The effect of ecdysis on DNA of the hepatopancreas and green gland of the Florida spiny lobster (Panulirus argus) Allen H. Altman, Russell J. Buono* and Margaret O. James The Whitney Laboratory (AHA, RJB, MOJ); and Department of Medicinal Chemistry (MOJ) University of Florida, 9505 Ocean Shore Blvd, St. Augustine, FL 32086-8623, U.S.A.
The DNA content per gram of wet hepatopancreas and green gland of Panulirus argus was found to vary with molt stage. In hepatopancreas of premoit and postmolt spiny lobsters, the DNA content was, respectively, 5-fold and 3-fold higher than in intermolt animals. Green gland DNA content showed a trend of varying in the same manner as hepatopancreas DNA. The DNA content per gram of wet green gland from intermolt lobsters averaged about 50 times higher than that of hepatopancreas. The changes in DNA content were not related to changes in wet tissue weights during the molting cycle. The ratio of hepatopancreas or green gland wet weights to total body weight remained constant. A ribosomal DNA probe hybridized to intermolt hepatopancreas DNA samples to a much greater extent than to premolt or postmolt samples, suggesting that rDNA sequences are under-represented in the amplified DNA which occurs prior to ecdysis.
Key words: Molting; Ecdysis; DNA amplification; Lobsters; Hepatopancreas; Green gland; Panulirus argus.
Comp. Biochem. Physiol. I07B, 419-426, 1994.
Introduction
The Florida spiny lobster (Panulirus argus), like most arthropods, undergoes ecdysis periodically by shedding its exoskeleton. This allows the animal to grow in size and regenerate lost appendages (Aiken, 1980; Skinner, 1985). In Panulirus, ecdysis spans just a few minutes (personal observation), but biochemical and morphological preparation for the event is ex- tensive and occurs prior to it (Travis, 1955b; Skinner, 1985).
Ecdysis is part of the molt cycle. In decapods, Drach (1939) divided the molt cycle into five major stages (A through E) based on the hard- ness of the exoskeleton. The exoskeleton is softest immediately after ecdysis and gradually
Correspondence to: A. H. Altman, The Whitney Labora- tory, 9505 Ocean Shore Boulevard, St Augustine, FL 32086-8623, U.S.A. Tel.: (904) 461-4021; Fax: (904) 461-4008.
*Current address: Thomas Jefferson University, Dept. of Anatomy, Philadelphia, PA 19107, U.S.A.
Received 2 July 1993; accepted 11 August 1993.
hardens. Travis (1955a) determined time inter- vals and described the stages for Panulirus ar- gus. A spiny lobster's (80-89mm carapace length) molt cycle spans approximately 65-70 days under summer conditions. Stage A begins immediately after ecdysis (Stage E), lasts for about 24 hr, and is characterized by a very soft exoskelton. The shell begins to harden in stage B which lasts for 4 or 5 days. Stages A and B are considered the postmolt period. Stage C, or intermolt, spans about 44 days and is character- ized by a hard single exoskeleton. A new exoskeleton under the old is constructed during stage D, or premolt, which lasts 10-14 days. Three of the stages have been futher divided into substages: A (two), C (four) and D (three or four). Dividing the molt cycle into distinct periods is somewhat artificial since the molt cycle is one continuous process accompanied by both external and internal changes (Skinner, 1985).
Numerous internal biochemical changes ac- company the molt cycle. Most of these changes
419
420 Allen H. Altman et al.
seem to peak around ecdysis. These involve both the synthesis and degradation of tissues and cellular components, enlargement and shrinkage of cells, and the production of hormones (Skinner, 1985). In Panulirus argus (Travis, 1955b), calcium, protein and phosphorus in the hemolymph and calcium in the urine vary during the molt cycle. Similar cyclical changes occur in the glycogen, mucopolysaccharide, lipid and calcium content of the hepatopancreas cells. High levels of active phenoloxidase are found in the cuticle of Panulirus argus just prior to ecdysis (Ferrer et al., 1984).
