Response of the American lobster to the stress of shell disease.

ABSTRACT Shell disease is a problem affecting lobsters in eastern Long Island Sound causing disfiguration of the shell, decreasing the lobsters' value, and whereas mild and medium levels of the disease are not lethal, ultimately, severe cases result in mortality. Levels of the molting hormone, ecdysone, were quantitated, using a radioimmunoassay (RIA), in hemolymph of animals exhibiting shell disease. Our results indicate that levels of ecdysone were increased in the hemolymph of shell-diseased lobsters, with a medium level of expression of the disease to 89 [+ or -] 32 ng/mL (n = 76), whereas unaffected, presumably healthy ones had 57 [+ or -] 16 ng/mL (n = 210). In 7 of 10 months of the year shell-diseased animals had higher ecdysone levels in their hemolymph than unaffected animals. In addition, ecdysone levels were abnormally high, 165 [+ or -] 53 ng/mL (n = 5), in shell-diseased ovigerous lobsters, whereas normal unaffected ovigerous ones had low levels of this hormone, 13 [+ or -] 4 ng/mL (n = 7). These results indicate that shell disease may induce lobsters to alter the systemic levels of ecdysone, possibly serving as a defensive measure, allowing the animals to ward off the effects of shell disease through induced molting.

KEY WORDS: Homarus americanus, lobster, shell disease, ecdysone, molting

INTRODUCTION

Shell disease (SD) in the American lobster (Homarus americanus H. Milne Edwards, 1837), is recognized as a serious problem resulting in threatened health, disfigurement, and ultimately, in severe cases, leads to mortality in lobsters found in the wild, reducing the catch and profitability (Smolowitz et al. 1992). The lobster industry in the northeastern United States supports one of the most valuable commercial fisheries, worth 301 million dollars in 2000 (National Marine Fisheries Service 2001). Castro and Angell (2000) reported that 20% of the lobsters captured in Rhode Island waters and offshore canyons were infected with SD, and over 50% of ovigerous animals were afflicted. Perhaps even more alarming is that the prevalence of SD in lobsters has steadily increased in recent years, with more than 67% of the lobsters collected around the Millstone Power Station in eastern Long Island Sound affected with SD in October of 2000 and 2001 (Millstone Environmental Laboratory 2001). Of these, the larger lobsters and females carrying eggs showed the highest incidence of SD, most likely because they molt less frequently. The pathogenicity of SD is beginning to be understood and is attributed to an assemblage of chitinolytic bacteria (Chistoserdov et al. 2003). Shell disease is recognized by ulcerated and necrotic pits on the shell, and five grades of severity have been described, depending on the extent and depth of cuticular erosion (Smolowitz et al. 1992).

Less well understood is the effect of SD on lobster molting and the control of the endocrine system that regulates molting, cuticle repair, shell regeneration, growth, reproduction and the involvement of the endocrine system in the repair of shell disease. Molting is essential for growth, maturation, cuticle production, regeneration, metamorphosis and replacement of damaged shell (Laufer et al. 2003). Individual lobsters molt with a frequency depending very much on size. For example, 350-450 g lobsters (shorts) molt about twice in a year. Mature ovigerous female lobsters of this size may only molt every other year (Aiken & Waddy 1980, Waddy et al. 1995). The molt cycle of the lobster occurs in 5 major stages, along with substages. In stage A, mineralization of the postecdysial cuticle (epicuticle and exocuticle) takes place; in stage B, striated endocuticle is deposited underneath the exocuticle; in stage [C.sub.3], lamellar endocuticle and in [C.sub.4], the membranous layer is deposited. [C.sub.4] is the start of the intermolt period and in D, there is a transition from intermolt to premolt or proecdysis, involving retraction of the epidermis (apolysis), dissolution of the membranous layer, absorption of calcium from the endocuticle and deposition of new epicuticle and exocuticle; and in stage E, ecdysis takes place (reviewed by Smolowitz et al. 1992, Waddy et al. 1995). Molting in the lobster, as well as other crustaceans, is regulated by a family of steroid hormones termed ecdysteroids. Ecdysone is produced mainly by the Y-organ and is converted into the active form, 20-hydroxyecdysone, by target tissues such as the epidermis. During the molt cycle of the lobster, ecdysone concentrations remain low until proecdysis, and gravid females delay ecdysis until after larval hatching and release (Chang 1984, Chang & O'Connor 1988). Ovigerous lobsters weighing 400 g and greater will incubate embryos as long as 11 too, never molting during egg bearing, and then molt at approximately 100 days posthatching/larval release (Chang 1984, Chang & O'Connor 1988). Molting during egg incubation would result in loss of the developing embryos. How this molt regulation is controlled while the animals are carrying eggs is currently not known, but Chang (1984) suggested that the presence of embryos is communicated to the mother by way of the pleopod glands, which may produce a molt-inhibiting factor.

