were starved until death occurred. (b) The calculated expected time of survival based on the spider lipid stores and basal metabolism of fasting spiders.
4. Discussion We tested the hypothesis that spiders fed more lipid-rich/ protein-poor prey would elevate metabolism in order to gain more protein through increased prey consumption. Our measurements of metabolic responses by the spiders did not support this hypothesis. Although spider body compositions were highly affected by the nutrient ratio of their prey, prey body compositions could not entirely predict spider body compositions (Fig. 1). This indicates that the spiders might have regulated either (1) the ratio of nutrients extracted from their prey (Mayntz et al., 2005), (2) the assimilation efficiency of the nutrients extracted from their prey (Lee et al., 2004), or (3) their metabolic response to consuming nutritionally different prey. Our results from the present study show that the latter was not the case. We found similar metabolic rates and respiratory exchange ratios of spiders from the three different dietary treatments, indicating that the spiders metabolized both the same type and amount of energy substrates. We measured gas exchange over 3-h intervals in feeding and digesting P. prativaga and found a 4-fold higher metabolism during the first 3-h period compared to immediately before feeding. It is likely that a finer time resolution would have disclosed an even larger increase in metabolism than revealed in our study. Thus, as documented extensively in many other animals (McCue, 2006), metabolism increased substantially during feeding and digestion. This specific dynamic action of food represents the energetic costs associated with subduing and handling the prey, digestive responses, and post-absorptive events (McCue, 2006). Protein synthesis appears to be a major contributor to the SDA response,
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which normally increases with protein content of the meal (Halton and Hu, 2004). However, this was not the case for P. prativaga as all three diets elicited very similar metabolic responses. In this respect it is important to remember that the diets used here were all predominantly protein-biased, and it is possible that larger differences in diet composition are needed to produce differences in the metabolic cost of feeding and digestion. Because we refilled the respiration chambers with new air after the spiders had captured their prey, the activity associated with capture cannot account for the rise in metabolism. Instead, we suggest that the excretion of digestive enzymes for extraoral digestion, sucking in the juices, and internal digestion and absorption caused the SDA response. We found that the total costs of consuming, digesting, and absorbing the prey fly was around 21% of the energy it contained, which is in the range measured for snakes fed different types of prey (McCue, 2006). While the respiratory exchange ratio (RE) did not differ between the three different diets, we found that RE increased during feeding and digestion, indicating that more protein and carbohydrates were metabolised in the postprandial period. Prey nutrient composition had a considerable effect on the growth rate of the spiders, which was significantly lower in spiders fed more lipid-rich prey. Other studies have found that survival and fecundity may also be reduced when spiders are fed prey of low protein and high lipid composition (Mayntz and Toft, 2001; Mayntz et al., 2003). When feeding on lipid-rich prey causes lower growth rates of the spiders, why have the wolf spiders not evolved the physiological adaptations to remove this excess of lipids and allow additional food intake to remedy protein deficiency? Our study suggests an explanation for this. While the spiders clearly lacked the ability to vary metabolism in response to nutrient imbalance, they reduced metabolism over prolonged periods of starvation, which has also been observed in other species of spiders (Tanaka and Itoˆ, 1982; Tanaka et al., 1985; Nakamura, 1987), resulting in longer survival than expected from lipid energy stores and BMR by the onset of fasting (Fig. 6). Indeed, the ability to lower metabolism during prolonged starvation seems to represent a general response among many species of invertebrates and vertebrates as it prolongs survival with a finite energy store (Wang et al., 2006). Thus, in addition to their flexible abdomens that allow spiders to gorge when food is available, the spiders have also adapted physiologically to survive extended periods of starvation. We suggest that the absence of metabolic adjustments to burn excessive lipids may be adaptive in spiders that are occasionally exposed to unpredictable food availability (Riechert and Harp, 1987; Wise, 1993). In such an environment, selection for increased metabolism to remove excessive lipids may be counteracted by selection for large lipid stores which enable the spiders to survive long and unpredictable starvation periods. Results from our survival experiment on the fasting spiders support this hypothesis (Fig. 6a). In conclusion, P. prativaga wolf spiders did not seem to regulate metabolism to allow higher protein intake, even though spiders were clearly protein deficient on a protein-poor diet. We propose that regulation of nutrient intake and assimilation by increasing metabolism might not be highly evolved in predators that experience occasional prey limitation and energy deprivation (Wise, 1993). Acknowledgements This study was supported by grants from the BBSRC in UK and The Danish Research Council. References AOAC, 2000. Official Methods of Analysis, 17th ed. AOAC International, Gaithersburg. Berthoud, H.R., Seeley, R.J., 2000. Neural and Metabolic Control of Macronutrient Intake. CRC Press, Boca Raton.
