Effect of meal type on specific dynamic action in the green shore crab, Carcinus maenas Iain J. McGaw & Chantelle M. Penney
Journal of Comparative Physiology B Biochemical, Systems, and Environmental Physiology ISSN 0174-1578 Volume 184 Number 4 J Comp Physiol B (2014) 184:425-436 DOI 10.1007/s00360-014-0812-5
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Author's personal copy J Comp Physiol B (2014) 184:425–436 DOI 10.1007/s00360-014-0812-5
Original Paper
Effect of meal type on specific dynamic action in the green shore crab, Carcinus maenas Iain J. McGaw · Chantelle M. Penney
Received: 14 December 2013 / Revised: 21 January 2014 / Accepted: 31 January 2014 / Published online: 15 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract The effect of meal type on specific dynamic action was investigated in the green shore crab, Carcinus maenas. When the crabs were offered a meal of fish, shrimp, or mussel of 3 % of their body mass the duration of the SDA response and thus the resultant SDA was lower for the mussel, compared with the shrimp or fish meals. In feeding behaviour experiments the crabs consumed almost twice as much mussel compared with fish or shrimp. When the animals were allowed to feed on each meal until satiated, the differences in the SDA response were abolished. The mussel was much softer (compression test) than the fish or shrimp meal, and meal texture is known to affect the SDA response in amphibians and reptiles. When the crabs were offered a meal of homogenized fish muscle or whole fish muscle, the SDA for the homogenized meal was approximately 35 % lower. This suggested that a significant portion of the SDA budget in decapod crustaceans may be related to mechanical digestion. This is not unexpected since the foregut is supplied by over forty muscles which control the cutting and grinding movements of the gastric mill apparatus. There were slight, but significant differences in protein, lipid, moisture and total energy content of each meal type. Three prepared meals that were high in either protein, lipid or carbohydrate were offered to the crabs to determine if the nutrient content was also a contributing factor to the observed differences in the SDA. The crabs did not eat the prepared meals as readily as the natural food items and as they are messy feeders there was
Communicated by I.D. Hume. I. J. McGaw (*) · C. M. Penney Department of Ocean Sciences, Memorial University, 0 Marine Lab Road, St John’s, NL A1C 5S7, Canada e-mail:
[email protected]
a large variation in the amount of food eaten. The lack of significant differences in the SDA response as a function of nutrient content was likely due to differences in amount of food eaten, which is a major factor determining the SDA response. The differences in SDA when consuming natural food items were likely due to a combination of the costs of mechanical digestion, variation in nutrient content and food preference: determining how each of these factors contributes to the overall SDA budget remains a pressing question for comparative physiologists. Keywords Carcinus maenas · Crab · Digestion · Feeding · Respiration · Specific dynamic action
Introduction The specific dynamic action of food or SDA describes a postprandial increase in metabolism. This increase in metabolic rate, which is usually measured as an increase in oxygen consumption, represents the sum of activities associated with the processing of the food, ingestion, mechanical breakdown in the gut and the subsequent transport and intracellular digestion of the nutrients (Mente 2003; Secor 2009). The characteristics of the SDA response measured include the time to reach peak oxygen consumption, the peak oxygen consumption and/or the scope of oxygen uptake (difference between resting and peak metabolism), and the duration that the postprandial metabolism remains elevated. The SDA or energy equivalent of the metabolic response is most often used to quantify energy expenditure. It represents the accumulated energy expended above the baseline for the duration of the SDA response (Secor 2009). When the meal size and environmental temperature are controlled, the nutrient content of the meal and the type
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of meal also affect some of the characteristics of the SDA response. The general pattern is that meals high in protein generate larger SDAs than those that have a higher lipid or carbohydrate content (reviewed in McCue 2006; Secor 2009). In the fish Oncorhynchus mykiss, Cyprinus carpio, Oreochromis niloticus and Silurus meridionalis, an increase in the percentage of protein leads to an increase in the SDA (LeGrow and Beamish 1986; Chakraborty et al. 1992; Ross et al. 1992; Fu et al. 2005). However, this pattern is not universal, for example, increased protein content does not produce an observable change in the SDA of the blue gill, Lepomis machrochirus, sea bass, Dicentrarchus labrax or horned