In the land crab, Gecarcinus lateralis, levels of rRNA in the muscle, hepatopancreas, and epi- dermis were higher during the premolt period coincident with the new exoskeleton synthesis (Skinner, 1966, 1968). Ribosomal RNA in- creased by nearly 5-fold in the epidermis, dou- bled in the hepatopancreas and increased four to five times in the muscle. In the crayfish (Orconectes virilis), the protein and RNA con- tent of the hepatopancreas increased while the DNA content remained the same as the animals progressed from intermolt to premolt (Gorell and Gilbert, 1971). During the course of a study on the binding of benzo(a)pyrene metabolites to hepatopancreas DNA (James et al., 1992) and green gland DNA (unpublished observations) in Panulirus, we noticed that the DNA content of the hepatopancreas varied with the molting stage. This observation led us to re-examine the data and to undertake additional studies on intermolt, premolt and postmolt lobsters.
The hepatopancreas or digestive gland is lo- cated along the gut, secretes digestive enzymes into the gut and absorbs digested food into the bloodstream. The green gland or antennal gland is the primary organ of urine production and is involved in water and ionic regulation (Phillips et al., 1980). The hepatopancreas accumulates organic reserves which are used for construction of the new exoskeleton and for energy during ecdysis (Yamaoka and Scheer, 1970). Both the hepatopancreas and green gland are involved in the metabolism of molting hormones (James and Shiverick, 1984; Snyder and Chang, 1991).
Here we report on the DNA content of the hepatopancreas and green gland during the molting cycle. The DNA content of the hepato- pancreas increases substantially just prior to ecdysis. This premolt DNA amplification is not general in nature and may involve specific parts of the genome.
Materials and Methods Chemicals
All chemicals were of reagent grade or better. Hydroxylapatite (DNA grade, Bio-Gel HTP)
was obtained from Bio-Rad Laboratories (Richmond, CA). Bisbenzimide dye (Hoechst 33258), calf thymus DNA and salmon testes DNA were purchased from Sigma Chemical Company (St Louis, MO).
Animals
Florida spiny lobsters (Panulirus argus) were collected in the Florida Keys. The lobsters were housed in flowing seawater tanks (22-26°C) and fed squid at least three times a week. Lobsters used in experiments weighed 250470 g.
Isolation of DNA from the hepatopancreas and green gland
Lobsters were placed on ice for 40 min and the hepatopancreas and green gland were then removed. The hepatopancreas was placed in 20mi of a KCl-phosphate buffer (1.15% KCI, 0.05 M K2PO4/KHPO 4, pH 7.4) and weighed. The green gland was weighed and frozen under nitrogen gas for later DNA analysis. A 5-10 g sample of hepatopancreas was homogenized and the lipid layer was removed from the nu- clear pellet by centrifuging at 600g for 10 min. All of these procedures were performed at 0-4°C.
DNA was isolated using procedures similar to those described by James et al. (1991) based on procedures developed by Andriaenssens et al. (1982) and Kanter and Schwartz (1979). Extra washes not required for DNA isolation were used to remove unbound benzo(a)pyrene and its metabolites as part of another study. The hepa- topancreas nuclear pellet or thawed green gland (total wet weight used was 0.24).45 g) were homogenized with 30ml of 0.16M sodium phosphate (pH 6.8) containing 8 M urea, 1 mM EDTA and 1% SDS. Following protein extrac- tion by stirring for 10min in 30mi phenol, chloroform, iso-amyl alcohol (25:25:1) equili- brated with 30 ml of the pellet homogenizing buffer, the mixture was centrifuged for 10 min at 12,000g and the nucleic acid supernatant sol- ution removed. This was extracted with ethyl acetate and twice with ethyl ether. Residual ether was removed by bubbling with nitrogen gas. Boiled hydroxylapatite suspension, 0.5 g, was added to the solution and nucleic acid was bound to the hydroxylapatite by vortexing periodically for 20 min. After centrifuging and decanting of the supernatant, the RNA was eluted from the hydroxylapatite by vortexing periodically first for 20 min with 20 ml and then for 10 min with 10 ml of 0.24 M sodium phos- phate (pH 7.4) containing 8 M urea. After two l-min constant vortexing washes with 0.01 M sodium phosphate buffer (pH 7.0), the DNA was eluted with 5 ml of 0.5 M sodium phosphate buffer (pH 7.0). In experiments to determine the
Ecdysis increases DNA in the spiny lobster
recovery of DNA through this procedure, pure calf thymus or salmon testes DNA (0.1, 0.5 or 1 mg) were added to the 0.16 M sodium phos- phate buffer and taken through the isolation procedure.
