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Ecdysone titers during the molt cycle of the crayfish Orconectes sanborni
GENERAL
AND
COMPARATlVE
Ecdysone
ENDOCRINOLOGY
Titers
J.Ross
during
STEVENSON,*
*Vepartment of Biological P.O. Box 247, Bodega
39, 20-25 (1979)
the Molt Cycle of the Crayfish sanborni
Orconectes
PAUL W. AMSTRONG,* ERNESTS.CHANG,~ JOHN D. O'CONNORS
AND
Sciences, Kent State University, Kent, Ohio 44242; Bay, California 94923; and Wepartment of Biology, Los Angeles, California 90024
tBodega University
Marine Laboratory, of California,
Accepted April 1, 1979 Hemolymph ecdysone titers were measured by radioimmunoassay at the different molt stages. One major peak was found at stage Dl”’ -D2 and a minor peak at stage B. Minima were found at stages A and C3-C4. No sex differences were found. Ecdysone levels were followed in individual animals over a period of days, and transitory high values, each followed by a return to low values, were found during premolt. It is suggested that ecdysone may be produced intermittently for precise control of premolt developmental changes. us (J.R.S.) has used since 1968 do not agree exactly with those published by Drach and Tchernigovtzeff at about the same time (1967) or with those published by Aiken (1973) for use with the lobster, Table 1 is presented for comparison of these different designations so that the results reported here may more readily be compared with those of other workers. Blood sampling. Animals of both sexes, but not egg-bearing females, were selected on the basis of their molting stage so that all stages would be represented. The animals were isolated in separate compartments in the aquaria so that they could be sampled and staged repeatedly over a period of some days. To obtain the samples, each crayfish was blotted dry and placed under two rubber bands stretched across a piece of plywood. Then, 15 ~1 of hemolymph was removed through the articular membrane at the base of the fourth walking leg using a 25~1 Hamilton microsyringe. Samples were placed in borosilicate RIA tubes (6 x 50 mm) and left uncovered in the refrigerator to dry overnight. Then they were sealed with Parafilm until the radioimmunoassays were performed. This storage has minimal effect upon the RIA activity. Radioimmunoassay. Aliquots of 100 ~1 of a solution of c#H]ecdysone (specific activity = 68 Ciimmol, generously provided by Dr. D. S. King, Zoecon Corp., Palo Alto, Calif.) in borate buffer were added to the assay tubes containing the dried blood samples. The buffer contained 0.1 M boric acid, 0.1 M sodium tetraborate, and 0.075 M NaCl, adjusted to pH 8.4. The aliquots contained approximately 3600 cpm each. Each tube was vortexed well to take up the dried unknown and then 100 ~1 of the M-20 ecdysone antiserum of Borst and O’Connor (1974) in borate buffer was added, and the tube was thoroughly vortexed again.
Ecdysone titers have been measured before in crayfish, by Adelung (1969), Keller and Adelung (1970), Carlisle and Connick (1973), and Willig and Keller (1973), all of whom used insect bioassays for the measurements. (The term “ecdysone” is used here in a generic sense and refers to any polyhydroxy steroid with molting hormone activity.) In this study, the much more sensitive radioimmunoassay (RIA) developed by Borst and O’Connor (1972, 1974) was used to assay the ecdysone titers in small samples of hemolymph. In addition to determinations of mean hemolymph titers at each stage of the molt cycle, determinations were also made repeatedly in individual crayfish so that the pattern of changes in individuals could be followed. METHODS Animals. Crayfish borni (Faxon) were
of the species Orconectes sancollected from Plum Creek and Breakneck Creek in Kent, Ohio and maintained in the laboratory at Kent State University in continuously aerated water over a bed of calcareous gravel in acrylic plastic aquaria. The temperature in the aquarium room was maintained at 21-23” and photoperiod at 16hr light: 8-hr dark. The crayfish were fed frozen fish three times a week. Molt stages were determined as described by Drach (1939, 1944) and as modified for Orconecres by Stevenson (1968, 1972) and Stevenson et al. (1968). Because the stage designations which one of 20 0016~6480/79/090820-06$01.00/0 Copyright @ 1979 by Academic press, Inc. All rights of reproduction in any form reserved.
ECDYSONE
COMPARISON
OF SOME PREMOLT
TITERS
OF
TABLE
1
21
CRAYFISH
STAGE DESIGNATIONS
BY DIFFERENT A&en,
Drach and Tchemigovtzeff, 1967
Stevenson, 1%8, 1972 Stevenson et al., 1968
Early DO Later DO Dl” Dl”’ D2
Do Dl’ Dl” Dl”’ D2
After incubating the tubes overnight at 4” or at room temperature for 4 hr, the antibody was precipitated with 200 ~1 saturated ammonium sulfate. The precipitation was performed at 4”, and each tube was mixed within I min after the ammonium sulfate addition. After sitting in the cold for 20 min, the tubes were centrifuged and the supematant was removed. Then 400 ~1 of 50% saturated ammonium sulfate in borate buffer was added to each pellet, the tubes were vortexed, allowed to sit in the cold for 15 min, and centrifuged, and the supematant solution was removed. The pellet was now vortexed with 25 ~1 of water and 600 ~1 of Aquasol (New England Nuclear). Then the vials were allowed to sit for 5-6 hr to allow the pellets to settle and the counting efficiency to stabilize. After this, they were placed into glass shell vial inserts (10 x 50 mm) which were then placed into standard glass scintillation vials and analyzed with a Beckman liquid scintillation spectrometer.