frog, Ceratophrys cranwelli (Schalle and Wissing 1976; Peres and Oliva-Teles 2001; Grayson et al. 2005). The SDA is also influenced by the relative proportions of lipids and proteins, and meals with a higher lipid to protein ration evoke larger SDAs because the lipids may have a protein sparing effect (LeGrow and Beamish 1986; Chakraborty et al. 1992; Fu et al. 2005; Luo and Xie 2008). The nutrient type is also important, digestion of complex proteins produces SDA’s of greater magnitude compared with simple proteins or those lacking specific amino acid residues (McCue et al. 2005). There is comparatively less information on the SDA responses of organisms consuming natural prey items. In the tortoise Kinixys spekii the highest SDA occurs after eating millipedes, while lower SDAs are measured when they consume leaves or fungi; this is related to the higher protein content of millipedes (Hailey 1998). In the skink Eumeces chinensis the SDA peak and magnitude are greater for a high protein meat meal compared with meal worms that have lower protein content (Pan et al. 2005). A similar pattern is observed for the ascidian Ciona intestinalis which exhibits a significantly higher SDA after feeding on flagellates compared to detritus (Sigsgaard et al. 2003). However, the nutrient content of a natural meal is not the only factor to affect the SDA. Consumption of vertebrate prey results in larger SDA’s in the garter snake Thamnopis sirtalis compared to consumption of soft bodied invertebrate prey (Bessler et al. 2010). The gila monster Heloderma suspectum, exhibits a lower SDA when feeding on chicken eggs compared to an equal sized meal of juvenile rats (Christel et al. 2007). Likewise, for a variety of anuran and salamander species fed hard bodied superworms, meal worms and crickets both the duration and the magnitude of the SDA response are higher than when these species consume soft bodied red worms, beetle larva or neonatal rodents (Secor and Faulkner 2002; Secor and Boehm 2006; Secor et al. 2007). These authors suggested that it takes more energy to break down and assimilate the hard-bodied prey items (Secor 2009). Related to this, the physical state of the meal can influence the SDA response. Indian pythons, Python molurus, expend more energy to digest a whole rat versus a ground rat. This is primarily
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due to the cost of transport of stomach acids and digestive enzymes to break down the intact meal, rather than energy required for mechanical breakdown by peristaltic contraction of the stomach muscles (Secor 2003; Boback et al. 2007). Different diets have been evaluated for commercially important species of shrimp and lobster; however, these articles tend to concentrate on short-term changes in oxygen consumption (1–6 h) and feed conversion efficiency ratios, rather than the SDA (Nelson et al. 1977, 1985; Rosas et al. 1995, 1996; Brito et al. 2000; Crear et al. 2002; Perera et al. 2005; Huang et al. 2008; Díaz-Iglesias et al. 2011). The general trend is metabolic rate increases with increasing protein content of the meal; however, the exact pattern is dependent on the species and the quality of both proteins and lipids. Other works have investigated the longer-term effects of diet on metabolism: The isopod Ligia pallasii exhibits higher oxygen consumption rates after 30 days of feeding on brown algae compared with green or red algae (Carefoot 1987), whereas Ligia exotica exhibits higher oxygen uptake rates when fed a mixed diet of algae and diatoms compared to either red or green algae (Carefoot 1989). Very few papers have directly addressed the effects of meal type on the SDA of crustaceans. The copepod Acartia tonsa has a higher SDA coefficient when feeding on the flagellate Tetraselmis impellucida versus Dunaliella tertiolecta, which is associated with high protein assimilation at the tissue level (Thor et al. 2002). The scope and magnitude of the SDA in Ligia pallasii are higher after a meal of brown algae compared to green algae. However, they ate twice as much brown algae (Carefoot 1989) and this, rather than diet likely accounted for the difference (Carefoot 1989; McGaw and Curtis 2013a). The species chosen for this study, the green crab Carcinus maenas, is an important invasive species in temperate marine environments; it occurs in the shallow subtidal and intertidal zones as well as in estuaries. As such it is an opportunistic predator on a wide variety of both fresh and decaying material including, but not limited to, bivalves, worms, crustaceans, carrion, detritus and algae (Behrens-Yamada 2001). Therefore, the aim of the present study was to investigate feeding behaviour on natural prey items and to determine how consumption of these may affect the characteristics of the SDA response. The nutrient content of the meal was also manipulated to determine if this would also affect feeding behaviour and the SDA response of Carcinus maenas.
