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Spawning cycle and oocyte maturation in laboratory-maintained giant freshwater prawns (Macrobrachium rosenbergii)
Aquaculture, 95 ( 1991) 347-357 Elsevier Science Publishers B.V., Amsterdam
347
Spawning cycle and oocyte maturation in laboratory-maintained giant freshwater prawns (Macrobrachium rosenbergii) Praneet Damrongphol, Nittaya Eangchuan and Boonserm Poolsanguan Department of Bioloa, Faculty of Science, Mahidol University,Bangkok 10400, Thailand (Accepted 7 November
1990)
ABSTRACT Damrongphol, P., Eangchuan, N. and Poolsanguan, B., 199 1. Spawning cycle and oocyte maturation in laboratory-maintained giant freshwater prawns (Macrobrachium rosenbergii). Aquaculture, 95: 341-357. Ovarian development of laboratory-maintained female Macrobrachium rosenbergii was classified into four stages, stages 0, 1,2 and 3, based on external examination of coloration and the relative size of the ovaries to the size of the cephalothorax. Ovaries at each stage consisted of oocytes at various developmental stages. Mature ovaries had a high proportion of mature oocytes. Chromosomes of the oocytes were arrested at diplotene of the first meiotic prophase during vitellogenesis. An increase in the number of oocytes undergoing germinal vesicle breakdown and entering the first meiotic metaphase was found after a premating molt. The oocytes persisted at this stage even after spawning. The intermolt period producing ovarian growth lasted longer than that producing somatic growth. In some females the emphasis in development was on somatic growth after spawning, so that a regular molt followed. On the other hand, development of reproductively active females still emphasized ovarian growth after spawning and a premating molt followed, resulting in a consecutive spawning. Spawning in unmated females commenced much later than that in mated females; and the spawning time was relatively predictable in mated females.
INTRODUCTION
Molt stages in Mucrobruchiumrosenbergiican generally be determined by examination of internal setal development within the antenna1 scale (Peebles, 1977). Development in female prawns involves two physiological phases, somatic and ovarian growth. During the phase of somatic growth, regular molts occur intermittently. When the emphasis in development is more on ovarian growth, a so-called premating molt results; and spawning eventually follows (Ling, 1969). The period leading to a premating molt can be identified by the presence of large orange-colored ovarian mass and the receptiveness for mating (Sagi and Ra’anan, 1985 ). 0044-8486/91/$03.50
0 1991 -
Elsevier Science Publishers
B.V.
P. DAMRONGPHOL
348
ET AL.
Laboratory-kept female M. rosenbergii are able to develop eggs and spawn repeatedly (Wickins and Beard, 1974). The ovaries continue to develop in berried females (O’Donovan et al., 1984). The ovarian cycle in the berried female M. rosenbergii with reference to the developing embryos has been reported previously (O’Donovan et al., 1984). No study on direct determination of developmental stages of the female M. rosenbergii has been described. The precise developmental stage must be taken into account in selection of females for experimental analysis. The present report describes the external examination of ovarian maturity, molting and spawning cycles, and oocyte maturation in the laboratory-maintained female M. rosenbergii in order to establish a method for determination of developmental stages and to understand the reproductive cycle of the female prawns. MATERIALS
AND METHODS
Mature giant freshwater prawns (M. rosenbergii), 7-8 months of age which weighed about 30-35 g per individual, were purchased from prawn farms in Nakorn Pratom, Thailand. The prawns were acclimated in a large fiberglass tank containing half original water and half newly-added tap water at 2526°C for l-2 days. Each prawn was transferred to a separate chamber measuring 22 x 33 x 30 cm deep containing about 18 1 of recirculating tap water at 25-26°C. The chambers were aerated and exposed to luminescent light of a photoperiod of 12D: 12L. The prawns were fed daily on mussels. Half of the recirculating water was gradually replaced every 3 days. Developmental stages of the females were determined daily from December 1988 to January 1990. External features of the ovaries of each female were examined by viewing laterally and dorsally through the carapace. Moreover, the external appearance of the ovaries was examined dorsally through the muscular connection between the cephalothorax and the first abdominal segment while the female was held with the cephalothorax flexing downward. Coloration and the relative length of the ovaries to the length of the cephalothorax were recorded. Molt stages determined by setal development in the antenna1 scale (Peebles, 1977 ) were also examined. Mating was usually done between 6.00 and 12.00 h. A mature male was placed with a female which had newly completed a premating molt. Day and time of molting, mating and spawning were recorded. Ovaries at various developmental stages were dissected, fixed in Bouins solution, dehydrated and then embedded in paraffin. The sections were stained with hematoxylin-eosin for histological observations. Oocytes immediately spawned from unmated females, and ovaries from females about 1 day prior to, just after and 7 h after a premating molt were fixed in Camoy’s fixative for several days. The specimens were stained with 2% aceto-orcein for chromosome observations.
