Changes in the body composition of adult male southern rock lobster, Jasus edwardsii, during starvation.

ABSTRACT This experiment determined the effect of starvation on the chemical composition and tissue histology of the abdominal muscle and digestive gland in adult male southern rock lobsters Jasus edwardsii during 14 and 28 days. Individual lobsters (n = 6) were stocked into 34-L tanks (17.5 [+ or -] 1.5[degrees]C) and either led or starved over 14 and 28 days and also compared with lobsters (n = 6) killed for initial samples at the start of the experiment. Starved lobsters showed a significant reduction in crude lipid from the digestive gland at day 14 and from the abdominal muscle at day 28 when compared with the initial population. Histologic investigation of the digestive gland showed a lower density of lipid droplets in both day 14 and day 28 starved lobsters, with this depletion apparently causing structural damage to the digestive tubule in lobsters starved for 28 days. Histologic investigation of glycogen levels in digestive tubules showed a decline with starvation. Available energy was significantly affected by the crude lipid content of both organs although the digestive gland contained significantly higher absolute amounts. Starvation forces the use of body reserves to maintain metabolic functions; the order and quantity of depletion indicated the minimum requirements for survival and highlighted differences in the strategies used by crustaceans.

KEY WORDS: glycogen, histology, Jasus edwardsii, lipid, southern rock lobster, starvation

INTRODUCTION

The southern rock lobster Jasus edwardsii (Hutton, 1875) (Palinuridae) is found along the coastlines of New Zealand and southern Australia and forms an important fishery in these regions (Thomas et al. 1998). Aquaculture is also under development in New Zealand and Australia: current strategies on grow wild caught puerulus, postpuerulus, and juveniles to market size (Jeffs & Hooker 2000, Thomas et al. 1998). Research into broodstock management and larval propagation is underway, and whereas there is a major focus on female broodstock and larval quality (Smith et al. 2003a, Smith et al. 2003b), little information is available on male rock lobsters.

Starved crustaceans, like all animals, must use body reserves to supply energy to maintain metabolism (Hervant et al. 1999, Lemmens 1994, Virtue et al. 1993), and the change in body composition indicates both the sequence and level of depletion of different body reserves. For decapod crustaceans there is conflict in the literature about the order and amount of reserve utilization and hence the role of protein, lipid, and carbohydrate during starvation. Many marine crustaceans metabolize lipid initially, using protein only after lipid reserves are depleted (Dawirs 1987, Regnault 1981). For example, rapid depletion of digestive gland lipid was documented in adult tiger prawns Penaeus esculentus (Barclay et al. 1983, Chandumpai et al. 1991), adult Crangon crangon (Regnault 1981), and in larval and adult shore crabs Carcinus maenas (Dawirs 1987, Heath & Barnes 1970). In contrast, larvae of the spider crab Hyas araneus (Anger & Dawirs 1982) and adult Antarctic krill Euphausia superba (Virtue et al. 1993) use body protein initially and lipids last. Therefore, assumptions about the use of body reserves as metabolic substrates by crustaceans during periods of nonfeeding or starvation should be avoided.

Lipid is used as the primary energy source to sustain onshore swimming activity of nonfeeding (starved) southern rock lobster puerulus prior to settlement (Jeffs et al. 1999, Jeffs et al. 2001, Lemmens 1994, Wells et al. 2001). Enzyme profiles also suggest that lipid as well as carbohydrate are used by puerulus and that protein may be used for energy following depletion of these reserves (Johnston, 2003). Lipid is also a major source of energy in phyllosoma larvae with polar lipids and polyunsaturated fatty acids particularly important (Nelson et al. 2003). Energy utilization in broodstock is less understood, and only recently has the importance of broodstock diet been linked with phyllosoma quality and survival in hatcheries (Smith et al. 2003a). This study will be the first to examine utilization of energy reserves in male southern rock lobster broodstock through dedicated starvation experiments and will complete our understanding of energy utilization in the life-cycle of the southern rock lobster. The aims of this study were to measure and compare the standard growth parameters and chemical composition of the abdominal muscle and digestive gland in adult male southern rock lobsters (J. edwardsii) from an initial population and those that were fed or starved for 14 (lays and 28 days. Histology of the digestive gland has proved a sensitive indicator of nutritional condition (Johnston et al. 2003) and was used in the current study.