Materials and methods Animal collection and housing Adult intermoult male green crabs, Carcinus maenas (65– 100 g), were collected from North Harbour, Newfoundland
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and maintained at 30–32 ‰ and 14–16 °C at the Department of Ocean Sciences, Memorial University. They were acclimated to laboratory conditions for at least 7 days, and all experiments were carried out at the holding temperature and salinity. The animals were fed fish twice a week and allowed to eat until satiated, but were isolated from the general population and fasted for 3–6 days prior to experimentation. This time period allowed all food to be evacuated from the digestive system, but avoided large-scale physiological changes associated with starvation (Wallace 1973). Behaviour The feeding behaviour was assessed to determine how much of each meal type a crab would consume. The food was soaked in seawater overnight to minimize any osmotic gain or loss of water and was patted dry in paper towels before being weighed. During feeding individual crabs were held in plastic boxes of 25 cm × 15 cm × 10 cm depth with 1 mm mesh screen on the sides. Fifteen crabs were offered an excess of either mussel, fish, or shrimp. The animals were allowed to consume the food and when they had ceased feeding for 1 h the experiment was terminated. The remaining food was collected with forceps, patted dry and weighed to calculate the wet mass consumed and was expressed as percent body weight eaten. The feeding experiment was then repeated with three prepared diets (high protein, fat or carbohydrate). Control samples of food were also placed in empty mesh cages, the samples were weighed at the start and end of the experiment and any weight gain or loss of the samples was adjusted for in the final calculation of mass of food eaten. Oxygen consumption Oxygen consumption (mg O2 kg/h) was measured using a L-DAQ intermittent flow respirometry system (Loligo systems, Copenhagen, Denmark). This fully automated system is equipped with two pumps: the first pump continually flushes seawater through the chamber while it is open. The chamber is sealed for measurements and a second pump recirculates the water through the chamber at a rate of 10 L/ min ensuring that oxygen gradients do not build up within the chamber. During experiments the animals were held in cylindrical chambers (20 cm diameter × 12 cm depth) and allowed to settle for 12 h. Oxygen consumption was calculated during a 20-min decline in oxygen levels while the chamber was sealed; then the chamber was continuously flushed for 10 min between readings. Data was recorded on a Loligo data acquisition system (Copenhagen, Denmark). The experiments were carried out in constant dim light, which helped reduce any diurnal rhythms and the apparatus
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were surrounded by black plastic sheeting to avoid visual disturbance to the animal. The resting metabolic rate (postabsorptive, minimal activity) was recorded for a 3-h control period. The animals were then fed the test meal; any animals that did not eat all the food offered were not used in the analyses. All the crabs had finished feeding by the time the first postprandial oxygen consumption reading (0.5 h) was completed. Oxygen consumption was recorded until it returned to pre-feeding levels. For each experiment the following parameters were calculated: (a) the time to reach peak oxygen consumption following feeding, (b) The scope of the SDA response—peak oxygen consumption divided by the basal pre-feeding rates (RMR) (c) the duration of the SDA response—until oxygen dropped back to pre-feeding levels and (d) the SDA of each animal was calculated from the total increase in oxygen uptake above baseline levels and standardized to kJ using the conversion factor of 1 mg O2 = 14 J (Secor 2009). Differences in parameters were compared using one-way ANOVAs or Student t tests. Data showing a significant effect were further analysed with a Fisher LSD post hoc test. Data that were not normally distributed were analysed with a Kruskal–Wallis non-parametric ANOVA on ranks followed by a Dunn post hoc test or two samples were analysed with Wilcoxon rank sum tests. In the first series of experiments the animals (n = 10–11 separate animals per meal type) were offered three natural prey items. The crabs were fed of 3 % of their body weight (wet mass) of mussel flesh (Mytilus edulis), fish muscle (sole), or shrimp muscle. The experiment was then repeated, allowing the animals to feed ad libitum on each meal; any excess was removed after they had finished feeding for 1 h. A second series of experiments were carried out to determine if the meal texture would affect the SDA. The crabs (n = 13) were fed 2.5–3.0 % of their body mass of sole flesh. The flesh was broken down to a pastelike texture with a tissue homogenizer and either compacted pieces of paste or whole pieces of fish were offered to the crabs. In a final series of experiments the effects of meals high in protein, lipids or carbohydrates on the SDA response were investigated. The crabs (n = 10) were offered prepared meals of 3 % of their body weight. The high-protein diet consisted of ground sole (70 % wet mass) in gelatin (30 % wet mass) and had an energetic content of 4.35 ± 0.15 kcal/g dry weight, the high lipid diet consisted of sole (45 % wet mass) blended with lard (15 % wet mass) and menhaden oil (10 % wet mass) in gelatin (30 % wet mass) with an energetic content of 7.05 ± 0.13 kcal/g and the high-carbohydrate diet consisted of sole (45 % wet mass), blended with potato starch paste (25 % wet mass) in agar (30 % wet mass) with an energetic content of 4.15 ± 0.05 kcal/g. A small amount of commercial crab attractant (Pautzke crab ‘n’ shrimp fuel®) was also added to each meal.