SPAWNING CYCLE OF GIANT FRESHWATER PRAWNS IN THE LABORATORY
349
The chi-squared test was used to evaluate statistical significance for the data of Tables 2 and 4. Student’s t-test was used to evaluate statistical significance for the data of Tables 3 and 5. RESULTS
I. Determination offemale developmental stages by ovarian development It was found that, in female M. rosenbergii, a pair of ovaries were fused at the anterior ends and were situated dorsally to the hepatopancreas. Immature ovaries were tiny transparent masses without coloration or with faint orange color overlain by two dark pigmented stripes. They could not be seen externally through the carapace, but they were visible dorsally at the connection between the cephalothorax and the first abdominal segment if the female was held with the cephalothorax bending downward. Developing ovaries became noticeable externally. They were bright orange and enlarged. The anterior ends extended toward the base of the last spine of the rostrum, and the posterior ends penetrated between the muscular masses in the first abdominal segment. Mature ovaries were bright orange and very large. The anterior ends reached the base of the second or third last spine of the rostrum; the posterior ends penetrated further. Ovarian development was then classified into four stages, stages 0, 1,2 and 3, based on coloration and the relative size of the ovaries to the size of the cephalothorax (see Fig. 1 and Table 1) . No seasonal variation in the ovarian development was observed. Similar percentages of females exhibited spawning each month (Table 2). Ovaries during the period leading to a regular molt were immature and were designated stage 0. Ovaries during the period leading to the last regular molt, the molt preceded a premating molt, were still immature but became faint orange and were designated stage 1. After stage 1, ovarian development progressed rapidly. It took an average of 18.5 + 10.9 s.d. days to enter stage 2 (Table 3 ), a stage in which developing ovarian masses began to be visible externally through the carapace. At the end of stage 2, an average of 9.6 + 4.0 s.d. days later (Table 3)) the anterior ends of the ovaries reached the base of TABLE 1 Coloration and the relative size ofthe ovaries in different developmental
stages
Stage
State of ovary
Coloration
Relative size
0 1 3
Immature Immature Developing
Clear, transparent Faint orange Bright orange
3
Mature
Bright orange
Small Small Large, extend anteriorly to the base of the last spine of the rostrum Very large, extend anteriorly to the base of the second or third last spine of the rostrum
350
Fig. 1. Diagram
P. DAMRONGPHOL ET AL.
0
1
2
3
showing the external
features of the ovaries in four different ovarian develop-
mental stages. TABLE 2 Percentages of laboratory-maintained to January 1990)
females which exhibited spawning each month (December
1988
Month
Year
Percentage of females exhibited spawning
No. of females exhibited spawning
Total no. of females
December January February March April May June July August September October November December January
1988 1989 1989 1989 1989 1989 1989 1989 1989 1989 1989 1989 1989 1990
50.0 40.0 45.1 33.3 44.8 30.8 51.5 41.9 32.0 36.0 52.5 41.7 40.0 45.0
11 10 16 8 13 8 17 18 8 9 21 10 14 9
22 25 35 24 29 26 33 43 25 25 40 24 35 20
the last spine of the rostrum. The ovaries grew furthur during stage 3. By the time of a premating molt, an average of 12.12 4.1 s-d. days later (Table 3 ), the ovaries were fully mature and marked the end of stage 3. The anterior ends of the ovaries reached the base of the second or the third last spine of
SPAWNING CYCLE OF GIANT FRESHWATER PRAWNS IN THE LABORATORY
351
TABLE 3 Intermolt periods between different types of molt Type of intermolt
dk s.d. (days ) Regular molt to regular molt Regular molt to last regular molt Last regular molt to premating molt Stage 1 to stage 2 Stage 2 to stage 3 Stage 3 to premating molt Premating molt to premating molt Berried female Unberried female Premating molt to regular molt Berried female Unberried female
No. of observations
Intermolt period Range (days)
30.9t
4.1
25-67
8
28.2f
5.3
21-43
5
9.8 10.9 4.0 4.1
27-56 8-42 3-22 7-22
14 12 37 61
35.2 k 10.2 38.0f 9.7 28.7& 8.2
21-59 28-59 21-59
34 24 10
26.82 27.0& 26.3+
22-31 22-3 1 26-27
10 7 3
38.7t 18.5& 9.6? 12.li
2.7 3.2 0.5
the rostrum. After spawning, the ovaries returned either to stage 0 or 1 depending on the type of the successive molt (see section III). Histological observations of the horizontal sections of the ovaries passing at the levels of the oviducts revealed that ovaries at each stage contained oocytes at various developmental stages. Ovaries in stage 0 were predominately filled with immature oocytes in early meiotic prophase and in previtellogenesis (Fig. 2a). Ovaries in stage 1 contained mainly oocytes in primary vitellogenesis in the central area and oocytes in secondary vitellogenesis at the periphery (Fig. 2b). Developing ovaries in stage 2 were occupied by many oocytes in secondary vitellogenesis (Fig. 2~). Mature ovaries in stage 3 were full of ripe eggs, but groups of immature oocytes were distributed around the central area of each ovary (Fig. 2d). II. Oocyte maturation Immature oocytes in previtellogenesis, about 40 pm diameter, were at the stage of pachytene of the first meiotic prophase (Fig. 3a). Oocytes in primary vitellogenesis, about 130 pm in diameter, were at the stage of diplotene (Fig. 3b). Oocytes in secondary vitellogenesis were still arrested at this stage. Most ripe eggs 1 day prior to a premating molt were advanced to the stage of diakinesis; the chromosomes were enclosed in a convoluted nucleoplasm (Fig. 3~). After a premating molt, some ripe eggs had undergone germinal vesicle
Fig. 2a. Horizontal section of the ovaries in stage 0. Each ovary contains predominately immature oocytes in early meiotic prophase (arrow) and in previtellogenesis. Scale bar= 200 pm. Fig. 2b. Horizontal section of the ovaries in stage 1. Most oocytes are in primary vitellogenesis; oocytes in secondary vitellogenesis (white arrow) are found at the periphery of each ovary. Scale bar = 200 /*m. Fig. 2c. Horizontal section of the ovaries in stage 2. Oocytes in secondary vitellogenesis are predominant; groups of oocytes in previtellogenesis and in primary vitellogenesis (arrow) are found in the central area of each ovary. Scale bar= 200 pm. Fig. 2d. Horizontal section of the ovaries in stage 3. Most oocytes are ripe and groups of immature oocytes (arrow ) are found in the central area of each ovary. Scale bar = 200 ,um.