RESULTS Ecdysone Titers
Figure 1 shows the mean ecdysone hemolymph titer at each molt stage. In general, the levels of ecdysones remained low during postmolt (stages A, B, Cl, C2, and C3) and intermolt (stage C4), with a small peak at stage B. Then, the levels began to rise during premolt stage DO, reaching the maximum during stages Dl”‘-D2. Then there was a drop again at stage D3-4, the last stage before the molt. To find whether animal size had any effect on ecdysone concentration in the hemolymph, the data were compared by three size classes during the stages when ecdysone concentration fluctuated the least. When the data were pooled for stages
AUTHORS 1973
Letter system
NUlTIbW system
Early DO Late DO Dl”’ D2 D2”-D3
1.5 2.5 4.0 4.5 5.0-5.5
45
40
35
30 3 iii $ 25 z Y g 20 i * tl: 15 P 10
FIG. 1. Mean ecdysone titer at each molt stage in the crayfish 0. sanborni. Data for stages Al and A2 and for stages D3 and D4 were combined because only a small number of samples was obtained in stages A2 and D4. Also, stages C3 and C4 were not distinguished because to do so would require injuring the animals, and because of the difftculty of determining the beginning of stage D2 with certainty, the values for stages Dl”’ and D2 were grouped together. Number of samples taken at each stage: A, 11; B, 5; Cl, 6; C2, 6; C3-4, 27; DO, 12; Dl’, 41; Dl”, 23; Dl”‘-D2, 40; D3-4, 15.
22
STEVENSON
A through C4, it was found that the mean concentration in crayfish having carapace lengths of 18-25 mm was 3.84 pg/pl; for carapace lengths of 26-30 mm it was 4.78 pgl& and for carapace lengths of 3 l-40 mm it was 5.33 pg/pl. The difference between the values for the smallest and largest size classes was significant (P < 0.05), according to Student’s t test. However, no significant differences were found between these same size classes at either stage DO or stage Dl ’ . Ecdysone titers were also compared by sex. No significant difference was found at any of the stages and no difference was found between the mean titer of all the females and that of all the males. The mean titer of all 99 of the samples taken from females was 12.344 ‘_ 2.039 pg/pl. The mean titer of all 87 of the samples taken from males was 16.416 f 4.498 pg/& An ecdysone titer of 352 pg/pl was recorded from a single sample from one male. This value was far higher than any other measured in any animal. If this one value is removed, the mean titer of the 86 remaining males is 12.421 & 2.030 pg/pl, which is virtually identical to the mean value obtained from the females. Some of the data obtained from individual animals are shown in Tables 2 and 3. Many of the animals displayed a transitory high ecdysone titer that appeared only during one of the days sampled. Table 2 lists (above the line) two such transitory high ecdysone levels that occurred in two animals during stage Dl’ together with the
ECDYSONE
AnimaI number
TABLE 2 LEVELS AND DURATION Ecdysone level (PW)
OF STAGE
Days before entering Dl”
1 3
17 16
2 4
2 4 5 11 17
7 9 8 6 9
6 2 9 17+ 17
Dl’
ET AL. TABLE
3
ECDYSONE LEVELS DURING STAGE Dl” DURATION OF STAGES Dl” AND SUCCEEDING STAGES
Animal number
Ecdysone level (pg/pl)
11 17 23 25 26
134 93 77 59 31
2 <4 2 <4 3
1 5 13 14 19 21 24 27 29
8 9 4
2 2 7 2 2 4 9 4 4
11
10 8 7 11
6
AND
Days before entering Dl”’
D2 or D3
Ecdysis
4 4 7
5 8
5 6 5 7 8 25+
number of days before each animal was found to have entered the succeeding molt stage, stage Dl”. For comparison, the initial ecdysone levels measured during stage Dl’ in several other crayfish are listed (below the line) together with the number of days before each of these was found to have entered stage Dl”. It can be seen that the two animals with the higher titers at stage Dl’ proceeded to the succeeding stage more rapidly than all the other animals except No. 4. Table 3 shows similar data for stage Dl”. Transitory high ecdysone titers (listed above the line) were detected in five crayfish during stage Dl”. These values were higher than the highest values recorded during stage Dl’ (Table 2), consistent with the general increase in titer during premolt as shown on Fig. 1. As shown in Table 3, the mean times for these animals to enter the succeeding molting stages were a little shorter than the times for the animals in which no such high titers were detected (shown below the line). Also, two of the animals that displayed high titers during
ECDYSONE
TITERS
stage Dl”, Nos. 25 and 26, were sampled on a sufficient number of days that a molt was detected and the animal continued to postmolt stage A (No. 26) or B (No. 25). None of the animals that did not display high titers of ecdysone molted during the same time period. In fact, one of them (No. 29) remained in stage Dl’ ” for at least 21 days. Thus, it appears again that the animals with the higher titers proceeded to the succeeding stages more rapidly than the other animals. Transitory high titers were also detected in several animals during stages Dl”‘, D2, and D3. They were about the same magnitude as the higher titers measured during stage Dl”. However, there were not enough data to determine whether these animals progressed to succeeding molt stages faster than animals not displaying such high titers. DISCUSSION
The radioimmunoassay used in this study is much more reliable than the bioassays used in previous studies of the ecdysone titers in the crayfish. The standard error for the bioassay may be as high as 50% vs less than 3% for the radioimmunoassay (Bollenbather, 1974). Also, the radioimmunoassay is more sensitive, allowing repeated measurements to be made on the same animal over a period of time. The M-20 antiserum does not distinguish between (Y- and pecdysone. The curve of ecdysone titer vs molt stage obtained in this study of 0. sanborni is very similar to that reported by Willig and Keller (1973) for 0. limosus. Both have a minimum at stage C (intermolt), begin to rise at stage DO (early premolt), and reach a maximum at Dl”’ -D2. (Their D2 corresponds to our late Dl”‘.) Then the titer drops during D3 and D4. The only difference between the two curves is found in early postmolt. In our study, the titer reached the same minimum at stage A as at C3-4 and then displayed a second peak at stage B, whereas Willig and Keller found