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Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Metabolic consequences of feeding and fasting on nutritionally different diets in the wolf spider Pardosa prativaga Kim Jensen a,b,*, David Mayntz c,d, Tobias Wang e, Stephen J. Simpson b, Johannes Overgaard e a
Department of Zoology, University of Oxford, South Parks Road, OX1 3PS, Oxford, UK School of Biological Sciences, University of Sydney, Heydon-Laurence Building A08, NSW 2006, Sydney, Australia c Ecology and Genetics, Department of Biological Sciences, University of Aarhus, Building 1540, 8000 A˚rhus C, Denmark d Department of Genetics and Biotechnology, University of Aarhus, Research Centre Foulum, 8830 Tjele, Denmark e Zoophysiology, Department of Biological Sciences, University of Aarhus, Building 1131, 8000 A˚rhus C, Denmark b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2009 Received in revised form 2 March 2010 Accepted 2 March 2010
We investigated whether spiders fed lipid-rich rather than protein-rich prey elevate metabolism to avoid carrying excessive lipid deposits, or whether they store ingested lipids as a buffer against possible future starvation. We fed wolf spiders (Pardosa prativaga) prey of different lipid:protein compositions and measured the metabolic rate of spiders using closed respirometry during feeding and fasting. After a 16day feeding period, spider lipid:protein composition was significantly affected by the lipid:protein composition of their prey. Feeding caused a large and fast increase in metabolism. The cost of feeding and digestion was estimated to average 21% of the ingested energy irrespective of diet. We found no difference in basal metabolic rate between dietary treatments. During starvation V˙ O2 and V˙ CO2 decreased gradually, and the larger lipid stores in spiders fed lipid-rich prey appeared to extend survival of these spiders under starvation compared to spiders fed protein-rich prey. The results show that these spiders do not adjust metabolism in order to maintain a constant body composition when prey nutrient composition varies. Instead, lipids are stored efficiently and help to prepare the spiders for the long periods of food deprivation that may occur as a consequence of their opportunistic feeding strategy. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Nutrient balancing Respiration Predator Starvation
1. Introduction All animals must balance energy intake with energy requirements, and must match the intake of protein, carbohydrates and lipid (i.e. the major macronutrients) to the requirements for metabolism, growth and reproduction. Predatory animals, such as spiders, may experience prolonged periods of food deprivation interrupted by sudden encounters with large prey items. Such animals are often endowed with physiological adaptations that enable them to maintain function during fasting and enable them to digest large meals when the opportunity arises (Wang et al., 2006). Although the infrequent encounters with appropriate prey items can limit the ability of predators to control the nutrient composition of their diet, it has been shown that predatory animals, like herbivores and omnivores (Kyriazakis and Emmans, 1991; Kyriazakis et al., 1991; Raubenheimer and Simpson, 1997;
* Corresponding author at: Department of Zoology, University of Oxford, Tinbergen Building, South Parks Road, OX1 3PS, Oxford, UK. Tel.: +44 1865 271253; fax: +44 1865 310447. E-mail addresses: [email protected], [email protected] (K. Jensen). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.03.001
Berthoud and Seeley, 2000; Raubenheimer and Jones, 2006; Simpson et al., 2006), can regulate the intake of macronutrients in their diets (Mayntz et al., 2005; Raubenheimer et al., 2007). If nutrient composition of the prey deviates from the optimal balance, animals may over-consume food to meet minimal requirements of a given macronutrient, even though the overall energy requirement is surpassed (Raubenheimer and Simpson, 1993). For example, herbivores and omnivores fed a proteindeficient diet typically increase their overall intake and, hence, overingest lipids and carbohydrates to meet their protein requirement (Simpson and Raubenheimer, 2005; Sørensen et al., 2008). Unless this surplus energy is voided, the excess energy intake is stored as body fat, which could result in obesity (Simpson and Raubenheimer, 2005; Sørensen et al., 2008). One way of voiding excess ingested energy when feeding on a low-protein diet is to increase metabolic rate to burn off excessive energy by a process known as facultative diet-induced thermogenesis, as is seen in some herbivores and omnivores (Zanotto et al., 1993, 1997; Stock, 1999; Trier and Mattson, 2003). The extent to which facultative diet-induced thermogenesis is employed might be expected to reflect the balance between the costs of fat storage and the longer term benefit of fat stores as energy reserves in times of food shortage (Warbrick-Smith et al., 2006). As for most spiders,
