GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
25, 9-13 (1975)
Ovary-inhibiting Hormone Activity in Shrimp (Crangon crangon) Eyestalks during the Annual Reproductive Cycle’ EL~BIETA KL@K-KAWI~ISKA~ AND ANDRZEJ BOMIRSKI Department of Biology and Parasitology, Institute of Medical Biology, Medical School, Gdarisk, Poland: and Biological Station, Gdatisk University, G&ki Wschodnie, Poland
Received April 28, 1974 Activity of the ovary-inhibiting hormone in the eyestalks of female shrimps (Crangon L.) was measured during the course of the annual reproductive cycle. In the early part of the breeding season, after the first oviposition, ovary-inhibiting hormone activity is apparently absent. During the latter part of the breeding season, after the second oviposition, it is present, but at a much lower level than after the breeding season when its activity is highest. Ovary-inhibiting hormone appears to be responsible for the phase of ovarian rest and to control the rate of vitellogenesis before the breeding season. crangon
The eyestalks of female decapod crustaceans inhibit vitellogenesis in the ovaries by producing and secreting ovaryinhibiting hormone (OIH).3 However, experiments involving eyestalk removal have revealed that in some phases of the female annual reproductive cycle this operation fails to accelerate vitellogenesis (Aoto and Nishida, 1956; Demeusy, 1964; Weitzman, 1964; Adiyodi, 1968). Thus, it appears that some of the following parameters are likely to be cyclic: production of OIH, release of OIH, response of the ovaries to OIH (Adiyodi and Adiyodi, 1970). In our previous work (Bomirski and Klek, 1974) a test for OIH activity was
used to study the ovary-inhibiting activity in the eyestalks of the female shrimps, Crangon crangon L., in different phases of their annual reproductive cycle. OIH activity was found to be present in the non-breeding season, but was not detectable during the breeding season. The current work investigates in a more detailed manner OIH activity in the eyestalks of female Crangon crangon during the annual breeding cycle. The reproductive cycle of female Crangon crungon in British coastal waters was described by Lloyd and Yonge ( 1947). Our own observations (Bomirski and Klek, 1974) have shown that in Gdarisk Bay the breeding season starts in May with a molt, which is followed by the appearance of r Partially supported by a grant from the Polish Academy of Sciences (PAN). egg-carrying setae on the pleopods and by * Present address: Department of Biology and Paraoviposition. Active vitellogenesis takes sitology, Institute of Medical Biology, Medical place in the egg-carrying females. After School, Al. Zwyciestwa 42a, 80-210 Gdansk, Poland. 3 List of abbreviations: PAN, Polish Academy of hatching of the larvae the females undergo another molt, followed by a second ovipoSciences; OIH, ovary-inhibiting hormone: GI, gonad index; DOI, degree of ovarian inhibition: GI*, gonad sition. The ovaries are inactive during the index of eyestalkless shrimps; GP, gonad index of second incubation of eggs. Hatching of eyestalked shrimps; GIT, gonad index of the eyethe larvae is followed by a molt during stalkless shrimps injected with eyestalks under test; W, which the egg-carrying setae disappear. range of the gonad indices within a test group; SE, standard error of the mean. These molts take place in the second half 9 Copyright 0 1975 by Academic Printed in the United States.
Press. Inc. All rights of reproduction
in any form reserved
10
KLEK-KAWINSKA
AND BOMIRSKI
of August and the month of September and they terminate the breeding season. After the molt the ovaries remained in the resting state until the end of observations (the end of October). Because of technical problems, observations could not be carried out between the end of October and the beginning of May and we were unable to determine when the resting phase ended, and vitellogenesis, preceding the spring oviposition, started.
season, when their ovaries were in resting condition. Such females may be recognized externally by the absence of ovigerous setae on the pleopods. The tests were carried out in the period from August 27 to October 12, 1973. Each test lasted 4 days. The principle of the test for OIH activity is the comparison of the gonad index (GI) in eyestalkless females, receiving injections of the tested eyestalk extract, with the gonad indices in two kinds of control females: eyestalkless and intact. Gonad index is calculated according to the formula: GI = (Ovarian weight x lOO)/Body weight.