MATERIALS AND METHODS

Experimental Procedure

The experiment was conducted at the School of Aquaculture, University of Tasmania. Captive male southern rock lobsters (J. edwardsii) were obtained from Marine Research Laboratories (MRL), Hobart, Tasmania. This experiment was run during two time blocks with a total of 30 lobsters used. Each time block had an initial group, day 14 fed, day 14 starved, day 28 fed, and day 28 starved; each treatment consisted of 3 lobsters randomly allocated to individual experimental tanks. Prior to the commencement of each time block, lobsters were acclimatized to the system for 7 days. During acclimatization, any mortalities or molts were replaced with similar-sized animals from the stock population.

The system was maintained at 17.5 [+ or -] 1.5[degrees]C and kept dark to increase feeding time. Water exited the 12 experimental tanks via a standpipe and was returned to the biofilter with 3-5% replacement every 2 days. Water quality parameters (salinity. dissolved oxygen, ammonia, nitrate, and nitrite) were monitored to ensure water quality remained within limits recommended for spiny lobsters (Jeffs and Hooker 2000). At the start of each time block, an initial sample of three lobsters were placed on ice to induce a chill-coma and used for assessment of weight, carapace length, organ size, and chemical and histologic composition (see below). At the same time, weight and carapace length were measured in 12 lobsters that were then stocked individually into experimental tanks and randomly assigned into treatments (fed or starved over 14 or 28 days). Single animals were stocked to prevent effects of agonistic behavior and cannibalism (Thomas et al. 2003). Lobsters were fed mussel (Mytilus edulis) every 3 days at 1330, and uneaten food and waste was siphoned out the next morning. At the end of the experiment, lobsters were induced into a chill-coma and the carapace removed to reveal the digestive gland. Samples of digestive gland tissue were fixed immediately for histology. Digestive gland and abdominal muscle tissue was wrapped in foil and frozen at -40[degrees]C for freeze-drying and analysis of chemical composition (discussed in following section).

Chemical Analysis

Digestive glands and abdominal muscle were analyzed for chemical composition using standard methods to determine dry matter (freeze dry to constant weight); nitrogen (Kjeldahl using a selenium catalyst) (AOAC 1995); crude protein calculated using % nitrogen x 6.25; crude lipid [Soxhlet apparatus and PET extraction (AOAC 1995)]; ash [muffle furnace at 550[degrees]C for 16 h (AOAC, 1995)] ; and carbohydrate (nitrogen-free extract, NFE), determined by the difference between dry matter and sum of crude protein, lipid, and ash. Energy was calculated from the chemical composition using values of 23.6 kJ [g.sup.-1] for protein, 38.6 kJ [g.sup.-1] for lipid, and 17.2 kJ [g.sup.-1] for carbohydrate (Jobling 1994).

Histology

Samples of digestive gland were dissected and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature. Following washes in 0.1 M phosphate buffer (pH 7.4), samples were dehydrated in a graded series of ethanol and half the tissue post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h. Tissue was washed in 0.1 M phosphate buffer, embedded in glycolmethacrylate (JB4) resin, sectioned at 2 [micro]m, and then stained with polychrome blue. The other half of each tissue sample was processed routinely for wax histology, sectioned at 5 [micro]m, then stained for glycogen using the periodic acid-Schiff (PAS) technique (Bancroft and Cook 1994). A negative diastase control was used to obtain more specificity in detecting glycogen (Humason 1972), and a positive control of trout mucus was initially stained with samples.

Statistical Analysis

Mean values are reported [+ or -] standard error (SE). Each lobster sampled was assessed as a single replicate. The chemical composition values for digestive gland and abdominal muscle were the mean of two duplicates. Data were pooled to give six replicates for each of the five treatments (day 0, day 14 fed, day 14 starved, day 28 fed, and day 28 starved). Normality and homogeneity of variance were confirmed (SPPS version 8). Comparison between means was by one-way analysis of variance (ANOVA) and post-hoc comparison using Tukey's HSD (SPSS version 8). Significance was accepted at probabilities of 0.05 or less.