OF
CRAYFISH
23
the titer still dropping at stage A and no second peak at stage B. Our peak at B agrees with earlier data published by Adelung (1969) and by Keller and Adelung (1970). Our results differ from those of Carlisle and Connick (1973), who found the peak titer in 0. propinquus to occur in late proecdysis and the titer at ecdysis to be still almost half the maximum. Our maximum mean titer of 30 pg/pl hemolymph (= 30 &ml) is only half the maximum mean titer of 60 rig/g found by Willig and Keller (1973) in whole animal extracts. This difference is surprising because it suggests that ecdysone is more concentrated in the tissues than in the hemolymph. Carlisle and Connick (1973) found just the opposite in 0. propinquus, a maximum of 230 Calliphora units in hemolymph, 170 in an antennary gland, and 32 in the remaining tissues. Of course we used a different species and a different technique than Willig and Keller (1973). Using the same technique as was used here, Chang et al. (1976) found much higher ecdysone titers, up to 425 pgIp1, in the hemolymph of Pachygrapsus crassipes than the maximum of 30 pg/pl we found in 0. sanborni. As Willig and Keller (1973) have already pointed out, the crayfish seems to have much lower titers than other crustaceans studied. For example, some reported maxima are about 300 nglg in Callinectes sapidus (Faux et al., 1969), about 100 rig/g in Carcinus maenas (Adelung, 1969; Spindler et al., 1974), and 275 rig/g in Gecarcinus lateralis (McCarthy and Skinner, 1977). However, Carcinus and Gecarcinus were induced to enter premolt by autotomy of several limbs and Pachygrapsus by having its eyestalks removed. It may be that the high titers partly reflect ecdysone secretion related to injury and regeneration. However, injury and regeneration cannot be the only cause of these high titers because the Callinectes in which over 300 rig/g total ecdysones were found were normally molting animals collected in the molt stages in which they were measured
24
STEVENSON
(Faux et al., 1969). When ecdysone was measured in normal Jasus lalandei and Homarus americanus, much lower values were recorded: 2 nglg in Jaws in stage C (Hampshire and Horn, 1%6) and 6 rig/g in Homarus in stage A (Gagosian et al., 1974). It may be that ecdysone levels are lower generally in Macrura than in Brachyura. Cirripedes may also have lower titers. Extraction of a population of B&anus balanoides that included molting individuals yielded only 40 ng ecdysterone/g fresh wt (after deducting the weight of the shells) (Bebbington and Morgan, 1977). The discovery of transitory high titers during premolt suggests that the crayfish does not experience a steadily rising ecdysone titer during premolt as one would expect from graphs of mean titer vs molt stage. Instead, it may be that each crayfish controls its preparations for ecdysis by producing ecdysone intermittently. This production may involve secretion, (Y- to @-ecdysone conversion, liberation from some storage reservoir, or some combination of these. In the crab, Pachygrapsus crassipes, it was found (Chang and O’Connor, 1978) that an increased secretion of ol-ecdysone by the molting gland does in fact precede the increase in hemolymph ecdysone titer seen in premolt, and therefore these transitory high titers seen in the crayfish very likely represent secretion also. The information in Table 2 suggests that during stage Dl ’ the animal may produce extra ecdysone which terminates stage Dl’ and causes the changes characteristic of stages Dl” and Dl”‘. The data do not show whether an animal may proceed beyond stage Dl’ without such extra ecdysone because where no transitory high titer is recorded, we do not know whether there was none or whether it was missed. Table 3 and the additional data presented suggest that each animal produces a larger amount of ecdysone, usually during stage Dl”‘, D2, or D3, but sometimes during Dl” instead, and these cause the final changes which lead to molting. It is already
ET AL.
known that a crustacean may stop preparation for molt if the environment is unfavorable (Passano, 1960; Mobberly, 1963; Bliss and Boyer, 1964). The mechanism for this control may be the production of these transitory high titers of ecdysone when the internal and external environment become favorable. That injections of extra ecdysone can stimulate the progression of molt stages in the crayfish has been shown by Stevenson and Tschantz (1973). Further evidence for the existence of transitory high titers is found in Keller and Adelung (1970). In their study, ecdysone was found in only some crayfish during each premolt stage. In other specimens, even in stages D2 and D3, the level of hormone was below the limit of detection. Dutkowski et al. (1977) found more complete cuticle produced in cultured wing discs of the insect Plodia after a pulse of ecdysone than after continuous incubation. This suggests that a capacity to produce ecdysone intermittently and to respond best to the transitory high titers that result may be widespread among the arthropods. REFERENCES Adelung, D. (1969). Die Ausschiittung and Funktion von Hautungshormon wlhrend eines Zwischenhlutungs-Intervalls bei der Strandkrabbe Carcinus maenas L.. Z. Natcrrforsch. B 24,
1447-
1455.