The purity of the isolated DNA was deter- mined by the A26o/A28 o ratio and the amount quantified by the fluorimetric method of Ce- sarone et al. (1979). Salmon testes DNA was used as a standard for the fluorimetric assay.
Determination of molting stage
Molt stage was determined for spiny lobsters using morphological characters of the carapace and feeding behavior as described by Travis (1955a) and a modification of a molt prediction method developed by Aiken (1973) for the American lobster using setal development and cuticular changes in the pleopods. Pleopods or swimmerets are located on the first five segments of the abdomen or tail. Our intent was to separate intermolt hardshelled stage C, Do, DI, and early D2 lobsters from premolt late D2 and D 3 lobsters, and softshelled postmolt A and B lobsters.
Intermolt C, Do, D~ and early D2 lobsters feed vigorously. Their pleopods show very little epi- dermis retraction resulting in narrow clear mar- gins. Premolt late D 2 and D3 lobsters do not feed and their pleopods exhibit a very wide clear margin resulting from epidermis retraction. New setae begin to form in the margin. When dissected, a newly forming exoskeleton is clearly visible. The new future exoskeleton is located just below the old exoskeleton. Postmolt A and B lobsters have very soft shells, do not feed, and exhibit pleopods with very narrow clear mar- gins.
Pleopods were removed from live lobsters with scissors and examined with a Nikon FX35A or Nikon Optophot under seawater on a microscopic slide and photographed.
DNA dot-blots
DNA from each lobster was alkaline-de- natured (0.3 M NaOH final concentration, 1 hr 68°), then cooled to room temperature, and neutralized by addition of an equal volume of 2 M ammonium acetate (pH 7.0). The samples were then vacuum-blotted on to nitrocellulose membrane (Bethesda Research Laboratories dot-blot apparatus and Schleicher and Schuell membrane), and covalently linked to the mem- brane by ultraviolet irradiation (Stratagene UV Stratalinker). Prehybridization was with 0.25% non-fat dried milk, 0.9 M NaCI, 0.09 M sodium citrate, 0.01% sodium azide (Sambrook et al., 1989) for 2hr at 68°C (high stringency). A 29-base synthetic oligonucleotide complemen- tary to a sequence (5'ATCCGCTAAGGA-
421
GTGTGTAACAACTCACC3') representing a conserved portion of the 28s ribosomal DNA (HiUis and Dixon, 1991) was synthesized (Uni- versity of Florida Interdisciplinary Center for Biotechnology Research DNA CORE Facility) and used as a probe. The complementary oligonucleotide was also synthesized and used as a positive control (250 ng/sample). Plasmid DNA (5 #g pBR322) was used as a negative control. The oligonucleotide was radiolabeled by end-labeling using P32-ATP and T4 polynu- cleotide kinase according to the manufacturer's protocols (United States Biochemical Corp., Cleveland, OH). About 1 x 106 counts/min la- beled probe were added per milliliter to the prehybridization solution and allowed to hy- bridize overnight at 68°C. Dot-blots were washed several times the following morning; the temperature was increased and salt concen- trations decreased to remove nonspecific bind- ing as previously described (Sambrook et al., 1989). The blots were then covered with plastic wrap and placed on Kodak X-OMAT AR5 X-ray film. Autoradiograms were kept at -85°C in cassettes until developed. The inten- sity of the signals generated on autoradiograms was quantitated using scanning densitometry (ISCO UA-5 Absorbance/Fluorescence Detec- tor with an ISCO Model 312 Gel Scanner). Peak heights or integration under the curves (peaks were cut out and weighed) gave similar hybrid- ization values. Three separate dot-blots using 5, 8 and 10 #g of lobster DNA were performed and resulted in nearly identical results.
Statistical analysis
Data were analyzed for statistical significance using Student's t-test for a difference between two independent means.
Results Identification of lobster molting stages
Representative pleopods of the three molting groups studied are shown in Fig. 1. Panel A shows a pleopod with a very narrow clear margin which is typically present in intermolt stage C, D 0, D~ and early DE lobsters. This margin enlarges and newly formed setae become visible (panel B) as the epidermis retracts in premolt lobsters (late D2, D3 and D4 stages). Postmolt stages A and B lobsters have extremely soft shells and exhibit pleopods with very narrow margins (panel C).
