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Growth and enzyme production in blue crabs (Callinectes sapidus) fed cellulose and chitin supplemented diets.

ABSTRACT The blue crab [Callinectes sapidus (Rathbun. 1896)] is a benthic decapod with a varied diet. The diet includes invertebrates and detrital material that can have relatively large amounts of chitin and cellulose, both of which can be difficult to digest for many organisms and often require the aid of specific bacteria in the gut microbiome. In this study, juvenile blue crabs were fed an optimized defined pelleted diet with a 20% replacement of wheat flour filler with either chitin, cellulose, or a 14%/6% mix of both, followed by a diet switch to the opposing ingredient. Crabs had increasing growth performance with increasing amounts of cellulose in the diet versus chitin and had an additional molt in most cases. This occurred during the initial phase and following the switch, indicating that performance can be recovered. Subsequently, cellulose and chitin digestion assays were used to show that the foregut. midgut, and hindgut were all able to significantly digest more cellulose than chitin with the majority of activity in the foregut and midgut. Implications for rearing and diet formulations as well as the role of cellulose and chitin digestion in the natural diet are discussed.

KEY WORDS: Callinectes sapidus. blue crab. diet, growth, fiber, chitin, cellulose, recirculating, lecithin, menhaden oil

INTRODUCTION

Although the blue crab (Callinectes sapidus) can migrate over large distances during its life cycle, it spends much of its time as a benthic forager (Forward et al. 2003, Carr et al. 2005). Its diet consists mainly of other crabs, polychaete worms, and razor clams; as well as plant material and detritus (Laughlin 1982, Stoner & Buchanan 1990). This means that a substantial portion of the food intake consists of chitin and cellulose, which require specific enzymes to break the linkages of the polymer so that absorbable units can be obtained. Endogenous cellulases and chitinases have been discovered in a variety of crustaceans and molluscs, including the prawns Macrohrachium rosenbergii (de Man, 1879), Penaeus japonicas (Spence Bate, 1888), and Pandalus borealis (Kroyer, 1838) (Kono et al. 1990, Esaiassen et al. 1996, Watanabe et al. 1998, Gonzalez-Pena et al. 2002); six species of Tasmanian crabs (Johnston & Freeman 2005); the lobsters Jasus edwardsii (Hutton. 1875) and Homarus americanus (H. Milne-Edwards, 1837) (Lynn 1990, Johnston 2003); the red claw crayfish Cherax quadricarinatus (Von Martens, 1868) (Byrne et al. 1999, Xue et al. 1999); the abalone Haliotis discus (Reeve, 1846) (Suzuki et al. 2003); and the blue mussel Mytilus edidis (Linnaeus, 1758) (Xu et al. 2000, Xu et al. 2001).

However, chitinases and cellulases can perform a variety of roles not linked to digestion. Cellulases have been implicated in root nodule infections (Chalifour & Benhamou. 1989) and biofilms (Costerton et al. 1987). Many vertebrates, including humans, have been shown to express chitinases in response to fungal infections (Vega & Kalkum 2012), and in shellfish, chitinases have been associated both with mantle formation (Huning et al. 2013) and during an immune response (Badariotti et al. 2007). Thus, detection of chitinases and cellulases by molecular methods or even demonstration of enzymatic activity does not necessarily mean that chitin or cellulose can provide a nutritive benefit to the animal. To do that, a diet challenge is required and performance metrics must be assessed. For example, following detection of a cellulase in the mud crab Scylla serrata (Forsskal. 1775), individuals were challenged with a 29% starch diet as well as two diets where the major protein source was replaced with starch or cellulose to 47% (Pavasovic et al. 2004). All three diets performed equally and an increase in dietary starch or cellulose resulted in an increase in cellulase specific activity. This positive correlation between cellulose content and cellulose activity has also been demonstrated in the prawn Macrobrachium rosenbergii (GonzalezPena et al. 2002). Given the amount of cellulose in the natural diet of Callinectes sapidus, it is possible that endogenous cellulase activity may be present, and that cellulose is a potential energy source. Also, a chitinase is known to exist in this species, probably for the purpose of molting, but activity in the gut or a role in digestion remains to be shown.

From an aquaculture perspective, cellulose and chitin represent potentially cheap carbon sources for blue crab production, but the digestibility and performance of each ingredient in a feed is an important consideration for a successful operation, both for restocking and commercial production. Despite recent recovery of the blue crab populations in the Chesapeake Bay (Lipcius et al. 2012), the population is still low compared with historic levels. Thus, restocking and commercial production through aquaculture may both serve to ease pressures on this valuable fishery. To test whether cellulose or chitin can be used as a feed ingredient in the aquaculture of Callinectes sapidus, optimization for cage size and ration of a commercial shrimp diet was first performed to standardize experimental conditions. The wheat flour portion of the diet (used as a filler) was then replaced with chitin, cellulose, or a mix of the two to determine growth performance. Finally, gastric fluids were extracted from the foregut, midgut, and hindgut as well as the hepatopancreas and gastric ceca and assayed for the ability to degrade cellulose or chitin. Growth performance increased with the addition of cellulose, and cellulase activity was detected mainly in the foregut and hepatopancreas of individuals at a constant level. Surprisingly, chitin addition had deleterious effects on growth and there was minimal chitinase activity in the gastric juices.