Aiken, D. E. (1973). Proecdysis, setal development, and molt prediction in the American lobster (Homarus
americanus).
J. Fish.
Res. Bd. Canad.
30, 1337- 1344. Bebbington, P. M., and Morgan, E. D. (1977). Detection and identification of molting hormone (ecdysones) in the barnacle Balanus balanoides. Comp.
Biochem.
Physiol.
B S&77-79.
Bliss, D. E., and Boyer, J. R. (1964). Environmental regulation of growth in the decapod crustacean Gecarcinus
lateralis.
Gen.
Comp.
Endocrinof.
4,
15-41. Bollenbacher, W. E. (1974). “Zn Vitro Secretions of Arthropod Ecdysial Glands: Correlation with the in Viva System.” Ph.D. dissertation, Department of Biology, UCLA, Los Angeles, Calif. Borst, D. W., and O’Connor, J. D. (1972). Arthropod molting hormone: Radioimmunoassay. Science 178, 418-419. Borst, D. W., and O’Connor, J. D. (1974). Trace
ECDYSONE
TITERS OF CRAYFISH
analysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay. Steroids 24, 637-656. Carlisle, D. B., and Connick, R. 0. (1973). Crustecdysone (20-hydroxyecdysone): Site of storage in the crayfish Orconectes propinquus. Canad. J. Zool. 51, 417-420. Chang, E. S., Sage, B. A., and O’Connor, J. D. (1976). The qualitative and quantitative determination of ecdysones in tissues of the crab, Pachygrapsus crassipes, following molt induction. Gen. Comp. Endocrinol. 30, 21-33. Chang, E. S., and O’Connor, J. D. (1978). In vitro secretion and hydroxylation of a-ecdysone as a function of the crustacean molt cycle. Gen. Comp.
Endocrinol.
36, 15 I- 160.
Drach, P. (1939). Mue et cycle d’intermue chez les crustach dtcapodes. Ann. Inst. OcPanogr. (Monaco) 19, 103-391. Drach, P. (1944). Etude prtliminaire sur le cycle d’intermue et son conditionnement hormonal chez Leander serratus (Pennant). Bull. Biol. Fr. Belg. 78, 40-62. Drach, P., and Tchernigovtzeff, C. (1967). Sur la mtthode de determination des stades d’intermue et son application g&&ale aux crustaces. Vie Milieu Ser. A. Biol. Mar. 18, 595-610. Dutkowski, A. B., Oberlander, H., and Leach, C. E. (1977). Ultrastructure of cuticle deposited in Pladia interpunctella wing discs after various pecdysone treatments in vitro. Wilhelm Roux’ Arch. Develop. Biol. 183, 155-164. Faux, A., Horn, D. H. S., Middleton, E. J., Fales, H. M., and Lowe, M. E. (1969). Moulting hormones of a crab during ecdysis. Chem. Commun. 1969, 175- 176. Gagosian, R. B., Bourbonniere, R. A., Smith, W. B., Couch, E. F., Blanton, C., and Novak, W. (1974). Lobster molting hormones: Isolation and biosynthesis of ecdysterone. Experientia 30, 723-724. Hampshire, F., and Horn, D. H. S. (1966). Structure of crustecdysone, a crustacean moulting hormone. Chem. Commun. 1966, 37-38. Keller. R. and Adelung, D. (1970). Vergleichende morphologische und physiologische Unter-
25
suchungen des Integumentgewebes und des Hautungshormongehaltes beim Fluszkrebs Orconectes limosus wahrend eines Hgutungszyklus. Wilhelm
Roux’
Arch.
Eniwicklungsmech.
Org.
164, 209-221. McCarthy, J. F., and Skinner, D. M. (1977). Proecdysial changes in serum ecdysone titers, gastrolith formation, and limb regeneration following molt induction by limb autotomy and/or eyestalk removal in the land crab. Gecarcinus lateralis. Gen. Comp.
Endocrinol.
33, 278-292.
Mobberly, W. C., Jr. (1963). Hormonal and environmental regulation of the molting cycle in the crayfish Faxonella clypeata. Tulane Stud. Zool. 11, 79-96. Passano, L. M. (1960). Molting and its control. In “The Physiology of Crustacea” (T. H. Waterman, ed.), Vol. 1, Chap. 15, pp. 473-536. Academic Press, New York/London. Spindler, K.-D., Adelung, D., and Tchernigovtzeff, C. (1974). A comparison of the methods of molt staging according to Drach and to Adelung in the common shore crab, Carcinus maenas. Z. Naturforsch.
C 29, 754-756.
Stevenson, J. R. (1968). Metecdysial molt staging and changes in the cuticle in the crayfish Orconectes sanborni (Faxon). Crustaceana 14, 1699177. Stevenson, J. R. (1972). Changing activities of the crustacean epidermis during the molting cycle. Amer. Zool. 12, 373-380. Stevenson, J. R., Guckert, R. H., and Cohen, J. D. (1968). Lack of correlation of some proecdysial growth and developmental processes in the crayfish. Biol. Bull. (Woods Hole, Mass.) 134, 160- 175. Stevenson, J. R., and Tschantz, J. A. (1973). Acceleration by ecdysterone of premoult substages in the crayfish. Nature (London) 242, 133- 134. Van Harreveld, A. (1936). A physiological solution for freshwater crustaceans. Proc. Sac. Exp. Biol. Med.