MATERIAL AND METHODS Animals. Females of the shrimp, Crangon crangon L. (Crustacea, Decapoda), were collected by bottom dredge in one location of the coastal waters of Gdarisk Bay (Poland). Water salinity in this area is 6-8%0. Sexually mature females 32-59 mm in body length were used throughout. Eyestnlks. Donors of the eyestalks were female shrimps in a definite phase of the annual reproductive cycle. A phase of the cycle was recognized on the basis of presence or absence of eggs attached to pleopods, presence or absence of ovigerous setae on pleopods and on the condition of the ovaries. Description of the females, from which the eyestalks were taken, is given in Table I. The eyestalks were excised at their bases and stored in acetone in a refrigerator. Directly before use the eyestalks were dried in air for 12 hr, ground in seawater from Gdat’rsk Bay, previously filtered and boiled, and then centrifuged at 2000 rpm for 5 min. For injections the supernatant was used in amounts of 0.02 ml per shrimp. Test for OIH activity. In the present work the test described earlier (Bomirski and Klek, 1974) was applied with some modifications. The test was made on female shrimps, Crungon crungon, after the breeding
TABLE DESCRIPTION
Stage of annual reproductive cycle Females prior to season Females after 1st oviposition Females after 2nd oviposition Females after reproductive season has ended
OF FEMALE
Crangon
Date of collection of eyestalks May 7-May 10, 1973
Having the gonad indices of the test shrimps (eyestalkless injected) and of both control shrimps one may calculate as a percentage, the degree of ovarian inhibition (DOI), produced by the eyestalks being tested, using the formula: DO1 = [(CIA - GIr) x lOO]/(GI”
- GIE).
where GIA is GI in control shrimps with eyestalks ablated; in these animals the inhibition of the ovaries is assumed to be 0, since the source of OIH is removed. GIE is Gl in the control shrimps with eyestalks intact: in these animals the inhibition of the ovaries is 100% because of the action of the endogenous OIH. GIr is GI in the test shrimps, i.e., eyestalkless shrimps injected with eyestalk extracts. The shrimps used in the tests were divided into groups of 20 animals. placed in separate glass tanks with filtered, aerated sea water from Gdarisk Bay, and kept at a constant temperature of 2 1“C and under normal day-night illumination for 4 days. During tests the animals were not fed. No molting was observed in intact, eyestalkless and eyestalkless-injetted shrimps. On day 0 of the test the shrimps comprising the test group had their eyestalks removed by ligation with a thin cotton thread. On the first, second, and third days each female received, into the muscles of the ab1 crungon
DONORS
Activity of ovaries
OF EYESTALKS
Eggs attached to pleopods
Egg-carrying setae on pleopods
Not present
Not present
Present
Present
Aug. IO-Sept. 2, 1973
Phase of vitellogenesis Phase of vitellogenesis Resting phase
Present
Present
1) Sept.-October, 1972 2) Sept. 16-Oct. 20, 1973
Resting phase Resting phase
Not present Not present
Not present Not present
June 12-July 20, 1973
OVARY-INHIBITING
HORMONE
domen, one injection of eyestalk extract. On the fourth day the shrimps that survived were killed and weighed, their ovaries were dissected out and weighed, and GI was calculated. Twelve groups of test animals were used. The results are expressed as mean GIT for each group. For calculation of GIA, six groups of 20 females were used. On day 0 their eyestalks were ligated and on the fourth day their Gl was calculated. GIA is the mean gonad index of 83 eyestalkless control shrimps that survived in all the groups. For calculation of GIE also six groups of 20 females were used. On day 0 the animals were placed in the tanks without any operation and on the fourth day their GI was calculated. GIE is the mean gonad index of 84 intact control females that survived until the end of the tests. Usually, at the same time two different test groups. one control eyestalkless group and one control eyestalked group were run. The significance of the difference between GIT and GIA on the one hand, and GIT and GI” on the other, was estimated, using Lord’s test based on the range. The values lGIA--GITl/~ and IGIT-GIEl/w, where w is the range of the gonad indices within a test group, were calculated and compared with appropriate tabulated values (Lord, 1947). The difference was deemed nonsignificant if P was greater than 0.05.