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wolf spiders are frequently exposed to periods of starvation interspersed with periods of plenty (Riechert and Harp, 1987; Wise, 1993). It is interesting, therefore, to study whether these spiders possess regulatory adaptations to handle nutrient imbalance and/or starvation. It has previously been shown that prey low in protein and rich in lipids reduce growth, survival and fecundity in wolf spiders (Mayntz and Toft, 2001). It is unclear, however, whether wolf spiders increase metabolic rate when exposed to protein-poor, lipid-rich diets. Such a response would aid the spiders in achieving a better nutritional balance, but could also potentially render the animals more vulnerable to depletion of energy stores during food deprivation. It has been shown that wolf spiders can reduce metabolism in response to starvation (Tanaka and Itoˆ, 1982; Tanaka et al., 1985; Nakamura, 1987), but the metabolic response to changes in body and prey nutrient composition, as well as its interaction with food deprivation, is unknown. To gain further insight into this question, the present study investigated how prey of variable macronutrient composition (lipids vs. protein) affects body composition, basal metabolic rate (BMR), metabolic response to feeding and digestion (SDA), and metabolism during fasting in the wolf spider Pardosa prativaga. 2. Materials and methods
Fig. 1. Lipid vs. crude protein content (mean SE) of Pardosa prativaga wolf spiders immediately after winter hibernation and after 16 days of ad libitum feeding on lipidrich, intermediate, or protein-rich Drosophila melanogaster fruit flies. The slopes of the inserted lines, indicated by the number at the end of each line, represent the lipid: crude protein ratio of the three D. melanogaster fly types. Crude protein content is calculated by multiplying the nitrogen content with a constant factor of 6.25 (AOAC, 2000). Data for fly body compositions are from similarly produced D. melanogaster (Jensen, 2010).
2.1. Experimental diets Fruit flies (Drosophila melanogaster) used as food for the spiders were raised on three different media based on mixtures (g:g) of a basic medium (Carolina Instant Drosophila Medium Formula 4-24, Burlington, NC, USA), casein (Sigma C-5890, Sigma–Aldrich, Steinheim, Germany) and sucrose (Fluka, 84097, Neu-Ulm, Germany). Lipid-rich flies were raised in a medium with a 1:4 ratio of sucrose and basic medium; intermediate flies were raised on a 1:9 ratio of casein and basic medium; and protein-rich flies were raised on a 3:2 ratio of casein and basic medium. All flies were reared in 3.4 cm Ø vials containing 2.5 g dry medium mixed with 15–22 ml water and a few drops of satiated yeast solution covering the surface. Cultures were inoculated with 40–50 adult D. melanogaster of mixed sexes for 3 days and the resulting larvae were raised at 24–26 8C until emergence. The different rearing media produce fruit flies with markedly different protein and lipid contents (Fig. 1). 2.2. Experimental animals Juvenile P. prativaga wolf spiders (Lycosidae) were collected in wet meadows around Aarhus (Jutland, Denmark) in February 2008 as they were hibernating under vegetation. They were kept in individual transparent plastic vials (2 cm Ø, 6 cm height) with moist Plaster-of-Paris bottoms and foam rubber stoppers at 5 8C in darkness without food for 1–2 months before experiments started. This mimicked the conditions in nature during winter when the spiders were caught, and experiments started in middle spring. Five days before initiation of feeding, the spiders were transferred to an incubator at 25 8C and a 16:8 h light:dark photoperiod. Each spider (N = 268) was weighed to the nearest 1 mg and 16 randomly selected spiders were frozen at 18 8C to determine initial body composition. The remaining spiders were distributed randomly between three dietary treatments: lipid-rich flies, intermediate flies, or protein-rich flies for 16 days. During the feeding period, the spiders were allowed to feed to satiety every second day. 2.3. Experimental protocol Basal metabolic rate (BMR) of 44 randomly selected spiders from each feeding regime was measured just before the feeding