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Digestion The clearance rates of the foregut were investigated using a serial slaughter technique (Hill 1976; Choy 1986). The crabs were fed mussel, shrimp or fish of approximately 3 % of the body mass and allowed 1 h to consume the food. Six crabs were removed from each feeding treatment at set intervals and immersed in iced seawater for 5 min to induce a chill coma and to halt the digestive processes. They were then frozen and stored at −20 °C. For analysis the animals were thawed, the foregut was dissected and the digesta was rinsed out with distilled water. These contents were dried to constant weight at 60 °C. A foregut clearance index was derived by multiplying the dry mass of the digesta by the reciprocal of the carapace width to standardize for size (Simon 2009). Regression lines were fitted to the data and the slopes of the lines compared (Zar 1984). In order to determine if there was any correlation between the characteristics of the SDA and digestion, the transit time of a radio-opaque meal was followed through the digestive system. The crabs were fed a radio-opaque meal consisting of electrolytic iron powder (10 % by mass) and gelatin (15 % by mass) added to whole pieces of mussel, fish or shrimp (McGaw 2006). During experiments the animals (n = 8 per food item) were housed in individual chambers where they were allowed to settle for 3 h before initiation of the experiment. The animals were offered the food and allowed 15 min to eat the meal; those that did not eat the entire meal were not used in the analysis. For X-ray analysis a plastic box was submerged in the chamber and the animals were coaxed into the box. The box was then placed in under a LIXI PS500 OS, X-ray system with LIXI image processing software. A still image of the gut system was captured at hourly intervals for the first 12 h and then at 3–12 h intervals following this period. Technical specifications for X-ray were 35 kV tube voltage and 155 μA tube current with a 5 cm focal window. The movement of the digesta and marker was followed until it had been voided in the faeces and the time of emptying of the foregut, midgut and hindgut regions was calculated (McGaw 2006). Differences in transit rates as a function of meal type were analysed using a 2-way repeated measures ANOVA. The digestive efficiency was assessed following the consumption of a mussel, fish or shrimp meal. During feeding the crabs (n = 6 per treatment) were held in plastic boxes of 25 cm × 15 cm × 10 cm depth with 1 mm mesh screen on the sides and were offered a pre-weighed meal of mussel, fish or shrimp. The animals were allowed to consume the food and when they had ceased feeding for 1 h the remaining food was collected with forceps, patted dry and weighed to calculate the mass consumed. At daily intervals (for a total of 3 days) faeces were removed and washed with distilled water to prevent salt crystallization. The faeces and samples of the food were dried at 60 °C to
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constant weight. The caloric energy value of the dried samples (0.1 g) was measured using a bomb calorimeter (Parr Instruments, SemiMicro, Moline, IL). Digestive efficiency was calculated using the equation outlined in Romero et al. (2006): AE = [EC food × dry mass (g) − EC faeces × dry mass (g)/EC food × dry mass (g)] × 100, where AE = assimilation efficiency and EC = energetic content. Differences in assimilation efficiencies were tested with a one-way ANOVA followed by Fishers LSD post hoc tests. Proximate analysis of food Samples of the meals were processed by the Centre for Aquaculture and Seafood Development at the Marine Institute, Memorial University. Moisture content was calculated by drying the sample to constant weight in an oven at 60 °C. The ash-free dry weight of the samples were determined by placing 3 g of dried sample in a crucible and heating to 550 °C in a muffle furnace for 15 h. The remaining inorganic material was weighed and percentage ash calculated. Fat was extracted from the dry samples by acid hydrolysis and the lipid content in each of the samples was calculated using a modified Soxhlet method (Booij and van den Berg 1994). The protein content of the samples was measured using the Kjeldahl method: this measures the total amount of nitrogen in the sample; the results therefore represent the crude protein content of the sample because some nitrogen may have come from non-protein components (Bradstreet 1954). Carbohydrate levels were calculated as the remaining percentage difference from totals of moisture, ash free weight, lipid and protein levels. The caloric energy value of 0.1 g dried sample was measured using a semi-micro bomb calorimeter (Parr Instruments). The compressibility of each food item was measured using a TA-XT Plus analyzer (Stable Micro Systems Ltd, Surrey, UK). Tests were performed using a flat cylindrical 9.5 mm probe using a 50 % compression test. Differences in parameters were compared using one-way ANOVAs. Data showing a significant effect were further analysed with a Fisher LSD post hoc test. Data that were not normally distributed were analysed with a Kruskal–Wallis non-parametric ANOVA on ranks followed by a Dunn post hoc test.