DNA isolation and yields
The isolation procedure used gave repro- ducible recoveries of added pure salmon testes or calf thymus DNA at the concentrations
422 Allen H. Altman e t al.
studied. The recovery did not change with in- creased added DNA in the range used, and was found to be 56 + 2%, mean + SD, N = 6 (see Fig. 2). The range of total DNA recovered experimentally was 100-2000#g DNA for he- patopancreas, and 120-850 #g DNA for green gland so that only a few points from the highest DNA content samples were above the concen- tration range for which recovery was tested. The values shown in Tables 1 and 2 have not been corrected for recovery.
Table 1 shows DNA yields from hepatopan- creas at different stages in the molt cycle. The DNA levels of intermolt bepatopancreas aver- aged 17.4 #g DNA/g wet tissue and ranged from 9.3/~g DNA/g to 41.4 #g DNA/g. Premolt lob- sters had elevated hepatopancreatic DNA con-
tent averaging 93.7/~g DNA/g and ranging from 36.1 to 197#g/g. Hepatopancreas DNA content dropped just prior to, or soon after, ecdysis as reflected in postmolt DNA contents which averaged 52.3 #g/g and ranged from 34.0 to 71.4#g/g. The differences observed were statistically significant.
Green gland DNA contents were not statisti- cally significant between the different molting stages, but showed a trend of varying in the same manner as hepatopancreas (Table 2). The mean DNA yield from intermolt lobsters was 39% lower than from premolt lobsters. DNA yields from individual green glands varied widely, resulting in considerable overlap be- tween the lobster groups. Intermolt green glands ranged from 414 to 1315#g DNA/g, premolt
Fig. 1. Identification of lobster molting stages by microscopic examination of pleopods. Pleopods were removed from live lobsters with scissors and examined with a light microscope under seawater on a microscopic slide. Pleopod photographs are: (A) Intermolt lobsters, stages C, D 0, D 1 or early D2; (B) Premolt lobsters, stages late D2, D 3 or 1)4; (C) Postmolt lobsters, stages A or B. Pleopod margins (big arrows) are widest in premolt lobsters and narrowest in postmolt lobsters. Intermolt lobster margins are intermediate in width. Small arrows in (B) point to new setae which develop in the pleopod margin just
prior to eedysis. Scale bar = 200/~m.
1000-
750-
i 500-
250-
0 0
Ecdysis increases DNA in the spiny lobster
y 2~o 500 750 ' 1000
DNA added, gg
Fig. 2. Recovery of standard salmon testes DNA by the hydroxylapatite batch method described in Materials and Methods. Each point represents one independent isolation. The mean recovery over the whole range was 56 + 2%,
mean + SD, N = 6.
green glands ranged from 829 to 2377#g DNA/g and postmolt green glands ranged from 878 to 1635/~g DNA/g.
Green gland DNA content per g tissue was always much greater than hepatopancreas DNA content. Intermolt lobster hepatopancreas DNA content averaged only about 2% of inter- molt green glands when compared on a per gram wet tissue basis. The total organ weight of hepatopancreas was about 25 times that of green gland (Tables 1 and 2).
Hepatopancreas and green gland wet weights during the molt cycle
The changes in hepatopancreas DNA content with molt cycle are not related to changes in the water content of hepatopancreas, since the wet weight of hepatopancreas expressed as a per- centage of wet body weight was similar at each molt stage (Table 1). This study also showed that the relationship between green gland wet weight and total body wet weight did not change with molt stage (Table 2),
Table 1. Yield of DNA isolated from spiny lobster hepato- pancreas
Molting Number of /~g DNA/g Percent wet stage* lobsters wet weight body weightt
*Intermolt lobsters are in molting stage C, D 0, D~, or early D2 (Fig. 1, panel A), premolt lobsters are in molting stage late D2 or D~ (Fig. I, panel B) and postmolt lobsters are in molting stage A or B (Fig. 1, panel C).
tWet hepatopancreas weight as a percentage of wet body weight.
:[:Values shown are mean + standard deviation. Statistical significance relative to intermolt values are shown
as **P < 0.001 or NS, not different, P > 0.05.
423
Table 2. Yield of DNA isolated from spiny lobster green gland
Molting Number of /~g DNA/g Percent wet stage* lobsters wet weight body weightt
*Molting stage as defined in Table 1. "l'Wet green gland weight as a percentage of total wet body
weight. Values shown are mean + standard deviation and no stat-
istically significant difference (95% confidence level) was found between molt stages for either DNA yield or percent wet body weight.