MATERIALS AND METHODS

Cage Size and Ration Optimization

Two hundred and ten hatchery-reared Callinectes sapidus individuals with an average carapace width (CW) of 24 mm (1839 mm range; standard error = 0.15 mm) and 1 g wet weight (WW) (0.5-4.5 g range; mean = 1.24 g) were held in individual cages in six of seven 297-1 (60 X 90 X 55 cm) tanks with air diffusers. The remaining tank was used for mechanical and biological filtration using denitrifying bacteria-coated plastic media and a flow rate to the six tanks of 1.3 1/min. Temperature was maintained at 21[degrees]C [+ or -] 1[degrees]C for 15 wk and raised to 26[degrees]C [+ or -] 1[degrees]C for the remainder of the 21-wk study to simulate seasonal change and facilitate molting. Salinity was maintained at 28 [+ or -] 2 ppt using artificial seawater (Table 1) and light was 300 lux at the tank surface with a 16 light cycle/8 dark cycle. Ammonia, nitrite, and pH were measured daily and nitrate was measured monthly to ensure adequate water quality.

Cages were maintained at a 5-cm depth and covered in a 1-mm nylon mesh topped with a weighted PVC grid to prevent escape. Cages were either 10 X 10 X 10 cm (small) or 15 x 15 X 10 cm (large) with 24 small and 12 large cages per tank. Feed consisted of a 1/8" diameter shrimp diet (Ziegler Bros, Inc., Gardners, PA) with a protein:fat:fiber ratio of 30:7:4. Rations were calculated as percent dry food per gram of biomass per day with the high, medium, and low rations based on the maximum, median, and minimum values from Guillaume (2001). Ration and cage combinations were randomly assigned to each half tank such that each combination occurred in two separate tanks. Crabs were acclimated to the diet via ad libitum feeding for 1 wk and then fed once a day, 6 days a week with the removal of excess food.

Wet weights and CW were measured biweekly and the dates of molting were recorded. Wet weight, CW, and dry weight (DW) following freeze drying were recorded for 13 unused individuals prior to the experiment start and all remaining individuals at the experiment end after 21 wk. Data were transformed as appropriate following analyses of normality and homogeneity. Analysis of variance (ANOVA) for initial WW and CW among treatments showed no significant difference (P = 0.17 and 0.21, respectively), but variance was surprisingly high within the brood and was found to covary with measured values during the study. Thus, analysis of covariance (ANCOVA) was performed for cage size and for each ration within each cage size with the initial values of either CW or WW as the covariate. This also alleviates the need to use repeated measures because all measurements go back to a known individual with known initial values. Pairwise comparisons using Tukey's HSD were performed to confirm significant ANCOVA results. Also, ANCOVA of the time to molt and the molt increment with the initial weight as the covariate was performed for each treatment following a significant ANCOVA result for WW or CW. A regression analysis was performed comparing WW, DW, and CW.

Dry weight gained was calculated from the regression relationship found between WW and DW:

DW = 0.3757[(WW).sup.0.9661]

This regression had an [r.sup.2] of 0.95. Final calculated DW was subtracted from initial calculated DW to estimate the DW gained. Feed conversion efficiency was calculated as grams of DW gained per gram dry feed fed using the water content by proximate analysis of the feed of 11.71%. Feed consumption was also performed for each ration by delivering mock feedings to three empty cages and then comparing the DW of the remaining food in the cage after 4 h to food remaining for ration-fed animals. Analysis of variance was used to determine significant differences in feed consumed between the rations.

After termination of the experiment, 68 of the freeze-dried crabs were used for genotyping. Deoxyribonucleic acid was extracted from claw muscle using the FastDNA kit (QBIOgene) according to the directions of the manufacturer. About 20 ng of extracted DNA was used as template to amplify microsatellites from the MIH locus according to Steven et al. (2005). Chromatograms were analyzed and binned with Genescan 3.1 and scored using Genotyper 2.1 (Applied Biosystems).

Feeding of Cellulose and Chitin Diets

One hundred and forty-four individuals with a 23 [+ or -] 0.2 mm average CW and 1.04 [+ or -] 0.02 g average WW were placed into 15 X 15 X 10 cm cages under the conditions described in the cage size and ration optimization methods with a temperature maintained at 25[degrees]C [+ or -] 4[degrees]C. Diets were manufactured by Melick Aquafeeds (Catawissa, PA) and based on their 1/8" shrimp pellets with a 1:2 lecithin:menhaden oil top coat to improve water stability. Diet formulations are presented in Table 2 and include a control diet as well as four experimental diets in which a portion of the wheat flour was substituted with 20% cellulose; 16% cellulose and 4% chitin; 4% cellulose and 16% chitin; and 20% chitin. Although blue crabs are able to digest maltose (McClintock et al. 1991) and are likely to derive energy from the wheat flour, it was decided that substitution of this ingredient was the least likely to remove essential nutrients. The cellulose used was food grade Solka-floc cellulose (International Fiber Corporation, North Tonawanda, NY), and the chitin was made from deproteinated, demineralized blue crab shells (ChitinWorks, Cambridge, MD). Fiber concentrations were confirmed by acid fiber digestion assays, and proximate analysis was used to determine moisture, protein, fat, and ash content (Table 3). Diet treatments were assigned to each cage using a random complete block design with each tank representing one block and used the middle ration size from the diet optimization portion.