There is evidence that ecdysteroids may also be produced in other tissues other than the Y-organ (reviewed by Chang & O'Connor 1988). Regeneration in the crab Pilumnus hirtellus is accompanied by an increase in ecdysone even in Y-organ ablated animals (Demeusy et al. 1988). Ecdysone has also been found in the testes of terminally molted Libinia emarginata, which lack Y-organs (Laufer et al. 1993). Ecdysone appears to be synthesized by testes and ovaries of L.emarginata, where ecdysone is incorporated into eggs for later embryonic development (Laufer, unpublished). Mature L.emarginata females lack Y-organs and have low ecdysone concentrations in their blood (Laufer et al. 1993). Because there are several forms and variants of the basic ecdysone molecule, we refer here to all of them as ecdysones or ecdysteroids (Kunieda et al. 1997).

Besides being involved in regulating growth and molting, it appears that ecdysones also serve a function in the regeneration of new cuticle and possibly wound healing. Studies by Hopkins (reviewed in Hopkins 2001) demonstrated an increase in circulating ecdysteroids during limb regeneration in the fiddler crab Uca pugilator after multiple limb autotomy. The transition from U. pugilator normal anecdysis to proecdysis and in proecdysis in limb bud regeneration is marked by large transient peaks in ecdysones. There is also an increase in ecdysone receptor mRNA in regenerating limb buds (Chung et al. 1998). Multiple limb autotomy in U. pugilator, like shell disease in lobsters, seems to stimulate a defense mechanism for repair and regeneration of cuticular structures. In insects, ecdysteroid synthesis is also induced in response to wound healing and cuticular regeneration (Kunieda et al. 1997).

We determined and report here systemic hemolymph concentrations of ecdysones in healthy unaffected and shell-diseased lobsters during most months of the year, as well as in several unaffected, presumably healthy, ovigerous and shell-diseased ovigerous specimens.

MATERIALS AND METHODS

Animals

Unaffected (n = 210) and shell-diseased (n = 76) lobsters both male and female, weighing 300-450 g (average 375 g) were collected in different months from Long Island Sound, Connecticut during 2002 to 2004. Seven unaffected and five shell-diseased ovigerous females were collected from offshore during July 2002 to 2003. The embryos from the ovigerous females were of similar developmental stage, close to hatching.

Measurement of Hemolymph Ecdysone

The animals were bled immediately on arrival in the laboratory of 0.5

mL hemolymph. Hemolymph was stored at -80[degrees]C until used. Ecdysone concentrations in the hemolymph were determined by radioimmunoassay (RIA), according to Chang (1984), by using a polyclonal antibody against ecdysones. The results of ecdysteroid levels throughout the year are expressed as ng equivalents of 20-hydroxyecdysone/mL of hemolymph. These results were analyzed for statistical significance by 2-way analysis of variance. The analyses of the ovigerous lobsters were analyzed statistically by the Student t-test. Grading of shell disease severity (in a total of 76 animals) included 3 levels by visual observation: mild (grades I and II) epicuticular erosion and erosion into the exocuticle, medium (grades III and IV) erosion into the striated endocuticle, the thin-lamellar endocuticle and erosion into the pseudomembrane and severe (grade V) total erosion of the cuticle and ulceration of the underlying cuticular epithelium, respectively, according to Smolowitz et al. (1992).