RESULTS
Table 2 records the mean gonad indices of the control eyestalked and eyestalkless groups that have been used for the calculation of GP and GIA. The values of GIE = 0.29 and of GIA = 0.99 were subsequently applied to calculate DOI of the test groups. TABLE GONAD
(GI)
INDICES
THAT
WERE
GI*.
USED
GROUPS
OF GIE AND AT THE BEGINNING OF THE
CONSISTED
Groupsof
control females, eyestalks intact
Number
CONTROL
FOR CALCULATIONS
EACH GROUP EXPERIMENT
2
OF FEMALE
of
OF
20
FEMALES
Groups of control females, eyestalks ablated
Mean GI
Number of survivors
Mean GI
16 14 11 14 15 14
0.25 ‘0.28 0.30 0.30 0.31 0.32
14 14 15 16 11 13
0.91 O.% 1.00 1.01 1.02 1.06
Total 84
GIe = 0.29 + 0.01
Total 83
GI” = 0.99 f 0.04
survivors
IN THE
SHRIMP
11
Table 3 contains GIT and DOI of the test groups of eyestalkless females injected with the eyestalks taken from Crangon crangon females in different phases of the annual reproductive cycle. Six test groups received extracts of the eyestalks collected in the same year in which the tests were made (1973). One group received the eyestalks from the females after the breeding season, collected in the preceding year (1972). The purpose of testing these eyestalks was to find out what the influence on OIH activity was after storage in acetone for a year. In view of the fact that some of the DO1 values shown in Table 3 are intermediate, we investigated the effect of dosage on DOI. For this purpose, the eyestalks collected in 1973 from females after the breeding season were used and the results are shown in Table 4. DISCUSSION
Measurement of ovary-inhibiting hormone activity in the eyestalks of females of the shrimp, Crangon crangon, reveals that this activity varies during the course of the annual reproductive cycle. The female reproductive cycle in the shrimps inhabiting Gdatisk Bay compromises the following phases: vitellogenesis before the breeding season, another vitellogenesis after the first oviposition, and ovarian rest. The resting phase starts in ovigerous females after the second oviposition, but persists beyond the ecdysis which terminates the breeding season (Bomirski and Klek, 1974). OIH activity was measured after the breeding season, when the ovaries are resting. Removal of the eyestalks during this period brings about a rapid onset of vitellogenesis and, consequently, an increase in ovarian weight. The highest activity of OIH in the eyestalks occurs after the breeding season. Under the conditions of the test, the dose of four eyestalks daily inhibits ovarian
12
KL&K-KAWIhKA
AND
TABLE
BOMIRSKI
3
GONAD INDICES (GIT) AND DEGREE OF OVARIAN INHIBITION (DOI) IN FEMALE TEST GROUPS INJECTED WITH EYESTALK EXTRACTS FROM FEMALES IN DIFFERENT PHASES OF THEIR REPRODUCTIVE CYCLE. EACH GROUP AT THE BEGINNING OF THE EXPERIMENT CONSISTED OF 20 FEMALES. NUMBER OF INJECTIONS ADMINISTERED WAS THREE”
Female donors of eyestalks
Number of eyestalksper injection per shrimp
Number of Significance of females in test difference group at end of experiment Mean GI + SE GIT vs GIE GIT vs GIA
Females prior to reproductive cycle (1973)
4 4
9 14
0.53 f 0.05 0.48 ” 0.04
P
AND
COMPARATIVE
ENDOCRINOLOGY
25, 9-13 (1975)
Ovary-inhibiting Hormone Activity in Shrimp (Crangon crangon) Eyestalks during the Annual Reproductive Cycle’ EL~BIETA KL@K-KAWI~ISKA~ AND ANDRZEJ BOMIRSKI Department of Biology and Parasitology, Institute of Medical Biology, Medical School, Gdarisk, Poland: and Biological Station, Gdatisk University, G&ki Wschodnie, Poland
Received April 28, 1974 Activity of the ovary-inhibiting hormone in the eyestalks of female shrimps (Crangon L.) was measured during the course of the annual reproductive cycle. In the early part of the breeding season, after the first oviposition, ovary-inhibiting hormone activity is apparently absent. During the latter part of the breeding season, after the second oviposition, it is present, but at a much lower level than after the breeding season when its activity is highest. Ovary-inhibiting hormone appears to be responsible for the phase of ovarian rest and to control the rate of vitellogenesis before the breeding season. crangon