34, 428-432.
Willig, A., and Keller, R. (1973). Molting hormone content, cuticle growth and gastrolith growth in the molt cycle of the crayfish Orconectes limosus. J. Camp.
Physiol.
86, 377-388.
AND
COMPARATlVE
Ecdysone
ENDOCRINOLOGY
Titers
J.Ross
during
STEVENSON,*
*Vepartment of Biological P.O. Box 247, Bodega
39, 20-25 (1979)
the Molt Cycle of the Crayfish sanborni
Orconectes
PAUL W. AMSTRONG,* ERNESTS.CHANG,~ JOHN D. O'CONNORS
AND
Sciences, Kent State University, Kent, Ohio 44242; Bay, California 94923; and Wepartment of Biology, Los Angeles, California 90024
tBodega University
Marine Laboratory, of California,
Accepted April 1, 1979 Hemolymph ecdysone titers were measured by radioimmunoassay at the different molt stages. One major peak was found at stage Dl”’ -D2 and a minor peak at stage B. Minima were found at stages A and C3-C4. No sex differences were found. Ecdysone levels were followed in individual animals over a period of days, and transitory high values, each followed by a return to low values, were found during premolt. It is suggested that ecdysone may be produced intermittently for precise control of premolt developmental changes. us (J.R.S.) has used since 1968 do not agree exactly with those published by Drach and Tchernigovtzeff at about the same time (1967) or with those published by Aiken (1973) for use with the lobster, Table 1 is presented for comparison of these different designations so that the results reported here may more readily be compared with those of other workers. Blood sampling. Animals of both sexes, but not egg-bearing females, were selected on the basis of their molting stage so that all stages would be represented. The animals were isolated in separate compartments in the aquaria so that they could be sampled and staged repeatedly over a period of some days. To obtain the samples, each crayfish was blotted dry and placed under two rubber bands stretched across a piece of plywood. Then, 15 ~1 of hemolymph was removed through the articular membrane at the base of the fourth walking leg using a 25~1 Hamilton microsyringe. Samples were placed in borosilicate RIA tubes (6 x 50 mm) and left uncovered in the refrigerator to dry overnight. Then they were sealed with Parafilm until the radioimmunoassays were performed. This storage has minimal effect upon the RIA activity. Radioimmunoassay. Aliquots of 100 ~1 of a solution of c#H]ecdysone (specific activity = 68 Ciimmol, generously provided by Dr. D. S. King, Zoecon Corp., Palo Alto, Calif.) in borate buffer were added to the assay tubes containing the dried blood samples. The buffer contained 0.1 M boric acid, 0.1 M sodium tetraborate, and 0.075 M NaCl, adjusted to pH 8.4. The aliquots contained approximately 3600 cpm each. Each tube was vortexed well to take up the dried unknown and then 100 ~1 of the M-20 ecdysone antiserum of Borst and O’Connor (1974) in borate buffer was added, and the tube was thoroughly vortexed again.
Ecdysone titers have been measured before in crayfish, by Adelung (1969), Keller and Adelung (1970), Carlisle and Connick (1973), and Willig and Keller (1973), all of whom used insect bioassays for the measurements. (The term “ecdysone” is used here in a generic sense and refers to any polyhydroxy steroid with molting hormone activity.) In this study, the much more sensitive radioimmunoassay (RIA) developed by Borst and O’Connor (1972, 1974) was used to assay the ecdysone titers in small samples of hemolymph. In addition to determinations of mean hemolymph titers at each stage of the molt cycle, determinations were also made repeatedly in individual crayfish so that the pattern of changes in individuals could be followed. METHODS Animals. Crayfish borni (Faxon) were
of the species Orconectes sancollected from Plum Creek and Breakneck Creek in Kent, Ohio and maintained in the laboratory at Kent State University in continuously aerated water over a bed of calcareous gravel in acrylic plastic aquaria. The temperature in the aquarium room was maintained at 21-23” and photoperiod at 16hr light: 8-hr dark. The crayfish were fed frozen fish three times a week. Molt stages were determined as described by Drach (1939, 1944) and as modified for Orconecres by Stevenson (1968, 1972) and Stevenson et al. (1968). Because the stage designations which one of 20 0016~6480/79/090820-06$01.00/0 Copyright @ 1979 by Academic press, Inc. All rights of reproduction in any form reserved.