period, and measurements of mass and BMR were repeated after the 16 days on the respective diets. Ten other spiders from each feeding regime were frozen to assess how the diets had influenced body composition. The remaining spiders were then fasted for 7 days, weighed, and BMR measurements were repeated. To measure the metabolic cost of feeding, often termed the specific dynamic action of a meal (SDA), we fed the spiders a single preweighed fly from their respective diet directly in the respiration chamber. This measure therefore includes the metabolic cost of handling, ingesting, digesting, and assimilating a prey item (McCue, 2006). When the fly was taken, the respiration chamber was closed immediately and gas exchange was measured over two consecutive 3-h periods. All spiders completed meal extraction within the first 3-h period. Gas exchange was measured again over the following 14 h after which respiration levels had returned to resting rates. The spiders were then returned to their vials and fed flies ad libitum for 24 h to ensure that they were fully fed before exposure to long-term starvation. During the starvation period, mass and BMR were measured after 5, 11, 17, and 35 days. 2.4. Measurements of metabolism Gas exchange was measured by closed respirometry, in which the spiders were enclosed in individual 10-ml glass syringes sealed with vacuum grease. Except during SDA measurements, enclosure ˙ periods were 24 h, after which the decrease in O2 volume VO2 and increase in CO2 volume V˙ CO2 were measured. The syringes contained a small drop of water to keep the air saturated, and were submerged in a water bath at 25 8C to stabilize temperature. Measurements of V˙ O2 and V˙ CO2 were performed by injecting 8 ml gas from the respiratory chambers into a CO2 and O2 analyser (Applied Electrochemistry, Sunnyvale, CA, USA). This gas sample was then drawn through the analyser at a known and constant rate so that the O2 decrease and CO2 increase could be calculated relative to injections of standard gas samples of known O2 and CO2 contents. V˙ O2 and V˙ CO2 were corrected to standard temperature and pressure under dry conditions (STPD), and mass specific metabolic rate was calculated using the latest measure of body mass prior to the measurement. In most cases, CO2 of the respiratory chamber did not exceed 2% and O2 did not decline below 18%. For comparison, standard concentrations of CO2 and O2 in the air were
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20.95% and 0.03%, respectively. The respiratory gas exchange ratio (RE) was calculated as V˙ CO2 =V˙ O2 . 2.5. Calculation of SDA The specific dynamic action (SDA) was calculated as the energy consumption during feeding and digestion that exceeded the BMR immediately prior to feeding (Overgaard et al., 2002). Thus, the increase in V˙ O2 caused by feeding and digestion was calculated by subtracting the resting levels measured before feeding from the levels integrated over the 20 h period following prey capture. This V˙ O2 was then converted to Joules using a conversion factor of 19.67 J/ ml O2 (Gessaman and Nagy, 1988). Fly lipid and protein contents were calculated using known values of protein and lipid compositions of similarly raised dry flies (Jensen, 2010). Dry masses of the provided flies were estimated using average fly mass/dry mass ratios from 20 reference flies collected from each diet. Using standard energetic contents of lipid and protein (AOAC, 2000) and fly body masses, the energetic content of the lipid-rich, intermediate, and protein-rich flies was estimated to 5.5, 4.7 and 4.6 J/mg wet mass, respectively. Finally, the SDA coefficient was calculated as energy expended/energy ingested (Jobling and Davies, 1980). 2.6. Starvation tolerance Spiders (N = 30) from each of the feeding regimes were used to assess the longevity of fasting spiders. These spiders had been allowed to feed on the three diets ad libitum for 16 days after which they were provided water but no food. Deaths were recorded every morning and evening. 2.7. Body measurements
Fig. 2. Relative body mass growth of Pardosa prativaga wolf spiders after feeding and fasting (mean SE). Spiders were fed one of three experimental diets that was either protein-rich (solid), intermediate (dashed) or lipid-rich (dotted). After a 16-day ad libitum feeding period, the spiders were fasted for 1 week before being fed ad libitum for 1 day. After this the spiders were fasted. Only the first 17 fasting days are included in the figure, and only data of spiders surviving this period are shown.