Hybridization of the rDNA probe to hepatopan- creas and green gland DNA
Densitometer scans of the autoradiographs shown in Fig. 3 showed that the rDNA probe hybridized to all samples of lobster hepatopan- creas and green gland DNA and to the comp- lement of the rDNA probe. There was no detectable hybridization to the negative control (plasmid pBR322 DNA). Studies with different dilutions of several samples showed that while the spot intensity increased when larger amounts of DNA were applied, the relationship between spot intensity and amount of DNA applied was not linear. Application of 5/~g of each sample per well gave a basis for compari- son of samples, but may have resulted in an underestimation of the amount of rDNA in intermolt samples with a high rDNA content. These limitations aside, Table 3 shows that hybridization of the rDNA probe was at least 50% higher to intermolt lobster hepatopancreas DNA than to premolt lobster DNA. Postmolt lobster DNA had intermediate hybridization values. This suggests that genes representing rDNA sequences are proportionally represented most in intermolt lobster DNA and under-rep- resented in premolt and postmolt lobsters. There was no significant difference between molt cycle stage in rDNA probe hybridization and green gland DNA samples.
Discussion
We report a substantial increase in the hepa- topancreas DNA content of Panulirus argus as ecdysis approaches. There are numerous cyclic biochemical changes which accompany the molting cycle in decapods, but, to our know- ledge, changes in the DNA content of internal tissues have not been reported.
Hepatopancreas DNA content increased over 5-fold in lobsters that were close to ecdysis, when compared to intermolt lobsters. These premolt lobsters had a newly forming exoskele- ton that could be clearly seen in dissection.
Allen H. Altman et al. 424
Fig. 3. Autoradiograph of a dot-blot showing a ribosomal DNA probe hybridized to DNA isolated from different molting stage spiny lobster bepatopancreas and green gland. A synthetic 29-base oligonucleotide probe, specific for genomic DNA sequences coding for rDNA was radiolabeled. The probe was hybridized against total DNA extracted from individual spiny lobster tissues. Except where noted below, 5 #g of DNA was applied to separate wells of a dot-blot apparatus. The bacterial plasmid pBR322 DNA was used as a negative control and 250 ng of the complementary oligonucleotide was used as a postive control. The autoradiograph was exposed for I 1 days at -80°C without intensifying screens. The sample positions are as follows. Row a: controls; positives in wells a2 and a8, negative in a5. Row b: wells b l - b l l, hepatopancreas DNA intermolt stage. Row c: wells cl-c7, hepatopancreas DNA premolt stage. Row d: wells dl-d4, hepatopancreas DNA postmolt stage. Row e: wells el-e3 are 1, 10, and 30 #g respectively of DNA from one green gland; wells e4-e6 are l, 10, and 30/~g respectively from another green gland; wells e7-e9 and el0-el2 are triplicate samples from two other green glands. Row f: wells fl-f'/, green gland DNA intermolt stage. Row g: wells gl~g5, green gland DNA premolt stage; wells g6-g9, green gland DNA
postmolt stage.
DNA contents were less in postmolt lobsters which averaged a 3-fold increase in their hepato- pancreas DNA when compared to intermolt lobsters. Spiny lobsters spend only a small fraction of their time in ecdysis and in the postmolt period. This suggests that, as ecdysis approaches, the DNA content of the hepato- pancreas increases in a short time and decreases
Table 3. Hybridization of the ribosomal DNA probe to DNA isolated from spiny lobster hepatopancreas and green
gland
Peak height (cm)* Molting stage Hepatopancreas DNA Green gland DNA
*The intensity of the signals generated on the autoradio- graph of the dot-blot shown in Fig. 2 were quantitated using scanning densitometry as described in Materials and Methods.
tValues are mean + standard deviation for (N) animals. Statistical significance relative to intermolt is shown as
**P < 0.01, ***P < 0.05 or NS not significantly differ- ent P > 0.05.
either shortly before ecdysis or soon after ecdy- sis. There was considerable variability in hepa- topancreas DNA content within each of the three molt groups studied. Possibly this vari- ation occurred because our studies divided the molt cycle only into three different stages in terms of DNA isolation from tissues. A finer distinction may be necessary in order to com- pletely characterize precisely when during the molt cycle DNA levels change due to this rapid increase in DNA content. The method we used to isolate DNA resulted in a very pure fraction with no RNA contamination, but did not give 100% recovery of the DNA present in the cells. When standard DNA was taken through the isolation procedure, the recovery was 56% over a 10-fold range of DNA added. The values reported in Tables 1 and 2 were not corrected for recovery, and therefore under-represent the total present. Since recovery was constant, how- ever, the differences between sets are valid.