ECDYSONE
COMPARISON
OF SOME PREMOLT
TITERS
OF
TABLE
1
21
CRAYFISH
STAGE DESIGNATIONS
BY DIFFERENT A&en,
Drach and Tchemigovtzeff, 1967
Stevenson, 1%8, 1972 Stevenson et al., 1968
Early DO Later DO Dl” Dl”’ D2
Do Dl’ Dl” Dl”’ D2
After incubating the tubes overnight at 4” or at room temperature for 4 hr, the antibody was precipitated with 200 ~1 saturated ammonium sulfate. The precipitation was performed at 4”, and each tube was mixed within I min after the ammonium sulfate addition. After sitting in the cold for 20 min, the tubes were centrifuged and the supematant was removed. Then 400 ~1 of 50% saturated ammonium sulfate in borate buffer was added to each pellet, the tubes were vortexed, allowed to sit in the cold for 15 min, and centrifuged, and the supematant solution was removed. The pellet was now vortexed with 25 ~1 of water and 600 ~1 of Aquasol (New England Nuclear). Then the vials were allowed to sit for 5-6 hr to allow the pellets to settle and the counting efficiency to stabilize. After this, they were placed into glass shell vial inserts (10 x 50 mm) which were then placed into standard glass scintillation vials and analyzed with a Beckman liquid scintillation spectrometer.
RESULTS Ecdysone Titers
Figure 1 shows the mean ecdysone hemolymph titer at each molt stage. In general, the levels of ecdysones remained low during postmolt (stages A, B, Cl, C2, and C3) and intermolt (stage C4), with a small peak at stage B. Then, the levels began to rise during premolt stage DO, reaching the maximum during stages Dl”‘-D2. Then there was a drop again at stage D3-4, the last stage before the molt. To find whether animal size had any effect on ecdysone concentration in the hemolymph, the data were compared by three size classes during the stages when ecdysone concentration fluctuated the least. When the data were pooled for stages
AUTHORS 1973
Letter system
NUlTIbW system
Early DO Late DO Dl”’ D2 D2”-D3
1.5 2.5 4.0 4.5 5.0-5.5
45
40
35
30 3 iii $ 25 z Y g 20 i * tl: 15 P 10
FIG. 1. Mean ecdysone titer at each molt stage in the crayfish 0. sanborni. Data for stages Al and A2 and for stages D3 and D4 were combined because only a small number of samples was obtained in stages A2 and D4. Also, stages C3 and C4 were not distinguished because to do so would require injuring the animals, and because of the difftculty of determining the beginning of stage D2 with certainty, the values for stages Dl”’ and D2 were grouped together. Number of samples taken at each stage: A, 11; B, 5; Cl, 6; C2, 6; C3-4, 27; DO, 12; Dl’, 41; Dl”, 23; Dl”‘-D2, 40; D3-4, 15.
22
STEVENSON
A through C4, it was found that the mean concentration in crayfish having carapace lengths of 18-25 mm was 3.84 pg/pl; for carapace lengths of 26-30 mm it was 4.78 pgl& and for carapace lengths of 3 l-40 mm it was 5.33 pg/pl. The difference between the values for the smallest and largest size classes was significant (P < 0.05), according to Student’s t test. However, no significant differences were found between these same size classes at either stage DO or stage Dl ’ . Ecdysone titers were also compared by sex. No significant difference was found at any of the stages and no difference was found between the mean titer of all the females and that of all the males. The mean titer of all 99 of the samples taken from females was 12.344 ‘_ 2.039 pg/pl. The mean titer of all 87 of the samples taken from males was 16.416 f 4.498 pg/& An ecdysone titer of 352 pg/pl was recorded from a single sample from one male. This value was far higher than any other measured in any animal. If this one value is removed, the mean titer of the 86 remaining males is 12.421 & 2.030 pg/pl, which is virtually identical to the mean value obtained from the females. Some of the data obtained from individual animals are shown in Tables 2 and 3. Many of the animals displayed a transitory high ecdysone titer that appeared only during one of the days sampled. Table 2 lists (above the line) two such transitory high ecdysone levels that occurred in two animals during stage Dl’ together with the
ECDYSONE
AnimaI number
TABLE 2 LEVELS AND DURATION Ecdysone level (PW)
OF STAGE
Days before entering Dl”
1 3
17 16
2 4
2 4 5 11 17
7 9 8 6 9
6 2 9 17+ 17
Dl’
ET AL. TABLE
3
ECDYSONE LEVELS DURING STAGE Dl” DURATION OF STAGES Dl” AND SUCCEEDING STAGES
Animal number
Ecdysone level (pg/pl)
11 17 23 25 26
134 93 77 59 31
2 <4 2 <4 3
1 5 13 14 19 21 24 27 29
8 9 4
2 2 7 2 2 4 9 4 4
11
10 8 7 11
6
AND
Days before entering Dl”’
D2 or D3
Ecdysis
4 4 7
5 8
5 6 5 7 8 25+
number of days before each animal was found to have entered the succeeding molt stage, stage Dl”. For comparison, the initial ecdysone levels measured during stage Dl’ in several other crayfish are listed (below the line) together with the number of days before each of these was found to have entered stage Dl”. It can be seen that the two animals with the higher titers at stage Dl’ proceeded to the succeeding stage more rapidly than all the other animals except No. 4. Table 3 shows similar data for stage Dl”. Transitory high ecdysone titers (listed above the line) were detected in five crayfish during stage Dl”. These values were higher than the highest values recorded during stage Dl’ (Table 2), consistent with the general increase in titer during premolt as shown on Fig. 1. As shown in Table 3, the mean times for these animals to enter the succeeding molting stages were a little shorter than the times for the animals in which no such high titers were detected (shown below the line). Also, two of the animals that displayed high titers during
ECDYSONE
TITERS