ANCOVA tests using initial mass as the covariate. Diet effects on the number of molts during the feeding period were analysed using Nominal Logistic Regression. Survival during starvation was compared using Wilcoxon Chi Square test. Statistical analyses were made in JMP 7.0 (SAS Institute 2007). 3. Results 3.1. Spider nutrient composition, mass, size, and developmental stage Body lipid vs. crude protein compositions of the spiders before and after feeding for 16 days on the three diets are shown in Fig. 1.
At the end of the experiment the spiders sampled before and after the 16-day feeding period were dried over 4 days at 60 8C and weighed to nearest 1 mg after measuring carapace lengths with a microscope eyepiece scale. Lipids were extracted over two 24-h washes in 2 ml petroleum ether, and the lean corpses were dried and weighed. The lipid mass of each spider was calculated by subtracting the lean dry body mass from the total dry body mass. Last, corpses were analysed for proportion of nitrogen relative to dry mass in a combustion analyser (Na 2000, Carlo Erba, Italy). Absolute nitrogen masses were calculated by multiplying nitrogen proportions with the respective dry masses, and crude protein masses were calculated using a protein:nitrogen factor of 6.25 (AOAC, 2000). Spider lipid energy stores were calculated by multiplying spider lipid masses with standard lipid energy content (AOAC, 2000). To get an estimate of predicted survivorship within each feeding treatment, we divided the calculated energy from lipid storages with the basal energy expenditure measured during the first 17 days of the last fasting period (Fig. 2), assuming constant BMR after the 17th fasting day. As metabolism is known to decline during fasting in wolf spiders (Tanaka and Itoˆ, 1982; Nakamura, 1987), this estimate expresses survivorship if no further adjustments to starvation were made after 17 days of starvation. 2.8. Statistical analysis Differences in nutritional composition and SDA coefficient across treatment groups were tested using ANOVA. ANOVA was also used to test for differences in V˙ O2 , V˙ CO2 , and RE across diet treatments at individual time points. Fractions and percentages were arcsine transformed before analysis. Tukey-Kramer tests were used to identify differences between individual dietary groups. Effects of diet on spider mass and size were compared with
Fig. 3. (a) Body mass and (b) carapace length of Pardosa prativaga wolf spiders (mean SE) after being fed 16 days ad libitum on lipid-rich, intermediate, or proteinrich fruit flies.