RESULTS

Chemical Composition

There were no significant differences in carapace length between the initial group and the different treatments nor were there were any significant differences in whole body wet weight, abdominal muscle index (ABI), or digestive gland index (DGI) between fed and starved lobsters (Table 1). However, starved lobsters showed a general trend of lower mean whole body weight and significantly different changes in wet body weight (Table 1). Starved lobsters tended to show a lower DGI, but multiple comparison was not sensitive enough to show which treatments were significantly different (Table 1).

Only crude lipid and gross energy content of abdominal muscle tissue were significantly different between treatments (Table 2). After 28 days of starvation, the abdominal muscle of lobsters had lower mean crude lipid than the abdominal muscle of lobsters in the initial population; no other significant changes were shown statistically. The correlations between chemical composition parameters of the abdominal muscle in fed and starved adult lobsters were investigated. Dry matter was positively correlated with crude protein (r = 0.50, P [less than or equal to] 0.01), and energy content (r = 0.69, P [less than or equal to] 0.01), and energy content was positively correlated with crude protein (r = 0.55, P [less than or equal to] 0.01) and crude lipid (r = 0.37, P [less than or equal to] 0.05).

Crude lipid content of the digestive gland was significantly lower in day 14 starved lobsters than at day 0 (Table 2). There were no other significant differences between treatments although crude lipid in day 28 lobsters was suggestive of the same trend. Carbohydrate was significantly higher in day 14 starved lobsters compared with day 14 fed and the day 0 group. There were no significant differences in dry matter, crude protein, ash, or energy. Dry matter was not correlated with crude lipid (r = 0.15, P > 0.05) or carbohydrate (r = 0.16, P > 0.05), and crude lipid was not correlated with digestive gland energy content (r = 0.15, P > 0.05). There was a significant negative correlation between crude lipid and carbohydrate (r = -0.43, P < 0.05).

Histologic Examination of tide Digestive Gland

Lipid droplets in epithelial cells of the digestive gland tubules from the initial lobster population were densely arranged in the apical cytoplasm and less dense adjacent to the basal lamina (Fig. 1A). The epithelia of tubules from lobsters fed for 14 days had densely arranged lipid droplets that were spaced throughout the cytoplasm (Fig. 1B). Epithelial cells in lobsters starved for 14 days had a large number of empty vacuoles concentrated apically with densely arranged lipid droplets in the cytoplasm adjacent to the basal lamina (Fig. 1C). This agrees with the reduction in crude lipid found in the chemical composition data. Lobsters fed for 28 days had tubules with densely arranged lipid droplets, similar to the 14-day fed lobsters. Epithelial cells of lobsters starved for 28 days had large amounts of empty vacuoles apically around the tubule lumen, with both loosely arranged lipid droplets and empty vacuoles adjacent to the basal lamina and miter membranes ruptured (Fig. 1E).

The digestive gland epithelial cells in the initial lobster population had minimal glycogen granular material (Fig. 2A). The negative control, diastase treatment was used over all treatments and all had less intense staining, indicating stained material was glycogen (Figs. 2B, 3D, 4B, and 4D). Epithelial cells from lobsters fed for 14 days had intense positive staining for glycogen (Fig. 3A) whereas starved lobsters had fewer positive staining granular bodies (Fig. 3C). Cells from lobsters fed for 28 days had less glycogen (Fig. 4A) than 14-day fed lobsters, although cells from lobsters starved 28 days had a much lower quantity of glycogen than 14 day starved lobsters (Fig. 4C).

DISCUSSION

Chemical Composition

The nonsignificant trend of lower whole body weight in starved lobsters is consistent with body tissue being used during food deprivation to maintain metabolism (Hervant et al. 1999, Lemmens 1994, Virtue et al. 1993). However, unlike vertebrates, crustacean body volume is set by their rigid exoskeleton (Stuck et al. 1996), therefore to maintain this set volume any change in body mass is counteracted by a change in water content (Barclay et al. 1983, Hervant et al. 1999, Stuck et al. 1996). The mass of digestive gland and abdominal muscle as a percentage of whole body weight showed opposite changes with starvation, which support conclusions that organs respond differently to nutritional stress (Schirf et al. 1987).