stage Dl”, Nos. 25 and 26, were sampled on a sufficient number of days that a molt was detected and the animal continued to postmolt stage A (No. 26) or B (No. 25). None of the animals that did not display high titers of ecdysone molted during the same time period. In fact, one of them (No. 29) remained in stage Dl’ ” for at least 21 days. Thus, it appears again that the animals with the higher titers proceeded to the succeeding stages more rapidly than the other animals. Transitory high titers were also detected in several animals during stages Dl”‘, D2, and D3. They were about the same magnitude as the higher titers measured during stage Dl”. However, there were not enough data to determine whether these animals progressed to succeeding molt stages faster than animals not displaying such high titers. DISCUSSION
The radioimmunoassay used in this study is much more reliable than the bioassays used in previous studies of the ecdysone titers in the crayfish. The standard error for the bioassay may be as high as 50% vs less than 3% for the radioimmunoassay (Bollenbather, 1974). Also, the radioimmunoassay is more sensitive, allowing repeated measurements to be made on the same animal over a period of time. The M-20 antiserum does not distinguish between (Y- and pecdysone. The curve of ecdysone titer vs molt stage obtained in this study of 0. sanborni is very similar to that reported by Willig and Keller (1973) for 0. limosus. Both have a minimum at stage C (intermolt), begin to rise at stage DO (early premolt), and reach a maximum at Dl”’ -D2. (Their D2 corresponds to our late Dl”‘.) Then the titer drops during D3 and D4. The only difference between the two curves is found in early postmolt. In our study, the titer reached the same minimum at stage A as at C3-4 and then displayed a second peak at stage B, whereas Willig and Keller found
OF
CRAYFISH
23
the titer still dropping at stage A and no second peak at stage B. Our peak at B agrees with earlier data published by Adelung (1969) and by Keller and Adelung (1970). Our results differ from those of Carlisle and Connick (1973), who found the peak titer in 0. propinquus to occur in late proecdysis and the titer at ecdysis to be still almost half the maximum. Our maximum mean titer of 30 pg/pl hemolymph (= 30 &ml) is only half the maximum mean titer of 60 rig/g found by Willig and Keller (1973) in whole animal extracts. This difference is surprising because it suggests that ecdysone is more concentrated in the tissues than in the hemolymph. Carlisle and Connick (1973) found just the opposite in 0. propinquus, a maximum of 230 Calliphora units in hemolymph, 170 in an antennary gland, and 32 in the remaining tissues. Of course we used a different species and a different technique than Willig and Keller (1973). Using the same technique as was used here, Chang et al. (1976) found much higher ecdysone titers, up to 425 pgIp1, in the hemolymph of Pachygrapsus crassipes than the maximum of 30 pg/pl we found in 0. sanborni. As Willig and Keller (1973) have already pointed out, the crayfish seems to have much lower titers than other crustaceans studied. For example, some reported maxima are about 300 nglg in Callinectes sapidus (Faux et al., 1969), about 100 rig/g in Carcinus maenas (Adelung, 1969; Spindler et al., 1974), and 275 rig/g in Gecarcinus lateralis (McCarthy and Skinner, 1977). However, Carcinus and Gecarcinus were induced to enter premolt by autotomy of several limbs and Pachygrapsus by having its eyestalks removed. It may be that the high titers partly reflect ecdysone secretion related to injury and regeneration. However, injury and regeneration cannot be the only cause of these high titers because the Callinectes in which over 300 rig/g total ecdysones were found were normally molting animals collected in the molt stages in which they were measured
24
STEVENSON
(Faux et al., 1969). When ecdysone was measured in normal Jasus lalandei and Homarus americanus, much lower values were recorded: 2 nglg in Jaws in stage C (Hampshire and Horn, 1%6) and 6 rig/g in Homarus in stage A (Gagosian et al., 1974). It may be that ecdysone levels are lower generally in Macrura than in Brachyura. Cirripedes may also have lower titers. Extraction of a population of B&anus balanoides that included molting individuals yielded only 40 ng ecdysterone/g fresh wt (after deducting the weight of the shells) (Bebbington and Morgan, 1977). The discovery of transitory high titers during premolt suggests that the crayfish does not experience a steadily rising ecdysone titer during premolt as one would expect from graphs of mean titer vs molt stage. Instead, it may be that each crayfish controls its preparations for ecdysis by producing ecdysone intermittently. This production may involve secretion, (Y- to @-ecdysone conversion, liberation from some storage reservoir, or some combination of these. In the crab, Pachygrapsus crassipes, it was found (Chang and O’Connor, 1978) that an increased secretion of ol-ecdysone by the molting gland does in fact precede the increase in hemolymph ecdysone titer seen in premolt, and therefore these transitory high titers seen in the crayfish very likely represent secretion also. The information in Table 2 suggests that during stage Dl ’ the animal may produce extra ecdysone which terminates stage Dl’ and causes the changes characteristic of stages Dl” and Dl”‘. The data do not show whether an animal may proceed beyond stage Dl’ without such extra ecdysone because where no transitory high titer is recorded, we do not know whether there was none or whether it was missed. Table 3 and the additional data presented suggest that each animal produces a larger amount of ecdysone, usually during stage Dl”‘, D2, or D3, but sometimes during Dl” instead, and these cause the final changes which lead to molting. It is already
ET AL.