Individuals were weighed and CW were measured following each molt during the trial. After 11 wk of feeding, all of the individuals fed the cellulose/chitin combination diets were weighed, CW were measured, and sacrificed. Half of these individuals were freeze-dried for DW. Five random individuals from the 20% cellulose, 20% chitin, and control diets were also sacrificed and taken for measurements and WW, and then two freeze-dried for DW. The remaining individuals from the 20% cellulose replacement and 20% chitin replacement diets were fed the opposing ration, switching from cellulose to chitin and vice versa. Feeding of the switched diets as well as the control diet continued for an additional 12 wk. At the termination of the experiment, all remaining individuals were weighed, sexed, measured for CW and freeze-dried. Linear regression was performed relating DW, WW, and CW.

Data collected were tested for normality and homogeneity and transformed as appropriate. Data were split into two separate datasets: the initial feeding period and the period following the diet switch. A significant difference was not found between individuals among treatments prior to the experiment start (WW f = 0.91, CW P = 0.93), but was found prior to the diet switch (WW P < 0.0001, CW P < 0.0001) using ANOVA. In both cases ANCOVA using the initial values of WW or CW as covariates was used to compare treatments, and results showing significant differences were confirmed using Tukey's HSD. Also, for diets with significant differences, ANCOVA for time to molt and molt increment using initial weight as a covariate was performed and ANOVA for the total number of molts per crab was performed. Specific growth rate for the initial grow-out as well as after the switch was calculated as follows, where the initial weight corresponds to the weight at either the start of the experiment or following the diet switch, respectively:

(Log(final weight) - Log(initial weight))/days

Quantification of Cellulose and C hit in-Degrading Enzymes

Ninety 167-day-old juvenile blue crabs from a single brood were placed into 15 X 15 X 10 cm cages under the conditions described in the cage size and ration optimization methods with a temperature maintained at 25[degrees]C [+ or -] 4[degrees]C. Individuals were split into three treatment groups using a random block method and fed 20% chitin replacement, 20% cellulose replacement, or the control diet as described in the methods for the chitin- and cellulose-diet feeding trials. Initial tissue samples from the hepatopancreas, foregut, midgut, hindgut, anterior gastric ceca, and posterior gastric cecum were removed from 21 individuals, 7 from each treatment group, and placed at -80[degrees]C following removal of contents. Remaining individuals were fed their respective diet for 12 wk, whereupon they were sacrificed and dissected. Also, five live crabs were purchased from a local market and were used to model the activity in individuals fed a "wild" diet.

Frozen tissues were suspended in 80 mM sodium acetate, pH 4.5 and homogenized with a micropestle for 5 min. Insoluble material was pelleted by centrifugation at 16,000 X g for 10 min. The supernatant was removed to a clean tube and used as substrate for following assays. Extracts from the midgut, hindgut, anterior gastric ceca, and posterior gastric cecum from all extracts for each treatment were combined to provide enough material for protein assays. Protein content was determined using the Micro BCA Protein Assay Kit (Pierce) according to the directions of the manufacturer with a blank excluding extract and a BSA standard curve. These values were used to express specific activity of cellulases and chitinases as rail per milligram of protein per minute.

Cellulase activity was quantified using the dye-linked substrate Azo-M-Cellulose (Megazyme. Ireland) according to the directions of the manufacturer. Briefly, extracts were combined with substrate and mixed at 25[degrees]C for 2 h, the reaction was terminated by addition of precipitant solution, precipitate was removed by centrifugation, and the activity indicator was assayed by optical density (OD) at 595 nm against a standard curve of Trichoderma reesei cellulase. The blank samples were prepared in the same manner except that the precipitant was added immediately following addition of extract. Resultant OD were averaged from triplicate reactions and converted to activity units (AU) where one unit will liberate 1 [micro]mol of glucose under the conditions of the assay.

Chitinase activity was determined using tritium-labeled chitin using methods described in the study by Molano et al. (1977). Briefly, 15 [micro]l of chitin was suspended in 80 [micro]l of extract and 5 [micro]l of 1 M ammonium acetate, pH 4.5, and mixed at 25[degrees]C for 2 h. Chitin was precipitated by the addition of 300 [micro]l trichloroacetate and pelleted by centrifugation at 20,800 X g for 5 min. The supernatant and the chitin pellet were each added to scintillation cocktail and disintegrations per minute were counted using a Beckman Coulter 1801 liquid scintillation counter. Disintegrations per minute were converted to fig of N-acetyl-D-glucosamine using a fully digested sample of a known amount of tritiated chitin. This was converted to activity where 1 unit will liberate 1 [micro]mol N-acetyl-D-glucosamine from chitin under the assay conditions.

Factorial ANOVA was used to determine significant differences using diet (20% cellulose replacement and 20% chitin replacement), enzymatic activity (cellulase and chitinase), and tissue type (foregut, midgut, hindgut. anterior gastric ceca, posterior gastric cecum, and hepatopancreas) as factors. Specific activity was log transformed to meet requirements for homogeneity and normality. Because of this transformation, one foregut sample, one hepatopancreas sample, and two samples each from the midgut, hindgut, and gastric ceca produced negative values and were omitted from analysis.