The eyestalks of female decapod crustaceans inhibit vitellogenesis in the ovaries by producing and secreting ovaryinhibiting hormone (OIH).3 However, experiments involving eyestalk removal have revealed that in some phases of the female annual reproductive cycle this operation fails to accelerate vitellogenesis (Aoto and Nishida, 1956; Demeusy, 1964; Weitzman, 1964; Adiyodi, 1968). Thus, it appears that some of the following parameters are likely to be cyclic: production of OIH, release of OIH, response of the ovaries to OIH (Adiyodi and Adiyodi, 1970). In our previous work (Bomirski and Klek, 1974) a test for OIH activity was
used to study the ovary-inhibiting activity in the eyestalks of the female shrimps, Crangon crangon L., in different phases of their annual reproductive cycle. OIH activity was found to be present in the non-breeding season, but was not detectable during the breeding season. The current work investigates in a more detailed manner OIH activity in the eyestalks of female Crangon crangon during the annual breeding cycle. The reproductive cycle of female Crangon crungon in British coastal waters was described by Lloyd and Yonge ( 1947). Our own observations (Bomirski and Klek, 1974) have shown that in Gdarisk Bay the breeding season starts in May with a molt, which is followed by the appearance of r Partially supported by a grant from the Polish Academy of Sciences (PAN). egg-carrying setae on the pleopods and by * Present address: Department of Biology and Paraoviposition. Active vitellogenesis takes sitology, Institute of Medical Biology, Medical place in the egg-carrying females. After School, Al. Zwyciestwa 42a, 80-210 Gdansk, Poland. 3 List of abbreviations: PAN, Polish Academy of hatching of the larvae the females undergo another molt, followed by a second ovipoSciences; OIH, ovary-inhibiting hormone: GI, gonad index; DOI, degree of ovarian inhibition: GI*, gonad sition. The ovaries are inactive during the index of eyestalkless shrimps; GP, gonad index of second incubation of eggs. Hatching of eyestalked shrimps; GIT, gonad index of the eyethe larvae is followed by a molt during stalkless shrimps injected with eyestalks under test; W, which the egg-carrying setae disappear. range of the gonad indices within a test group; SE, standard error of the mean. These molts take place in the second half 9 Copyright 0 1975 by Academic Printed in the United States.
Press. Inc. All rights of reproduction
in any form reserved
10
KLEK-KAWINSKA
AND BOMIRSKI
of August and the month of September and they terminate the breeding season. After the molt the ovaries remained in the resting state until the end of observations (the end of October). Because of technical problems, observations could not be carried out between the end of October and the beginning of May and we were unable to determine when the resting phase ended, and vitellogenesis, preceding the spring oviposition, started.
season, when their ovaries were in resting condition. Such females may be recognized externally by the absence of ovigerous setae on the pleopods. The tests were carried out in the period from August 27 to October 12, 1973. Each test lasted 4 days. The principle of the test for OIH activity is the comparison of the gonad index (GI) in eyestalkless females, receiving injections of the tested eyestalk extract, with the gonad indices in two kinds of control females: eyestalkless and intact. Gonad index is calculated according to the formula: GI = (Ovarian weight x lOO)/Body weight.