known that a crustacean may stop preparation for molt if the environment is unfavorable (Passano, 1960; Mobberly, 1963; Bliss and Boyer, 1964). The mechanism for this control may be the production of these transitory high titers of ecdysone when the internal and external environment become favorable. That injections of extra ecdysone can stimulate the progression of molt stages in the crayfish has been shown by Stevenson and Tschantz (1973). Further evidence for the existence of transitory high titers is found in Keller and Adelung (1970). In their study, ecdysone was found in only some crayfish during each premolt stage. In other specimens, even in stages D2 and D3, the level of hormone was below the limit of detection. Dutkowski et al. (1977) found more complete cuticle produced in cultured wing discs of the insect Plodia after a pulse of ecdysone than after continuous incubation. This suggests that a capacity to produce ecdysone intermittently and to respond best to the transitory high titers that result may be widespread among the arthropods. REFERENCES Adelung, D. (1969). Die Ausschiittung and Funktion von Hautungshormon wlhrend eines Zwischenhlutungs-Intervalls bei der Strandkrabbe Carcinus maenas L.. Z. Natcrrforsch. B 24,
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Aiken, D. E. (1973). Proecdysis, setal development, and molt prediction in the American lobster (Homarus
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ECDYSONE
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analysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay. Steroids 24, 637-656. Carlisle, D. B., and Connick, R. 0. (1973). Crustecdysone (20-hydroxyecdysone): Site of storage in the crayfish Orconectes propinquus. Canad. J. Zool. 51, 417-420. Chang, E. S., Sage, B. A., and O’Connor, J. D. (1976). The qualitative and quantitative determination of ecdysones in tissues of the crab, Pachygrapsus crassipes, following molt induction. Gen. Comp. Endocrinol. 30, 21-33. Chang, E. S., and O’Connor, J. D. (1978). In vitro secretion and hydroxylation of a-ecdysone as a function of the crustacean molt cycle. Gen. Comp.
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Drach, P. (1939). Mue et cycle d’intermue chez les crustach dtcapodes. Ann. Inst. OcPanogr. (Monaco) 19, 103-391. Drach, P. (1944). Etude prtliminaire sur le cycle d’intermue et son conditionnement hormonal chez Leander serratus (Pennant). Bull. Biol. Fr. Belg. 78, 40-62. Drach, P., and Tchernigovtzeff, C. (1967). Sur la mtthode de determination des stades d’intermue et son application g&&ale aux crustaces. Vie Milieu Ser. A. Biol. Mar. 18, 595-610. Dutkowski, A. B., Oberlander, H., and Leach, C. E. (1977). Ultrastructure of cuticle deposited in Pladia interpunctella wing discs after various pecdysone treatments in vitro. Wilhelm Roux’ Arch. Develop. Biol. 183, 155-164. Faux, A., Horn, D. H. S., Middleton, E. J., Fales, H. M., and Lowe, M. E. (1969). Moulting hormones of a crab during ecdysis. Chem. Commun. 1969, 175- 176. Gagosian, R. B., Bourbonniere, R. A., Smith, W. B., Couch, E. F., Blanton, C., and Novak, W. (1974). Lobster molting hormones: Isolation and biosynthesis of ecdysterone. Experientia 30, 723-724. Hampshire, F., and Horn, D. H. S. (1966). Structure of crustecdysone, a crustacean moulting hormone. Chem. Commun. 1966, 37-38. Keller. R. and Adelung, D. (1970). Vergleichende morphologische und physiologische Unter-
25
suchungen des Integumentgewebes und des Hautungshormongehaltes beim Fluszkrebs Orconectes limosus wahrend eines Hgutungszyklus. Wilhelm
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164, 209-221. McCarthy, J. F., and Skinner, D. M. (1977). Proecdysial changes in serum ecdysone titers, gastrolith formation, and limb regeneration following molt induction by limb autotomy and/or eyestalk removal in the land crab. Gecarcinus lateralis. Gen. Comp.
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Mobberly, W. C., Jr. (1963). Hormonal and environmental regulation of the molting cycle in the crayfish Faxonella clypeata. Tulane Stud. Zool. 11, 79-96. Passano, L. M. (1960). Molting and its control. In “The Physiology of Crustacea” (T. H. Waterman, ed.), Vol. 1, Chap. 15, pp. 473-536. Academic Press, New York/London. Spindler, K.-D., Adelung, D., and Tchernigovtzeff, C. (1974). A comparison of the methods of molt staging according to Drach and to Adelung in the common shore crab, Carcinus maenas. Z. Naturforsch.
C 29, 754-756.
Stevenson, J. R. (1968). Metecdysial molt staging and changes in the cuticle in the crayfish Orconectes sanborni (Faxon). Crustaceana 14, 1699177. Stevenson, J. R. (1972). Changing activities of the crustacean epidermis during the molting cycle. Amer. Zool. 12, 373-380. Stevenson, J. R., Guckert, R. H., and Cohen, J. D. (1968). Lack of correlation of some proecdysial growth and developmental processes in the crayfish. Biol. Bull. (Woods Hole, Mass.) 134, 160- 175. Stevenson, J. R., and Tschantz, J. A. (1973). Acceleration by ecdysterone of premoult substages in the crayfish. Nature (London) 242, 133- 134. Van Harreveld, A. (1936). A physiological solution for freshwater crustaceans. Proc. Sac. Exp. Biol. Med.
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Willig, A., and Keller, R. (1973). Molting hormone content, cuticle growth and gastrolith growth in the molt cycle of the crayfish Orconectes limosus. J. Camp.
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