RESULTS

Cage Size and Ration Optimization

The mean water chemistry values for the 21-wk trial were N[H.sub.3], 0.285 [+ or -] 0.023 mg/1; N[O.sub.2], 0.782 [+ or -] 0.107 mg/1; N[O.sub.3], 26.7 [+ or -] 2.6 mg/1; [O.sub.2], 6.40 [+ or -] 0.03 mg/1; and pH, 7.85 [+ or -] 0.01 mg/1. Of the 210 crabs used for this portion of the study, 16 escaped, 3 were cannibalized by escapees, 13 were used for initial DW, and 5 were outside an initial CW range of 20-30 mm and were removed from further analysis. For the remaining 173 crabs, 100% molted twice, 96.5% molted three times, 63.0% molted four times, and 7.5% reached the fifth molt. For each successive molt, the intermolt period lengthened but the molt increment remained relatively constant (Table 4). Regression analysis comparing WW and CW produced the following formula with an [R.sup.2] of 0.99:

WW = 7.7E'5([+ or -] 6.7E-6)([CW.sup.3.04([+]or -]0-2))

The comparison of DW and CW produced the following equation with an [R.sup.2] of 0.95:

DW = 9[E.sup.-5]([+ or -] 2[E.sup.-5])([CW.sup.2.74([+ or -] 0-074)])

There was an overall difference between cage sizes by nested ANCOVA [[F.sub.(1167)] = 7.59, P = 0.01] showing a negative effect on growth in the small cages. There were also significant differences [[F.sub.(4167)] = 3.44, P = 0.01] between the rations within each cage size (Fig. 1) where the low ration produced a lower final WW than the median ration (10.38 [+ or -] 0.07 g and 13.12 [+ or -] 1.01 g, respectively) in the small cages and the low ration produced a lower final WW than the high ration in the large cages (12.83 [+ or -] 1.22 g, 15.01 [+ or -] 1.54 g, respectively). However, molt increment was not significantly different in any treatment and the time to molt was only significantly different in molt 3 for the small cage size between the intermediate and low ration (Tukey's HSD P < 0.05). Thus, the deciding factor for differences in the final weight was the percentage of starting crabs that molted for a fourth and fifth time. There were also significant differences [[F.sub.(5168)] = 7.79, P < 0.0001] in feed conversion (Fig. 2), where the low and median rations outperformed the high ration in the small cages and the low ration outperformed the median and high rations in the large cages (Tukey's HSD P < 0.05). Also, the feed conversion ratio was lower in the large cages versus the small cages in general ([F.sub.(1168)] = 5.01, P = 0.03). Feed consumption was similar in all treatments with only the median ration in the small cages showing a significant difference [[F.sub.(4168)] = 3.36. P = 0.01], Variance was high in all cases and consumption ranged between 75% and 30%. There was no significant difference between growth of male and female crabs in any of the statistical tests performed. Of the 68 crabs genotyped, 53 were successfully discriminated using microsatellites at the MIH locus. Rather than finding four alleles in four combinations, five alleles in eight combinations resulted indicating multiple paternity from multiple inseminations of the same female (Jivoff 1997). Maternal ancestry was confirmed using nad2 sequences from the mitochondrial genome. A significant difference was found in feed conversion ratio (Table 5) using ANOVA comparing individuals from the two fathers [[F.sub.(1,51)] = 4.59, P = 0.04],

Growth with Cellulose and Chitin Replacement

Of the 144 crabs present at the start of the experiment, 25 died during the course of the experiment and 6 escaped. Of the 25 mortalities, 14 died during a molt and 6 were taken for initial measurements. Power regression analysis comparing DW to WW, DW to CW, and WW to CW had [R.sup.2] values of 0.82, 0.75, and 0.94, respectively, according to the following formulas:

DW = 0.402([+ or -] 0.044) [WW.sup.0.863([+ or -] 0.051)]

DW = 0.00055([+ or -]0.00035)[CW.sup.12([+ or -]0.17)]

WW = 0.00038 ([+ or -] 9 [E.sup.-5])[CW.sup.26([+ or -] 0.1)]

Significant differences were found between diets comparing the final WW by ANCOVA [F(4I09) = 18.05, P < 0.0001], Crabs fed the 20% chitin replacement diet (4.14 [+ or -] 0.25) had a significantly lower final WW than crabs fed the 20% cellulose (9.34 [+ or -] 0.73), 14% cellulose 6% chitin (9.00 [+ or -] 0.72), control (8.78 [+ or -] 0.87), or 14% chitin 6% cellulose (7.47 [+ or -] 0.55) replacement diets. There was no significant difference in the consumption of the experimental diets [[F.sub.(4125)] = 1.71, P = 0.15] with an average consumption of 73% ([+ or -] 0.03), but the consumption was higher than in the cage size trials (0.44 [+ or -] 0.03) and was probably due to increased palatability from the oil top coat. There were significant differences in both the time to molt [[F.sub.(4116)] = 4.64, P = 0.002] and the molt increment [[F.sub.(4116)] = 5.15, P = 0.0007] during molt 2, where the 20% chitin diet had a longer time to molt and a smaller molt increment (Table 6). There were no significant differences during the other molts or for total molts [[F.sub.(4117)] = 2.25, P = 0.07] but the 20% chitin diet failed to produce four molts and only the control diet reached five molts for two individuals. There were also specific growth rate differences [[F.sub.(4,64)] = 4.22, P = 0.004] with the 20% chitin diet resulting in a significantly lower specific growth rate than the 20% cellulose diet (Fig. 3)

Following the diet switch, there was a significant difference in specific growth rate [[F.sub.(2,39)] = 15.04, P < 0.0001] where the crabs fed the 20% cellulose replacement diet had a higher growth rate than either the 20% chitin replacement diet or the control (Fig. 4). The initial weights following the diet switch accounted for the majority of the variance as expected. There were no significant differences found by ANCOVA for the time to molt [[F.sub.(2,26)] = 2.56, P = 0.10] or molt increment [[F.sub.(2,26)] = 0.05, P = 0.95] for molt 1. but only crabs fed the 20% cellulose replacement diet molted a second time. Thus, the total number of molts between diets was significant [[F.sub.(2.37)] = 1 1.52, P = 0.0001] favoring the 20% cellulose replacement diet.