RESULTS

Ecdysone Concentrations in Unaffected Lobsters

Ecdysone levels in unaffected lobsters (n = 210) were lowest, (4 ng [+ or -] 2 ng/mL) in July. The standard error of the mean for ecdysone concentration for each month and category is indicated in Figure 1. The numbers along the x-axis represent the number of animals (n) assayed that month. Ecdysone levels in the unaffected animals increased in August, September and October to 52 [+ or -] 11 ng/mL (n = 31),44 [+ or -] 8 ng/mL (n = 20) and 49[+ or -] 21 ng/mL (n = 43), respectively (Fig. 1) and peaked in November at 141 [+ or -] 14 ng/mL (n = 10). In December and January ecdysone concentrations decreased to 64 [+ or -] 9 ng/mL (n = 10) and 14 [+ or -] 8 (n = 7) ng/mL, respectively. The February, March and April ecdysone concentrations then increased to 46 [+ or -] 20 ng/mL (n = 32), 75 [+ or -] 6 ng/mL (n = 10), and 56 [+ or -] 13 ng/mL (n = 10), respectively. May and June showed major increases in ecdysones to 144 [+ or -] 50 ng/mL (n = 10) and 159 [+ or -] 37 ng/mL (n = 24), respectively. Average ecdysone levels for 210 unaffected lobsters were 57 [+ or -] 16 ng/mL during 12 months of the year.

[FIGURE 1 OMITTED]

Ecdysone Concentrations in Shell-diseased Lobsters

Ecdysone levels in shell-diseased lobsters (n = 11) collected in July were 46 [+ or -] 27 ng/mL, compared with 4 ng/mL (n = 3) in unaffected animals (Fig. 1). In August, 7 shell-diseased animals had 134 [+ or -] 61 ng/mL compared with 52 ng/mL in 31 unaffected animals. In 6 shell-diseased lobsters collected in September, the ecdysone dropped to 55 [+ or -] 16 ng/mL, whereas the ecdysone concentration in 20 unaffected lobsters was 44 [+ or -] 8 ng/mL. The ecdysone concentration increased to 550 ng/mL in 1 shell-diseased lobster collected in October, whereas 43 unaffected lobsters had 49 [+ or -] 21 ng/mL. By December, January and February the ecdysone concentrations in shell-diseased lobsters were much higher than in unaffected animals. They were 151 [+ or -] 9 ng/mL (n = 4), 90 [+ or -] 90 ng/mL (n = 2) and 96 [+ or -] 25 ng/mL (n = 16), respectively, compared with 64 [+ or -] 9 ng/mL(n = 10), 14 [+ or -] 8 ng/mL(n = 7) and 46 [+ or -] 20 ng/mL (n = 32). In April shell-diseased animals had concentrations of 55 [+ or -] 9 ng/mL (n = 4) compared with 56 [+ or -] 13 ng/mL (n = 10) in unaffected animals. In May and June the ecdysone concentration in shell-diseased animals was 98 [+ or -] 60 ng/mL (n = 10) and 70 [+ or -] 25 ng/mL (n = 15) compared with unaffected lobsters with 144 [+ or -] 50 ng/mL (n = 10) and 159 [+ or -] 37 ng/mL (n = 24), respectively. No shell-diseased lobsters were collected in either November or March. The average grade of shell disease severity was medium (n = 76) according to severity levels by Smolowitz et al. (1992).

In 7 out of 10 months the shell-diseased animals showed ecdysone levels to be higher than in unaffected animals. The average ecdysone level for the 76 shell-diseased animals was 89 [+ or -] 32 ng/mL compared with 57 [+ or -] 16 ng/mL in unaffected animals (n = 210). This result is highly significant by 2-way analysis of variance, with P = 0.002.

Comparing Ovigerous Unaffected and Shell-diseased Lobsters

In 5 ovigerous lobsters with a medium severity of shell disease the ecdysone concentration averaged 165 [+ or -] 53 ng/mL. This exceeded that of 7 unaffected ovigerous lobsters, which had an average ecdysone concentration of 13 [+ or -] 4 ng/mL (Fig. 2). The ecdysone concentrations in hemolymph of ovigerous lobsters with shell disease were significantly higher (P < 0.005 by the Student t-test) than in ovigerous lobsters without shell disease.