In lobsters a steroid hormone (the ecdy- steroid, 20-hydroxyecdysone) has been ident- ified as mediating decapod molting (Chang, 1985). In lobsters hemolymph ecdysteroid
Ecdysis increases DNA in the spiny lobster 425
steroid titers are low until they reach a high premolt peak followed by a rapid decline just prior to ecdysis (Chang, 1989). Our high premolt DNA peak may be related to eedy- steroid levels since both of these seem to be coincident. 20-Hydroxyecdysone has an effect on the pattern of epidermal DNA synthesis, which peaks just prior to ecdysis during the molt cycle in the crayfish Orconectes sanborni (Wittig and Stevenson, 1975). Ecdysteroids stimulate DNA synthesis in Drosophila causing clearly observable chromosomal puffs (Richards, 1980).
We anticipated that the amplified DNA would be ribosomal since in related animals rRNA increases during the premolt period (Skinner, 1966b, 1968; Gorell and Gilbert, 1971). However, the amplified DNA does not appear to be predominantly ribosomal in nature. In fact, the amplified DNA present in premolt and postmolt lobsters had a substan- tially lower percentage of rDNA compared to intermolt lobsters as shown by the reduced binding of our rDNA probe to the amplified DNA. Thus ribosomal gene amplification which is known to occur in amphibian oocytes (Brown and Dawid, 1968) and house cricket oocytes (Trrster et al., 1990), does not appear to be occurring in premolt lobsters. The increased levels of DNA may be due to the amplification of another type of DNA. Genes responsible for chorion proteins are amplified in Drosophila melanogaster (Spradling and Mahowald, 1980) and genes responsible for cocoon proteins are amplified in Rhynchosciara americana (Glover et al., 1982). Our premolt lobster DNA amplifi- cation is not a general phenomenon as it may be in nematode-induced giant cells of the pea, Pisum sativum (Wiggers et al., 1991) since rDNA sequences seem to be under-represented in the amplified DNA. We conclude that the premolt DNA amplification may involve specific parts of the genome and could play a direct role in lobster ecdysis.
Acknowledgements--This work was supported in part by the U.S. Public Health Service Grant ES05781. We thank Scan Boyle for assistance in lobster handling and collection of some DNA samples, Debra Fadool for help in identifying lobster molting stages and Dr Robert Greenberg for techni- cal advice on DNA blots.
References
Aiken D. E. (1973) Proecdysis, semi development and molt prediction in the American lobster (Homarns amerieanus) injected with ecdysterone. J. Fish. Res. Bd Can. 32, 1337-1344.
Aiken D. E. (1980) Molting and growth. In The Biology and Management of Lobsters, Vol. 1, pp. 91-163. Academic Press, New York.
Andriaenssens P. I., Bixler C. J. and Anderson M. W. (1982) Isolation and quantitation of DNA-bound ben- zo(a)pyrene metabolites: comparison of hydroxyapatite and precipitation procedures. Analyt. Biochem. 123, 162-169.
Brown D. D. and Dawid I. B. (1968) Specific gene amplifi- cation in oocytes. Science 160, 272-280.
Cesarone C. F., Bolognesi C. and Santi L. (1979) Improved microfluorometric DNA determination in biological ma- terial using 33258 Hoechst. Analyt. Biochem. 100, 188-197.
Chang E. S. (1985) Hormonal control of molting in decapod Crustacea. Am. Zool. 25, 179-185.
Chang E. S. (1989) Endocrine regulation of molting in Crustacea. Rev. Aquat. Sci. 1, 131-157.
Drach P. (1939) Mue et cycle d'intermue chez les Crustac~s D~,.apodes. Ann. Inst. OcJanogr. 19, 103-391.
Ferrer O. J., Koburger J. A., Otwell W. S., Glceson R. A., Simpson B. K. and Marshall M. R. (1984) Phenoloxidase from the cuticle of Florida spiny lobster (Panulirus argus): mode of activation and characterization. J. Food Sci. 54, 63-67, 176.