Measurement of Chi tinase and Cellulose Activity

Specific activities of cellulase and chitinase from the individuals fed the 20% replacement diets are shown in Table 7. Factorial ANOVA for the foregut and hepatopancreas values showed an overall significance in the model [[F.sub.(7,31)] = 100, P < 0.0005]. Within the model, a significant difference was shown for both tissue type [[F.sub.(1,31)] = 6.94, P = 0.013] and enzyme [[F.sub.(1,31)] = 664, P < 0.0005], but also for the interaction of enzyme and tissue type [F[(1,31) = 43.4, P < 0.0005] due to the higher relative cellulase levels in the foregut versus higher relative chitinase levels in the hepatopancreas. The factorial ANOVA for the gastric ceca, midgut, and hindgut was also significant with respect to the model [[F.sub.(3,10)] = 8.15, P = 0.005], but this was limited to the enzyme type alone [[F.sub.(1,10)] = 21.0, P = 0.001], Diet was not a significant part of the model for either the foregut, hepatopancreas comparison or the midgut, hindgut, gastric ceca comparison. The "wild" control crabs also showed differing enzyme activities with an average of 1.92 mU/mg/min cellulase versus 0.104 mU/mg/min chitinase activity in the gastric juices and 1.47 mU/mg/min cellulase and 0.0638 mil/ mg/min chitinase activity in the hepatopancreas.

DISCUSSION

The cage and ration optimization portion of the study demonstrated that cage size can limit growth, making initial and estimated final size an important consideration when designing the experiment as well as for aquaculture of the blue crab. In this study, the initial k ([cm.sup.2] compartment space/[cm.sup.2] CW) was 17 and 38 for the small and large cages, respectively, and the final k was 4 and 8. These values are much smaller than the k threshold for inhibiting growth shown in Homarus americanus ([less than or equal to] 33) and Clierax quadricarinatus ([less than or equal to] 50) (Van Olst & Carlberg 1978, Manor et al. 2002), which makes it surprising that these individuals molted at all given the wide migratory range and aggressive nature of the blue crab. Generally, individuals in the large cages showed similar molt frequency to previous laboratory-rearing experiments (Millikin et al. 1980, Cadman & Weinstein 1988, Guerin & Stickle 1997), as well as similar molt increments (Leffler 1972, Winget 1977, Guerin & Stickle 1997). Higher molt frequencies have been observed in the wild (Brylawski & Miller 2003), but because individuals were able to be kept for many months without disease and with a constant appetite, the conditions used in this study were determined to be adequate for feeding trials. Although the median ration had a higher feed conversion ratio than the small ration when using the large cage size (Fig. 2), the median ration produced a similar final WW to the large ration (Fig. 1) and was chosen for its overall performance for subsequent trials. Also, because the blue crab consumes food slowly and incompletely, the large ration may contribute to poor water quality when using more or larger individuals. The different feed conversion efficiencies shown by the offspring of different fathers in the cage and ration optimization portion present an interesting consideration for aquaculture and show the importance of breeding for efficient production. The estimated cost for this feeding regimen was $0.04 per crab starting at 25 mm CW over 5 mo.

One of the rationales of this study was to use the diet of wild blue crabs to create a more efficient manufactured diet for aquaculture purposes. Along with estimated proportions of protein, fatty acids, vitamins, and minerals, cellulose and chitin were included due to the high amounts of segmented invertebrates and detritus in the natural blue crab diet. Although individuals did quite well with large amounts of cellulose in the diet, increasing amounts of chitin produced a decrease in overall growth and the number of molts (Fig. 3). This is quite surprising given the cannibalistic nature of this species. Crabs grown in an aquaculture setting readily consume each other, especially during molting, with excellent growth performance (Zmora et al. 2005). Cannibalism is indeed one of the great hurdles in the advancement of blue crab aquaculture. Also, blue crabs have been shown to eat their exoskeleton after final larval stage molts (Sandoz & Rogers 1944) as well as during juvenile molts in this study. Calcium carbonate is absorbed directly from the surrounding water through the gills (Cameron 1985) and the exoskeleton is otherwise devoid of essential nutrients except for a small amount of protein (Vigh & Dendinger 1982). Thus, chitin resorption is the most likely reason behind consumption of the shed exuviae. Switching from the chitin replacement to the cellulose replacement diet was able to recover normal growth, indicating that the cellulose replacement diet was able to compensate for an inhibitory characteristic of the chitin diet. It is likely that the processed form of chitin used in this study is inhibiting the resorption of chitin or otherwise preventing nutrient uptake. Cellulose on the other hand did not appear to have deleterious effects and gave similar growth results to the wheat flour filler in the control diet. This leads to the conclusion that blue crabs can digest cellulose, which has been shown many times when investigating omnivorous benthic invertebrates. From an aquaculture perspective, however, these results indicate that the use of grains as filler in blue crab diets can be replaced with the much more abundant ingredient of cellulose.