MATERIAL AND METHODS Animals. Females of the shrimp, Crangon crangon L. (Crustacea, Decapoda), were collected by bottom dredge in one location of the coastal waters of Gdarisk Bay (Poland). Water salinity in this area is 6-8%0. Sexually mature females 32-59 mm in body length were used throughout. Eyestnlks. Donors of the eyestalks were female shrimps in a definite phase of the annual reproductive cycle. A phase of the cycle was recognized on the basis of presence or absence of eggs attached to pleopods, presence or absence of ovigerous setae on pleopods and on the condition of the ovaries. Description of the females, from which the eyestalks were taken, is given in Table I. The eyestalks were excised at their bases and stored in acetone in a refrigerator. Directly before use the eyestalks were dried in air for 12 hr, ground in seawater from Gdat’rsk Bay, previously filtered and boiled, and then centrifuged at 2000 rpm for 5 min. For injections the supernatant was used in amounts of 0.02 ml per shrimp. Test for OIH activity. In the present work the test described earlier (Bomirski and Klek, 1974) was applied with some modifications. The test was made on female shrimps, Crungon crungon, after the breeding
TABLE DESCRIPTION
Stage of annual reproductive cycle Females prior to season Females after 1st oviposition Females after 2nd oviposition Females after reproductive season has ended
OF FEMALE
Crangon
Date of collection of eyestalks May 7-May 10, 1973
Having the gonad indices of the test shrimps (eyestalkless injected) and of both control shrimps one may calculate as a percentage, the degree of ovarian inhibition (DOI), produced by the eyestalks being tested, using the formula: DO1 = [(CIA - GIr) x lOO]/(GI”
- GIE).
where GIA is GI in control shrimps with eyestalks ablated; in these animals the inhibition of the ovaries is assumed to be 0, since the source of OIH is removed. GIE is Gl in the control shrimps with eyestalks intact: in these animals the inhibition of the ovaries is 100% because of the action of the endogenous OIH. GIr is GI in the test shrimps, i.e., eyestalkless shrimps injected with eyestalk extracts. The shrimps used in the tests were divided into groups of 20 animals. placed in separate glass tanks with filtered, aerated sea water from Gdarisk Bay, and kept at a constant temperature of 2 1“C and under normal day-night illumination for 4 days. During tests the animals were not fed. No molting was observed in intact, eyestalkless and eyestalkless-injetted shrimps. On day 0 of the test the shrimps comprising the test group had their eyestalks removed by ligation with a thin cotton thread. On the first, second, and third days each female received, into the muscles of the ab1 crungon
DONORS
Activity of ovaries
OF EYESTALKS
Eggs attached to pleopods
Egg-carrying setae on pleopods
Not present
Not present
Present
Present
Aug. IO-Sept. 2, 1973
Phase of vitellogenesis Phase of vitellogenesis Resting phase
Present
Present
1) Sept.-October, 1972 2) Sept. 16-Oct. 20, 1973
Resting phase Resting phase
Not present Not present
Not present Not present
June 12-July 20, 1973
OVARY-INHIBITING
HORMONE
domen, one injection of eyestalk extract. On the fourth day the shrimps that survived were killed and weighed, their ovaries were dissected out and weighed, and GI was calculated. Twelve groups of test animals were used. The results are expressed as mean GIT for each group. For calculation of GIA, six groups of 20 females were used. On day 0 their eyestalks were ligated and on the fourth day their Gl was calculated. GIA is the mean gonad index of 83 eyestalkless control shrimps that survived in all the groups. For calculation of GIE also six groups of 20 females were used. On day 0 the animals were placed in the tanks without any operation and on the fourth day their GI was calculated. GIE is the mean gonad index of 84 intact control females that survived until the end of the tests. Usually, at the same time two different test groups. one control eyestalkless group and one control eyestalked group were run. The significance of the difference between GIT and GIA on the one hand, and GIT and GI” on the other, was estimated, using Lord’s test based on the range. The values lGIA--GITl/~ and IGIT-GIEl/w, where w is the range of the gonad indices within a test group, were calculated and compared with appropriate tabulated values (Lord, 1947). The difference was deemed nonsignificant if P was greater than 0.05.