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The spiders had a relative lipid composition of 8.9 1.1% (mean SE) at the onset of experimental feeding. However, after 16 days of feeding on the different prey types, the relative lipid contents of the spiders were significantly affected by the nutrient composition of their diet (ANOVA, F3,46 = 23.35, P < 0.0001). A post hoc test revealed that the relative lipid composition of the spiders differed significantly between all three diets after the 16-day feeding period (TukeyKramer, P < 0.05). The relative protein composition was also significantly affected by feeding on different diets (ANOVA, F3,46 = 14.69, P < 0.0001). Post hoc analysis showed that spiders on the protein-rich diet had significantly higher relative crude protein content than spiders on the two other diets (Tukey-Kramer, P < 0.05), but crude protein content did not differ significantly between the intermediate and the lipid-rich diet (Tukey-Kramer, P > 0.05). Spiders on all diets in most cases increased body mass by at least 100% during the 16-day feeding period, and gradually lost body mass during the subsequent fasting period (Fig. 2). After the 16-day feeding period, we found a significant dietary treatment effect on both body mass (ANCOVA, F2,27 = 7.93, P = 0.002) and carapace length (ANCOVA, F2,27 = 5.32, P = 0.013). The general tendency was higher body mass (Fig. 3a) and longer carapace (Fig. 3b) in spiders fed more protein-rich prey. Spiders went through either none, one, two or three molts during the
feeding period, but there was no significant effect of diet on number of molts (Nominal Logistic Regression, F2,224 = 12.50, P > 0.05). Most spiders were subadult after one molt and adult after two molts. 3.2. Basal metabolic rate and the metabolic response to feeding and digestion Diet composition did not affect V˙ O2 or V˙ CO2 before or after the 16-day feeding period (Fig. 4). Furthermore, the maximal metabolic response during feeding and digestion did not differ among diets. The metabolic response to feeding and digestion caused a large increase in gas exchange in all three experimental groups and was associated with an increase in the respiratory exchange ratio (RE) from a fasting value of 0.79 0.005 (mean SE) to a value of 0.85 0.005 (mean SE) during feeding and digestion (Fig. 4). Following ingestion of meals corresponding to an average of 8.5% of spider mass, V˙ O2 and V˙ CO2 increased to more than 4-fold above fasting values within the 3-h period after prey capture. This cumulative SDA response did not differ among diets (ANOVA, F2,49 = 0.28, P = 0.76, Fig. 5a). Similarly, we found no difference between dietary treatments when the SDA response was calculated relative to the amount of energy ingested (SDA coefficients). The SDA coefficient was approximately 21% in all three groups (ANOVA, F2,49 = 0.08, P = 0.92, Fig. 5b). Thus, 21% of the ingested energy was respired as a consequence of feeding and digestion in all groups. 3.3. Survival during fasting Survival during fasting differed significantly across the three diets (P = 0.001, x290 ¼ 13:3, Wilcoxon). The fasting duration causing 50% mortality was 34 days for spiders fed a protein-rich diet, 40 days for intermediate spiders, and 46 days for lipid-rich spiders (Fig. 6a). When calculated solely from lipid storage and BMR, spiders from the three diet types were estimated to survive approximately 18, 23 and 32 days (Fig. 6b). Survival time thus correlated well with lipid storage.
Fig. 4. Gas exchange of digesting and fasting Pardosa prativaga wolf spiders. (a) Oxygen consumption (V˙ O2 ), (b) carbon dioxide production (V˙ CO2 ), and (c) respiratory exchange ratio (RE). The spiders were maintained at 25 8C. In order to visualize the short period associated with consumption and digestion of a single fly (1 fly), the x-axis is extended during this period. Data are presented as mean SE.
Fig. 5. Metabolic increase (SDA response) of Pardosa prativaga wolf spiders during feeding and digestion of a single D. melanogaster varying in lipid:protein content (mean SE). (a) The SDA response presented as the total oxygen consumption in excess of the basal metabolism. (b) The SDA response presented as the amount of energy used for feeding and digestion relative to the ingested energy (SDA coefficient).
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Fig. 6. (a) Survival of Pardosa prativaga wolf spiders during starvation. The spiders were fed either protein-rich (N = 30), intermediate (N = 30) or lipid-rich (N = 30) fruit flies ad libitum for 16 days immediately after winter hibernation, after which they were starved until death occurred. (b) The calculated expected time of survival based on the spider lipid stores and basal metabolism of fasting spiders.