The identification of cellulase activity in the foregut and hepatopancreas of the blue crab corroborates the growth data for the cellulose replacement diet. This was also found in the gastric juices of wild-caught crabs at similar levels. McClintock et al. (1991) previously showed low levels (0.0007 mU/mg/ml) of B-glucosidase using cellobiose as a substrate, indicating that much of the activity demonstrated in this study may be against larger or more complex oligomers. Cellulase activity in the blue crab was similar to what has been shown in the Tasmanian crab (1.4-19 mU/mg/ml, Johnston & Freeman 2005) and the spiny lobster (2.1 [+ or -] 0.6 mU/mg/ml, Johnston 2003), but these are mainly carnivorous species. The foraging crayfish Cherax quadricarinatus is known to have an endogenous cellulase and has a specific activity of 70 mU/mg/ml in its digestive gland (Xue et al. 1999). Although it is unclear if the blue crab has an endogenous cellulase or if its gut microbiome is responsible for cellulase activity, the lack of differentiation of activity depending on the presence of cellulose is an indicator of a constitutive endogenous cellulase with low expression. Chitinase activity also did not change with respect to diet and was low in all cases. In this case, the blue crab is known to have an endogenous chitinase, but it is not clear whether it is expressed in the gut, leaving open the possibility for chitin-degrading bacteria in the gut.

The highest levels of cellulase activity were found in the blue crab foregut, implicating this region as the primary site of secretion. Carbohydrases have been shown to originate in the hepatopancreas of Penaeus monodon (Fabricius, 1798) by in situ hybridization (Lehnert & Johnson 2002). Cellulase is then secreted into the foregut, which has been shown in Cherax quadricarinatus by a higher relative activity than the hepatopancreas (Xue et al. 1999). Given the similarities in results between the blue crab and these shrimp and crayfish species, it is likely that cellulases originate in the hepatopancreas and are excreted from the foregut. Chitinase, however, had its highest activity in the hepatopancreas. This may be due to a minor role in digestion resulting in storage in the hepatopancreas. The amount of chitinase may then increase during molting, as has been shown for Palaemon serratus (Pennant, 1777), which otherwise has a constant low activity during intermolt (Spindler-Barth et al. 1990). This may also explain a benefit to the consumption of the shed exoskeleton after molting, if chitinase activity and secretion increase during this period in the blue crab. It may also be possible that the foregut becomes the primary site for chitinase activity during molting, allowing for more efficient digestion of the polymer. The fact that both levels were lower in the wild-caught crabs may be linked to their age. because they are adults versus the juveniles used in the feeding trials. It may be that the amount measured is lower in the adults because the relative volume of the digestive organs is so much larger than the surface area available for secretion of enzymes. It could also be due to a transition to a more carnivorous diet resulting in lower enzyme production. However, the lack of change in cellulase activity from the presence or absence of cellulose makes this unlikely.

ACKNOWLEDGMENTS

Thanks to the knowledgeable staff of the Aquaculture Research Facility at I MET for their help in system design and animal rearing. This is contribution #16-188 from the Institute of Marine and Environmental Technology and #5282 from the University of Maryland Center for Environmental Science. This work was funded by a grant from NOAA Award #NA 17FU2841 Blue Crab Advanced Research Consortium Project to A.R.P.

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ANDREA L. ALLMAN, ERNEST P. WILLIAMS AND ALLEN R. PLACE *

Institute of Marine and Environmental Technologies, University of Maryland Center for Environmental Science, 701 E. Pratt Street, Baltimore, MD 21202

* Corresponding author. E-mail: cushmane@dnr.sc.gov

DOI: 10.2983/035.036.0132

Caption: Figure 1. Plot of final WW for individuals fed either the small, median, or large ration size housed in the small (10 cm wide) or large (15 cm wide) cages. Values presented are least square means and error bars are least significant differences. Bars with different letters are significantly different from each other by Tukey's HSD (P < 0.05).

Caption: Figure 2. Feed conversion ratios for the rations within each cage are presented as DW of feed fed versus DW gained. Values shown are least square means with error bars showing least significant differences. Different letters above each bar denote significantly different treatments by Tukey's HSD with a P value < 0.05 performed on WW values for individuals and feed.

Caption: Figure 3. Specific growth rate [LOG(Final wet weight) LOG(initial wet weight)] [days.sup.-1] for individuals fed each diet prior to the diet switch. Values used are least square means and error bars are least significant differences. Bars with different letters on top are significantly different from each other by Tukey's HSD at P < 0.05.

Caption: Figure 4. Specific growth rate [LOG(Final wet weight) LOG(initial wet weight)] days 1 of juvenile blue crabs following diet switch from 20% cellulose to 20% chitin and vice versa. Values used are least square means and error bars are least significant differences. Different letters over the bars indicate significant differences between diets based on Tukey's HSD with a P < 0.05.

TABLE 1.
Artificial seawater major ion constituents.

Element   Species                                 Concentration
                                                   (mg/l) (1)

CI        [Cl.sup.-]                              19574
Na        [N.sup.a+]                               9560
Mg        [Mg.sup.2+]                              2135
S         S[O.sub.4.sup.2-], NaS[O.sub.4.sup.-]    1066
Ca        [Ca.sup.2+]                               462
k         [k.sup.+]                                 343
C         HC[O.sub.3.sup.-],                         39.7
            C[O.sub.3.sup.2-], C[O.sub.2]
Sr        [Sr.sup.2+]                                 8.3
b         b[(OH).sub.3], B[(OH).sub.-]                8.5
Li        [Li.sup.+]                                  0.018
Mo        Mo[O.sub.4.sup.-2-]                         0.006

TABLE 2.
Composition of control and replacement diets with ingredients given in
percent.