Glover D. M., Zaha A., Stocker A. J., Santelli R. V., Pueyo M. T., De Toledo S. M. and Lara F. J. S. (1982) Gene amplification in Rhynchosciara salivary gland chromosomes. Proc. natn. Acad. Sci. U.S.A. 79, 2947-2951.
Gorell T. A. and Gilbert F. J. S. (1971) Protein and RNA synthesis in the premolt crayfish, Orconectes virilis. Z. vergl. Physiol. 73, 345-356.
Hillis D. M. and Dixon M. T. (1991) Ribosomal DNA: molecular evolution and phylogenetic inference. Q. Rev. Biol. 66, 411--453.
James M. O., Altman A. H., Li C.-L. J. and Boyle S. M. (1992) Dose- and time-dependent formation of benzo(a)pyrene metabolite DNA adducts in the spiny lobster, Panulirus argus. Mar. Environ. Res. 34, 299-302.
James M. O., Schell J. D., Boyle S. M., Altman A. H. and Cromer E. A. (1991) Southern flounder hepatic and intestinal metabolism and DNA binding of bcnzo(a)- pyrene (BaP) metabolites following dietary adminis- tration of low doses of BaP, BaP-7,8-dihydrodiol or a BaP metabolite mixture. Chem.-Biol. Interact. 79, 305-321.
James M. O., Shiverick K. T. (1984) Cytochrome P-450-de- pendent oxidation of progesterone, testerone and ecdysone in the spiny lobster, Panulirus argus. Archs Biochem. Biophys. 233, 1-9.
Kanter P. M. and Schwartz H. S. (1979) A hydroxylapatite batch assay for quantitation of cellular DNA damage. Analyt. Biochem. 97, 77-84.
Phillips B. F., Cobb J. S. and George R. W. (1980) General biology. In The Biology and Management of Lobsters, Vol. 1, pp. 1-82. Academic Press, New York.
Richards G. (1980) Ecdysteroids and puffing in Drosophila melanogaster. In Developments in Endocrinology: Progress in Ecdysone Research, Vol. 7, pp. 363-378. Elsevier/ North-Holland Biomedical Press, Amsterdam, The Netherlands.
Sambrook J., Fritsch E. F. and Maniatis T. (1989) Analysis and cloning of eukaryotic genomic DNA. Chapt. 9 in Molecular Cloning, 2nd edn, Book 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Skinner D. M. (1966) Breakdown and reformation of somatic muscle during the molt cycle of the land crab, Gecarcinus lateralis. J. exp. Zool. 163, 115-124.
Skinner D. M. (1968) Isolation and characterization of ribosomal ribonucleic acid from the crustacean, Gecarci- nus lateralis. J. exp. Zool. 169, 347-356.
Skinner D. M. (1985) Molting and regeneration. In The Biology of Crustacea, Vol. 9, pp. 43-146. Academic Press, New York.
426 Allen H. Altman et al.
Snyder M. J. and Chang E. S. (1991) Metabolism and excretion of injected [3H]-ecdysone by female lobsters, Homarus americanus. Biol. Bull. 180, 475-484.
Spradling A. C. and Mahowald A. P. (1980) Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc. natn. Acad. Sci. U.S.A. 77, 1096-1100.
Travis D. F. (1955a) The molting cycle of the spiny lobster, Panulirus argus Latreille. II. Pre-ecdysial histological and histochemical changes in the hepatopancreas and integu- mental tissues. Biol. Bull. 108, 88-112.
Travis D. F. (1955b) The molting cycle of the spiny lobster, Panulirus argus Latreille. III. Physiological changes which occur in the blood and urine during the normal molting cycle. Biol. Bull. 109, 484-503.
Tr6ster H., Edstr6m Jan-Erik, Trendelenburg M. F. and Hofmann A. (1990) Structural organization of Acheta rDNA: evidence for differential amplification of soma and germ-line specific rDNA sequences. J. Molec. Biol. 216, 533-543.
Wiggers K. J., Magill C. W., Start J. L. and Price H. J. (1991) Evidence against amplification of four genes in giant cells induced by Meloidogyne incognita. J. Nematol. 23, 421-424.
Wittig K. and Stevenson J. R. (1975) DNA synthesis in the crayfish epidermis and its modification by ecdysterone. J. comp. Physiol. 99, 279-286.
Yamaoka L. H. and Scheer B. T. (1970) Chemistry of growth and development in crustaceans. Chem. Zool. 5, (Part A) 321-341.