Results Feeding behaviour When the crabs were allowed to feed ad libitum on mussel, fish or shrimp flesh they had all stopped feeding within 1 h. The crabs consumed on average 6.91 ± 0.6 % of their body mass of mussel flesh; this was significantly greater than the 3.39 ± 0.55 % body mass of fish or the 4.34 ± 0.3 %
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body mass of shrimp flesh eaten (ANOVA, F = 13.34, P 98 %) and there was no significant difference between each of the three meals (Table 4).
resting metabolic levels and can remain elevated for between 12 and 48 h, depending on the species and the size of the meal (McGaw and Curtis 2013a). The characteristics of the SDA response obtained here for Carcinus maenas were similar to those recorded for other species (Houlihan et al. 1990; McGaw 2006; Curtis and McGaw 2010; McGaw and Curtis 2013a); however, unlike many of the other species there was a significant variation in oxygen uptake rates between and within individual animals. The crabs exhibited spontaneous periods of activity which appeared to be unrelated to endogenous rhythms or exogenous cues; such behaviour is common for this species and likely accounted for differences in resting metabolic rate between groups (Reid and Naylor 1990; Warman et al. 1993). When Carcinus maenas was allowed to feed ad libitum on the three natural prey items they consumed more mussel flesh (almost 7 % BW) than shrimp or fish. This species has a broad diet ranging from small fish to algae and detritus (Behrens-Yamada 2001), which would tend to argue against a simple preference (Kittaka and Booth 2000). The mussel flesh had the lowest protein and energy content and the highest moisture content and there was a relationship between energy content and amount of food eaten, with more of the lower energy food consumed. A number of other invertebrate species can select meals of different nutritional quality depending on their metabolic needs (Loo and Bitterman 1992; Behmer et al. 2005; Behmer 2009; Lee et al. 2012). However, when the crabs were offered artificial foods with more pronounced differences in
Proximate analysis There were a number of significant differences in nutrient content of the three meals (Table 5). The caloric energy content of 1 g of dry mass of the fish meal was higher than that of the shrimp, which in turn was higher than that of mussel. The fish also had a slightly higher protein and fat content and a lower ash and moisture content than either the mussel or shrimp. When the caloric content of the three meals was calculated taking into account the differences in moisture and ash content, the meals were still in the same order with fish having 1.1 kcal/g, shrimp 0.74 kcal/g, and mussel having an energetic content of 0.69 kcal/g per 1 g of wet flesh. The compressibility values (softness) of the fish and shrimp flesh were both significantly higher than that of the mussel (Table 5).
Discussion The postprandial increase in oxygen consumption in decapod crustaceans typically peaks at 1.5- to threefold above
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Table 3 Characteristics of the SDA response of Carcinus maenas Meal type Protein Meal = Fixed Body mass (g) RMR (mg O2 kg/h) Peak VO2 (mg O2 kg/h)
78.6 ± 3.1 48.11 ± 1.34a
Carbohydrate
78.9 ± 4.8 42.87 ± 1.29b
78.8 ± 1.1 45.82 ± 1.50ab
Statistics
F = 0.21, P = 0.998 F = 3.76, P = 0.026*
143.27 ± 13.55
139.81 ± 10.22
F = 0.10, P = 0.909
Time to peak (h)
5.25 ± 1.25a
1.65 ± 0.57b
3.1 ± 0.75ab
F = 4.20, P = 0.03*
Scope (peak MO2/RMR)
2.88 ± 0.24
3.43 ± 0.33
3.18 ± 0.34
F = 0.82, P = 0.45
Duration (h)
42.7 ± 5.77
42.60 ± 3.73
32.9 ± 4.05
F = 1.49, P = 0.243
SDA (kJ)
1.09 ± 0.18
1.39 ± 0.35
0.71 ± 0.12
F = 2.06, P = 0.147
13.85 ± 2.16
17.52 ± 4.09
9.20 ± 1.61
H = 2.82, P = 0.244
SDA (kJ/kg)
136.39 ± 9.06
Fat
The crabs (n = 10 per treatment) were offered prepared meals of 3 % of their body mass that were high in carbohydrate, lipid or protein. Values represent the mean ± SEM. Data were analysed with student t tests, or Mann–Whitney U tests and significance reported at the P