The total wet weight and percent dry matter of abdominal muscle showed no obvious or significant trends when lobsters were starved. However, the reduction in percent dry matter of abdominal muscle in lobsters fed for 28 days indicates possible catabolism of muscle tissue. This, in addition to the increases in uneaten feed recovered from tanks of day 28 fed lobsters, indicated they had entered premolt shortly after day 14 and were close to ecdysis at time of sampling (day 28). Crustaceans stop feeding during early premolt and use body reserves until the shell hardens (Barclay et al. 1983, Chang & O'Connor 1983, Factor 1995, Harrison 1997, Hiller-Adams & Childress 1983, Jones et al. 1997).

Although abdominal muscle crude protein showed a small range (22.0-23.7% wet weight) between treatments, it was positively correlated with the percent dry matter (P < 0.01); a similar correlation was also found in Pacific white shrimp Penaeus vannamei (Stuck et al. 1996). Starved lobsters tended to have a lower abdominal muscle percent crude protein than lobsters fed over the same time period, a trend also found in adult tiger prawns when starved for 14 days (Barclay et al. 1983) and in small adult J. edwardsii lobsters (80-90 mm carapace length) fed once every 10 days (Musgrove 1997). Lobsters fed for 28 days showed lower percent crude protein than lobsters from day 0 and day 14 fed, and this reduction is explained by muscle protein decreasing to permit the escape of lobster from the old exoskeleton during the molt cycle (Chang & O'Connor, 1983). The significantly lower lipid level in the abdominal muscle of starved lobsters at 28 days compared with the initial population is consistent with findings in starved adult tiger prawns (Chandumpai et al. 1991). The significant correlation between percent crude lipid and abdominal muscle energy content indicates that although the mean absolute wet weight of crude lipid stored in the abdominal muscle was less than 0.4% whole body wet weight), changes still affected the energy matrix within the abdominal muscle during starvation, which somewhat conflicts with suggestions that the low levels of lipid in the lobster muscle are mostly structural and are not normally available for energy production (Dall 1981).

The trend of lower percent dry matter in the digestive gland of starved lobsters, indicating a reduction of total body stores, further supports the digestive gland being the primary organ for energy storage in decapod crustaceans (Dall 1981, Gibson & Barker 1979, Icely & Nott 1992). The percent crude protein of digestive gland increased in starved lobsters as has been found in the digestive gland of the marine carnivorous prawn Pandalus platycero (Whyte et al. 1986). However, the apparent increase in crude protein content is due to the reduction in digestive gland tissue mass rather than an increase in the amount of protein. In general, decapod crustaceans have higher digestive gland lipid than most other groups (Gibson & Barker 1979). This study found large adults (117.42 119.34 mm carapace length) had a crude lipid content of 47-71% digestive gland dry weight and within the range for juvenile southern rock lobsters in some studies (Crear et al., 2001) but not others (Johnston et al. 2003, Ward et al. 2003). Generally, the digestive gland lipid content in the current study was considerably higher than in other decapod crustaceans (Barclay et al. 1983, Chandumpai et al. 1991). Digestive gland crude lipid also appears to vary within the lobster family: 40-50% for adult J. lalandii (carapace lengths 70-89 mm) (Cockcroft 1997); 17-65% for adult Homarus americanus during 102 days of starvation (Stewart et al. 1972); and <12% for adult J. edwardsii (Musgrove 1997). The absolute amount of crude lipid was higher in the digestive gland (6.41-12.96 g) than in the abdominal muscle (1.17-2.94 g), which further emphasizes the importance of the digestive gland as an energy store in crustaceans (Cockcroft 1997, Dall 1981, Jeffs et al. 2001).