RESULTS
Table 2 records the mean gonad indices of the control eyestalked and eyestalkless groups that have been used for the calculation of GP and GIA. The values of GIE = 0.29 and of GIA = 0.99 were subsequently applied to calculate DOI of the test groups. TABLE GONAD
(GI)
INDICES
THAT
WERE
GI*.
USED
GROUPS
OF GIE AND AT THE BEGINNING OF THE
CONSISTED
Groupsof
control females, eyestalks intact
Number
CONTROL
FOR CALCULATIONS
EACH GROUP EXPERIMENT
2
OF FEMALE
of
OF
20
FEMALES
Groups of control females, eyestalks ablated
Mean GI
Number of survivors
Mean GI
16 14 11 14 15 14
0.25 ‘0.28 0.30 0.30 0.31 0.32
14 14 15 16 11 13
0.91 O.% 1.00 1.01 1.02 1.06
Total 84
GIe = 0.29 + 0.01
Total 83
GI” = 0.99 f 0.04
survivors
IN THE
SHRIMP
11
Table 3 contains GIT and DOI of the test groups of eyestalkless females injected with the eyestalks taken from Crangon crangon females in different phases of the annual reproductive cycle. Six test groups received extracts of the eyestalks collected in the same year in which the tests were made (1973). One group received the eyestalks from the females after the breeding season, collected in the preceding year (1972). The purpose of testing these eyestalks was to find out what the influence on OIH activity was after storage in acetone for a year. In view of the fact that some of the DO1 values shown in Table 3 are intermediate, we investigated the effect of dosage on DOI. For this purpose, the eyestalks collected in 1973 from females after the breeding season were used and the results are shown in Table 4. DISCUSSION
Measurement of ovary-inhibiting hormone activity in the eyestalks of females of the shrimp, Crangon crangon, reveals that this activity varies during the course of the annual reproductive cycle. The female reproductive cycle in the shrimps inhabiting Gdatisk Bay compromises the following phases: vitellogenesis before the breeding season, another vitellogenesis after the first oviposition, and ovarian rest. The resting phase starts in ovigerous females after the second oviposition, but persists beyond the ecdysis which terminates the breeding season (Bomirski and Klek, 1974). OIH activity was measured after the breeding season, when the ovaries are resting. Removal of the eyestalks during this period brings about a rapid onset of vitellogenesis and, consequently, an increase in ovarian weight. The highest activity of OIH in the eyestalks occurs after the breeding season. Under the conditions of the test, the dose of four eyestalks daily inhibits ovarian
12
KL&K-KAWIhKA
AND
TABLE
BOMIRSKI
3
GONAD INDICES (GIT) AND DEGREE OF OVARIAN INHIBITION (DOI) IN FEMALE TEST GROUPS INJECTED WITH EYESTALK EXTRACTS FROM FEMALES IN DIFFERENT PHASES OF THEIR REPRODUCTIVE CYCLE. EACH GROUP AT THE BEGINNING OF THE EXPERIMENT CONSISTED OF 20 FEMALES. NUMBER OF INJECTIONS ADMINISTERED WAS THREE”
Female donors of eyestalks
Number of eyestalksper injection per shrimp
Number of Significance of females in test difference group at end of experiment Mean GI + SE GIT vs GIE GIT vs GIA
Females prior to reproductive cycle (1973)
4 4
9 14
0.53 f 0.05 0.48 ” 0.04
P