                                                Diet 3

                   Diet 1       Diet 2       14% cellulose

Ingredient         Control   20% cellulose     6% chitin

Menhaden oil        2.425     2.425           2.425
Corn grain         25        25              25
Menhaden meal      10        10              10
Poultry feathers    5         5               5
Soybean meal       30        30              30
Pegabind            1.25      1.25            1.25
Wheat flour        20         2.5             2.5
Mineral mix         0.1       0.1             0.1
Dical               1.25      1.25            1.25
DL methionine       0.75      0.75            0.75
Vitamin mix         0.3       0.3             0.3
Choline CL 70%      0.15      0.15            0.15
Ascorbic acid       0.025     0.025           0.025
Limestone           1.25      1.25            1.25
Red Dog (fiber)     2.5       0               0
Crab chitin         0         0               6
Solka-Hoc           0        20              14

                      Diet 4

                   6% cellulose     Diet 5

Ingredient          14% chitin     20% chitin

Menhaden oil        2.425           2.425
Corn grain         25              25
Menhaden meal      10              10
Poultry feathers    5               5
Soybean meal       30              30
Pegabind            1.25            1.25
Wheat flour         2.5             2.5
Mineral mix         0.1             0.1
Dical               1.25            1.25
DL methionine       0.75            0.75
Vitamin mix         0.3             0.3
Choline CL 70%      0.15            0.15
Ascorbic acid       0.025           0.025
Limestone           1.25            1.25
Red Dog (fiber)     0               0
Crab chitin        14              20
Solka-Hoc           6               0

TABLE 3.

Proximate analysis of the control and experimental diets as
well as results from acid fiber digestion.

Diet             Moisture        Protein         Fat

20% cellulose    3.27            32.3            6.59
                 [+ or -] 0.21   [+ or -] 0.12   [+ or -] 0.05
14% cellulose    3.22            37.4            8.22
6% chitin        [+ or -] 0.16   [+ or -] 0.15   [+ or -] 0.06
6% cellulose     2.81            42.3            8.88
14% chitin       [+ or -] 0.04   [+ or -] 0.10   [+ or -] 0.03
20% chitin       3.15            36.7            7.18
                 [+ or -] 0.35   [+ or -] 0.06   [+ or -] 0.07
Control          3.6             34.43           7.99
                 [+ or -] 0.15   [+ or -] 0.09   [+ or -] 0.05

Diet             Ash             Fiber

20% cellulose    6.49            18.21
                 [+ or -] 0.01   [+ or -] 0.23
14% cellulose    6.72            14.18
6% chitin        [+ or -] 0.05   [+ or -] 0.07
6% cellulose     6.81            13.39
14% chitin       [+ or -] 0.03   [+ or -] 0.65
20% chitin       6.55            19.9
                 [+ or -] 0.03   [+ or -] 0.33
Control          6.77            7.72
                 [+ or -] 0.05   [+ or -] 0.42

TABLE 4.
Molt characteristics for each niolt for all cage and ration sizes.

Molt     Cage                          Time to
no.      size     Ration size            molt

Molt 1   Small   High             11 [+ or -] 1.2
                 Intermediate     10 [+ or -] 1.2
                 Low               9 [+ or -] 0.8
         Large   High              9 [+ or -] 1.3
                 Intermediate      9 [+ or -] 1.6
                 Low              11 [+ or -] 1.8
Molt 2   Small   High             25 [+ or -] 2
                 Intermediate     22 [+ or -] 2
                 Low              22 [+ or -] 1
         Large   High             20 [+ or -] 1
                 Intermediate     28 [+ or -] 4
                 Low              24 [+ or -] 2
Molt 3   Small   High             49 [+ or -] 3
                 Intermediate     42 [+ or -] 4
                 Low              57 [+ or -] 4
         Large   High             40 [+ or -] 5
                 Intermediate     44 [+ or -] 5
                 Low              50 [+ or -] 4
Molt 4   Small   High             56 [+ or -] 2
                 Intermediate     59 [+ or -] 3
                 Low              60 [+ or -] 3
         Large   High             51 [+ or -] 4
                 Intermediate     58 [+ or -] 4
                 Low              54 [+ or -] 2
Molt 5   Small   High                     42
                 Intermediate     54 [+ or -] 7
                 Low                      --
         Large   High             40 [+ or -] 2
                 Intermediate     47 [+ or -] 7
                 Low                      --

Molt     Cage                            Molt
no.      size     Ration size         increment        N