4. Discussion We tested the hypothesis that spiders fed more lipid-rich/ protein-poor prey would elevate metabolism in order to gain more protein through increased prey consumption. Our measurements of metabolic responses by the spiders did not support this hypothesis. Although spider body compositions were highly affected by the nutrient ratio of their prey, prey body compositions could not entirely predict spider body compositions (Fig. 1). This indicates that the spiders might have regulated either (1) the ratio of nutrients extracted from their prey (Mayntz et al., 2005), (2) the assimilation efficiency of the nutrients extracted from their prey (Lee et al., 2004), or (3) their metabolic response to consuming nutritionally different prey. Our results from the present study show that the latter was not the case. We found similar metabolic rates and respiratory exchange ratios of spiders from the three different dietary treatments, indicating that the spiders metabolized both the same type and amount of energy substrates. We measured gas exchange over 3-h intervals in feeding and digesting P. prativaga and found a 4-fold higher metabolism during the first 3-h period compared to immediately before feeding. It is likely that a finer time resolution would have disclosed an even larger increase in metabolism than revealed in our study. Thus, as documented extensively in many other animals (McCue, 2006), metabolism increased substantially during feeding and digestion. This specific dynamic action of food represents the energetic costs associated with subduing and handling the prey, digestive responses, and post-absorptive events (McCue, 2006). Protein synthesis appears to be a major contributor to the SDA response,
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which normally increases with protein content of the meal (Halton and Hu, 2004). However, this was not the case for P. prativaga as all three diets elicited very similar metabolic responses. In this respect it is important to remember that the diets used here were all predominantly protein-biased, and it is possible that larger differences in diet composition are needed to produce differences in the metabolic cost of feeding and digestion. Because we refilled the respiration chambers with new air after the spiders had captured their prey, the activity associated with capture cannot account for the rise in metabolism. Instead, we suggest that the excretion of digestive enzymes for extraoral digestion, sucking in the juices, and internal digestion and absorption caused the SDA response. We found that the total costs of consuming, digesting, and absorbing the prey fly was around 21% of the energy it contained, which is in the range measured for snakes fed different types of prey (McCue, 2006). While the respiratory exchange ratio (RE) did not differ between the three different diets, we found that RE increased during feeding and digestion, indicating that more protein and carbohydrates were metabolised in the postprandial period. Prey nutrient composition had a considerable effect on the growth rate of the spiders, which was significantly lower in spiders fed more lipid-rich prey. Other studies have found that survival and fecundity may also be reduced when spiders are fed prey of low protein and high lipid composition (Mayntz and Toft, 2001; Mayntz et al., 2003). When feeding on lipid-rich prey causes lower growth rates of the spiders, why have the wolf spiders not evolved the physiological adaptations to remove this excess of lipids and allow additional food intake to remedy protein deficiency? Our study suggests an explanation for this. While the spiders clearly lacked the ability to vary metabolism in response to nutrient imbalance, they reduced metabolism over prolonged periods of starvation, which has also been observed in other species of spiders (Tanaka and Itoˆ, 1982; Tanaka et al., 1985; Nakamura, 1987), resulting in longer survival than expected from lipid energy stores and BMR by the onset of fasting (Fig. 6). Indeed, the ability to lower metabolism during prolonged starvation seems to represent a general response among many species of invertebrates and vertebrates as it prolongs survival with a finite energy store (Wang et al., 2006). Thus, in addition to their flexible abdomens that allow spiders to gorge when food is available, the spiders have also adapted physiologically to survive extended periods of starvation. We suggest that the absence of metabolic adjustments to burn excessive lipids may be adaptive in spiders that are occasionally exposed to unpredictable food availability (Riechert and Harp, 1987; Wise, 1993). In such an environment, selection for increased metabolism to remove excessive lipids may be counteracted by selection for large lipid stores which enable the spiders to survive long and unpredictable starvation periods. Results from our survival experiment on the fasting spiders support this hypothesis (Fig. 6a). In conclusion, P. prativaga wolf spiders did not seem to regulate metabolism to allow higher protein intake, even though spiders were clearly protein deficient on a protein-poor diet. We propose that regulation of nutrient intake and assimilation by increasing metabolism might not be highly evolved in predators that experience occasional prey limitation and energy deprivation (Wise, 1993). Acknowledgements This study was supported by grants from the BBSRC in UK and The Danish Research Council. References AOAC, 2000. Official Methods of Analysis, 17th ed. AOAC International, Gaithersburg. Berthoud, H.R., Seeley, R.J., 2000. Neural and Metabolic Control of Macronutrient Intake. CRC Press, Boca Raton.
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