[FIGURE 2 OMITTED]

DISCUSSION

Chang (1984) and Chang and O'Connor (1988) state that non-molting lobsters had low levels of ecdysone, usually in the range of 10 ng/mL, over a period of more than 100-200 days, followed by a peak as high as 350-400 ng/mL for about 20 days. The lobsters we examined (n = 210) showed a major ecdysone build-up in the population of unaffected specimens with a low of 14 [+ or -] 8 ng/mL in January to a major peak in May and June of 144 and 159 ng/mL, respectively, with a sharp drop of ecdysone levels in July to 4 ng/mL. These results strongly suggest that the normal population of lobsters unaffected by shell disease molted mostly in July. Ecdysone also peaked in November, suggesting that some lobsters may have molted in the fall. Aiken and Waddy (1980) suggested that lobsters fitting the size and age characteristics of the population studied here may molt more frequently than once a year. Our data are consistent with that view. Whereas Chang (1984) and Chang and O'Connor (1988) followed individual specimens with multiple assays on each specimen and determined that the ecdysone peak preceding a molt is sharp and may reach more than 300 ng/mL, our data represent averages of single samples taken from multiple individuals. The lobsters examined in this study show broader ecdysone increases over time. The May, June and November values of unaffected lobsters represent close to half of the peak concentrations achieved by Chang's individual lobsters just prior to an actual molt. The May-June peak is then followed by a sharp drop in ecdysone concentration in July, further supporting the view that a molt probably occurred in this population.

In 76 shell-diseased animals the concentration of ecdysone remained relatively high at an average value of 89 ng/mL when compared with 210 normal-appearing animals, which averaged 57 ng/mL during the year. Shell disease appears to lead to increased molting frequencies. In support of this view, we also point out that many of the lobsters we examined had residual scars on their exoskeletons from SD endured prior to the last molt. It appears that although they were able to escape an earlier infection by shedding their shells, they were confronted with the disease once again. This concept is supported by others (Chistoserdov et al. 2003, Landers et al. 2001). Presumably, either the lobsters continue with this defensive molting strategy or ultimately succumb to the disease. In support of this view we cite SD severity data (Long Island Sound Lobster Health News 2003), which reported SD mostly mild in severity in 2001. Following a molt in August the frequency of SD was very low, but by February and April of 2002 the severity of shell disease was mostly severe with a high frequency of SD. This cycle was followed by a molt in August, which again showed a low incidence of SD. These data, along with our results, suggest that molting can relieve the frequency of occurrence of SD, however, over time it seems to become more severe.

The ecdysone concentrations found in five shell-diseased ovigerous lobsters were significantly higher than in seven unaffected ovigerous lobsters. The embryos of all females were close to hatching, but under normal conditions many months before a molt. The results suggest that shell-diseased ovigerous lobsters may go through the molting process prematurely, leading to loss of the brood. This compares with results by Chang (1984) that ecdysone levels in healthy ovigerous lobsters carrying developing embryos are very low and are correlated with decreased molting in ovigerous females. This low ecdysone prevents loss of embryos through molting.

Ecdysone synthesis is under the control of MIH (Molt Inhibitory Hormone) produced by the X-organ-sinus gland complex. MIH is a Crustacean Hyperglycemic Hormone (CHH), a neuropeptide that, among other functions, regulates glucose metabolism (Liu et al. 1997). During stress such as anoxia or elevated temperatures, levels of CHHs can increase, thus inhibiting molting (Chang et al. 1998, Chang et al. 1999). It is possible that the stress of shell disease causes lobsters to have developed an adaptive defense mechanism to overcome either MIH synthesis or release, resulting in increased ecdysone levels needed to induce the formation of reparative tissue regeneration and subsequent molting to generate a new exoskeleton.

ACKNOWLEDGMENTS

The authors thank Ms. Penny Howell of the Connecticut Department of Environmental Protection, Dr. Mike Syslo of the Massachusetts Lobster Hatchery on Martha's Vineyard and Captain Bro Cote for collecting animals. The authors also thank Dr. E. Chang for supplying ecdysteroid antibodies and Prof. Uwe Koehn for assistance with statistical analyses. This research was supported by the Connecticut Sea Grant College Program, NOAA, and the Connecticut Department of Environmental Protection's Long Island Sound Research Fund.

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HANS LAUFER, (1,2) * NESLIHAN DEMIR (1) AND WILLIAM J. BIGGERS (1,3)

(1) Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269; (2) The Marine Biological Laboratory, Woods Hole, Massachusetts, 02543

(3) Present address: Department of Biology, Wilkes University, Wilkes-Barre,Pennsylvania 18766

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