Molt 1   Small   High             0.18 [+ or -] 0.01   38
                 Intermediate     0.19 [+ or -] 0.01   34
                 Low              0.20 [+ or -] 0.02   36
         Large   High             0.18 [+ or -] 0.01   20
                 Intermediate      0.2 [+ or -] 0.01   23
                 Low               0.2 [+ or -] 0.02   22
Molt 2   Small   High             0.26 [+ or -] 0.01   38
                 Intermediate     0.26 [+ or -] 0.01   34
                 Low              0.25 [+ or -] 0.01   36
         Large   High             0.27 [+ or -] 0.02   20
                 Intermediate     0.25 [+ or -] 0.01   23
                 Low              0.27 [+ or -] 0.02   22
Molt 3   Small   High             0.21 [+ or -] 0.01   36
                 Intermediate     0.25 [+ or -] 0.01   33
                 Low              0.21 [+ or -] 0.01   36
         Large   High             0.21 [+ or -] 0.01   19
                 Intermediate     0.20 [+ or -] 0.01   21
                 Low              0.20 [+ or -] 0.01   22
Molt 4   Small   High             0.16 [+ or -] 0.01   23
                 Intermediate     0.19 [+ or -] 0.01   24
                 Low              0.19 [+ or -] 0.02   18
         Large   High             0.17 [+ or -] 0.02   16
                 Intermediate     0.19 [+ or -] 0.01   15
                 Low              0.20 [+ or -] 0.01   13
Molt 5   Small   High                    0.21           1
                 Intermediate     0.21 [+ or -] 0.01    4
                 Low                      --            0
         Large   High             0.20 0.02             6
                 Intermediate     0.15                  2
                 Low                      --            0

Time to molt is in days and molt increment is in millimeters. Values
shown are means [+ or -] standard error.

TABLE 5.
Mean feed conversion ratio for juvenile Callinectes sapidus
individuals assigned parentage using the MIH locus
micro-satellite marker.

Parent     Allele   Feed conversion ratio   N

Mother      146                             53
            171
Father-1    144              7.14           32
            146     [+ or -] 0.01
Father-2    160              5.88           21
            200     [+ or -] 0.01

TABLE 6.
Molt increment and time to molt for the five diets as well as the
number of individuals that reached that molt.

                                                       14% cellulose

               Molt          Control   20% cellulose     6% chitin

Molt 1    Time to molt      12.86         15.59           13.26
          [+ or -] LSD       4.05          3.61            3.83
          Molt increment     0.28          0.31            0.30
          [+ or -] LSD       0.07          0.07            0.07
          N                 26            28              28
Molt 2    Time to molt      19.11         21.15           23.30
          [+ or -] LSD       3.48          2.82            3.57
          Molt increment     0.26          0.30            0.27
          [+ or -] LSD       0.06          0.06            0.05
          N                 23            25              26
Molt 3    Time to molt      24.63         25.99           25.68
          [+ or -] LSD       3.74          4.36            1.85
          Molt increment     0.26          0.28            0.23
          [+ or -] LSD       0.02          0.06            0.05
          N                 21            22              22
Molt 4    Time to molt      25.77         27.40           26.34
          [+ or -] LSD       1.54          3.35            7.53
          Molt increment     0.28          0.30            0.22
          [+ or -] LSD       0.05          0.02            0.10
          N                  7             9              10
Molt 5    Time to molt      19.56         --              --
          [+ or -] LSD      13.00         --              --
          Molt increment     0.10         --              --
          [+ or -] LSD       0.12         --              --
          N                  2            --              --

                           6% cellulose

               Molt         14% chitin    20% chitin

Molt 1    Time to molt     16.70           19.60
          [+ or -] LSD      4.71            4.36
          Molt increment    0.26            0.23
          [+ or -] LSD      0.05            0.04
          N                28              28
Molt 2    Time to molt     26.48           30.57
          [+ or -] LSD      4.32            4.36
          Molt increment    0.30            0.19
          [+ or -] LSD      0.06            0.04
          N                24              23
Molt 3    Time to molt     26.12           30.02
          [+ or -] LSD      4.45            2.91
          Molt increment    0.31            0.20
          [+ or -] LSD      0.09            0.04
          N                21              18
Molt 4    Time to molt     28.68           --
          [+ or -] LSD      6.56           --
          Molt increment    0.24           --
          [+ or -] LSD      0.04           --
          N                 6              --
Molt 5    Time to molt     --              --
          [+ or -] LSD     --              --
          Molt increment   --              --
          [+ or -] LSD     --              --
          N                --              --

Values presented are least square means with errors shown as least
significant difference.

TABLE 7.
Cellulase and chitinase activity from each tissue group from the five
individuals fed either the 20% cellulose or 20% chitin replacement
diet.

Assay          Region            Tissue          20% cellulose

Cellulase   Digestive gland   Hepatopancreas   2.71 [+ or -] 0.53
            Digestive tract   Foregut          4.29 [+ or -] 0.40
                              Midgut                  1.72
                              Hindgut                0.144
            Gastric ceca      Anterior               0.764
                              Posterior                0
Chitinase   Digestive gland   Hepatopancreas   0.128 [+ or -] 0.026
            Digestive tract   Foregul          0.019 [+ or -] 0.011
                              Midgut                 0.041
                              Hindgut                0.003
            Gastric ceca      Anterior               0.024
                              Posterior              0.004

Assay          Region            Tissue            20% chitin

Cellulase   Digestive gland   Hepatopancreas   2.29 [+ or -] 0.033
            Digestive tract   Foregut          4.79 [+ or -] 0.050
                              Midgut                 0.086
                              Hindgut                 0.15
            Gastric ceca      Anterior                1.86
                              Posterior              0.468
Chitinase   Digestive gland   Hepatopancreas   0.106 [+ or -] 0.027
            Digestive tract   Foregul          0.035 [+ or -] 0.010
                              Midgut                 0.014
                              Hindgut                0.082
            Gastric ceca      Anterior                 0
                              Posterior              0.051

Values shown are specific activity mU/mg/min either as a single value
or as a mean with standard error when replicates were available.
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