The increase in the estimated carbohydrate levels of digestive gland in lobsters fed for 28 days may be explained by the increase in total glycogen deposited in digestive gland and epidermis during the transition of spiny lobsters into premolt (Schwabe et al. 1952). Although later publications have challenged some of the conclusions front this paper (Dall 1981, Stewart et al. 1972), the increase in digestive gland glycogen was also found to be significantly higher in prawn P. kerathurus before molting (Hilmy et al. 1986). The negative correlation between carbohydrate and crude lipid in the digestive gland may be explained by the suggestion that carbohydrates act as an intermediate energy substrate following the breakdown of other storage products such as lipids (Jeffs et al. 1999) and may also explain increased content due to the breakdown of lipid. The method of carbohydrate quantification used was to calculate the difference between dry matter and the other measured tissue macro chemical components and gave estimated ranges of 1.6-42.7 and 56.4-212.0 mg/g dry tissue for the abdominal muscle and digestive gland tissues, respectively. The carbohydrate content of the digestive glands from the day 14 and day 28 starved lobsters were 212 and 140 mg/g dry tissue, respectively, and appeared very high in comparison to other samples in the current study and to some other studies on crustaceans (Hilmy et al. 1986, Whyte et al. 1986). Digestive gland carbohydrate varied between 17 and 47 mg/g dry material, depending on dietary carbohydrate, in small (<10 g) J. edwardsii (Johnston et al. 2003). However, higher values have been reported in the digestive gland of lobsters including 168 mg/g dry tissue for adult (470 g) Homarus americanus (Floreto et al. 2000). The results from the current study indicate that further investigation of carbohydrate dynamics using direct measurements would be informative and also that the histologic approach used in the current study went some way to addressing this issue. The histologic assessment of glycogen is clearly more sensitive than estimating carbohydrate by difference (see below).

Histology

The prevalence of empty lipid vacuoles in digestive gland epithelial cells of unfed lobsters (Figs. 1C and E) confirms that lipid is used by J. edwardsii broodstock during periods of starvation and supports the reduction of lipid content in the digestive gland of starved lobsters as determined by chemical analysis. Tissue histology also shows that short-term depletion of lipid reserves has no effect on structure of the digestive gland tubules, which remain unchanged in lobsters starved for 14 days. But after 28 days of starvation, when lipid reserves are near exhausted, outer cell membranes are degraded and the cytoplasm is diffuse. Such tissue damage was also observed during periods of nutritional deprivation in adult freshwater crayfish matron Cherax tenuimanus (Jussila 1997). Although change in the number and size of B-cells or R-cells is used as an indication of nutritional stress (Al-Mohanna and Nott 1987, Niles et al. 1993), no such differences were apparent between the treatments in this trial.

Glycogen was also depleted from digestive gland epithelia during starvation with greater depletion from tissues the longer lobsters were starved (Figs. 3C and 4C). Although it is widely accepted that digestive gland glycogen reserves are rapidly depleted over a period of nonfeeding in crustaceans (Dall 1986, Gibson & Barker 1979, Hilmy et al. 1986, Jeffs et al. 2001), this is one of the few studies to demonstrate this fact using tissue histology. The higher intensity staining in the day 14 fed lobsters indicates they arc in better nutritional condition than lobsters from the initial population and agrees with chemical composition data. It is unclear why lobsters fed for 28 days had lower glycogen than lobsters fed for 14 days, as 28 days fed animals had higher levels of carbohydrate, as determined by difference. It is possible this discrepancy may be due to resorption of glycogen by day 28 lobsters for chitin synthesis as they were entering premolt during the latter stages of the experiment (Aiken 1980, Chang, 1995). This physiologic mechanism may not be apparent from the chemical composition data, which only estimated total carbohydrate.

CONCLUSIONS

This study has confirmed the importance of lipid and glycogen as energy sources for southern rock lobster broodstock using the combination of chemical analyses and tissue histology. Research on starvation effects in southern rock lobster over longer lime periods may show more of the significant differences between fed and starved animals found in other crustaceans. There appears to be a relationship between crustacean body mass and the energy reserve composition, this may indicate a switch in dietary needs with carapace length. The increase in the weight of apparent carbohydrate in both the abdominal muscle and digestive gland during starvation is cause for investigation as these results conflict with the redaction of body reserves during starvation. The results from this study, together with further research into the effect of starvation on southern rock lobster biochemical composition, should aid in the development of understanding of the importance of individual energy reserves.

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L. E. MCLEOD, C. G. CARTER * AND D. J. JOHNSTON School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1370, Launceston 7250, Tasmania, Australia

* Corresponding author. E-mail: [e-mail]