SPAWNING
CYCLE
OF GIANT
FRESHWATER
PRAWNS
353
IN THE LABORATORY
c
e-'
,.
“,
Fig. 3a. Oocyte in previtellogenesis at the stage of pachytene. Scale bar = 10 pm. Fig. 3b. Oocyte in primary vitellogenesis at the stage of diplotene. Scale bar=20pm. Fig. 3c. Chromosomes at the stage of diakinesis in the ripe egg found 1 day prior to a premating molt. Scale bar= 20 pm. Fig. 3d. Chromosomes at the first meiotic metaphase in the mature egg found after a premating molt. Scale bar= 20 pm. Fig. 3e. Chromosomes at the first meiotic metaphase in the egg immediately spawned from the unmated female. Scale bar = IOpm.
breakdown and entered the first meiotic metaphase (Fig. 3d). About 7 h after a premating molt, most ripe eggs were at this mature chromosomal stage. Eggs immediately spawned from unmated females were also at the first meiotic metaphase (Fig. 3e). III. Molting and spawning cycles Various intermolt periods in the female giant freshwater prawns were listed in Table 3. The intermolt period between regular molts, which places stress
354
P. DAMRONGPHOL
ET AL.
on somatic growth, took 30.9 ? 4.1 s.d. days, whereas the intermolt period between the last regular molt and a premating molt, which emphasized ovarian growth, lasted 38.759.8 s.d. days. Moreover, the intermolt period between consecutive premating molts in berried females was about 10 days longer than that in unberried females. After spawning, the ovaries of reproductively active females returned to stage 1 and, thus, a premating molt followed, resulting in consecutive spawning. However, for some females, the ovaries were in stage 0 after spawning. Therefore, a regular molt followed. The general scheme of molting cycles is depicted in Fig. 4A. Relation of time span in the molt stages as described by Peebles ( 1977) and the ovarian stages described in the present report is shown in Fig. 4B. Most premating molts (51.4% of observations) and spawning (52.9% of A
B MOLT STAGES
ABC
DA
D ABC
D ABC
111 1111 1111 OVARIAN
STAGES
0
1
0
0
40
20
60
11 23
PM
100 80 TIME IN DAYS
Fig. 4. A. Scheme of molting cycles. B. Relation of time span in the ovarian and the molt stages. M, regular molt; ML, the last regular molt; PM, premating molt. TABLE 4 Percentages of observations riods of the day Event
Premating molt: Spawning: Mated female Unmated female
showing occurrence of a premating molt and spawning at different pe-
Percentage of observation
No. of observations
0.00-6.00 h
6.00-l 2.00 h
12.00-18.00 h
18.00-24.00 h
11.4 3.8 3.5 5.9
14.3 10.6 4.6 41.2
22.9 32.1 29.9 47.0
51.4 52.9 62.0 5.9
35 104 87 17
SPAWNING CYCLE OF GIANT FRESHWATER PRAWNS IN THE LABORATORY
355
TABLE 5 Hour intervals between a premating molt and spawning, and between mating and spawning State of female
Hour interval (2ks.d.)
No. of observations
Premating moltSpawning Mated female: Productive eggs Unproductive eggs Unmated female:
19.8k5.8 19.9+51. 20.6 t 7.4 40.0+ 5.4
Hour interval (8+ s.d.)
No. of observations
Mating-Spawning
30 20 10 5
9.6 * 5.4 8.5k4.1 14.2f7.3
87 70 17
observations) occurred during 18.00-24.00 h (Table 4). Spawning time in the mated female was relatively predictable; it was 19.8? 5.8 s.d. h after a premating molt (Table 5 ), while in unmated females, spawning time was relatively variable and delayed to 40.0 2 5.4 s.d. h after a premating molt (Table 5 ). Eggs spawned from unmated females were usually discarded within l-2 days. Most of the eggs spawned from mated females were productive, i.e., they developed to hatching. The rest, the unproductive eggs, were found to be discarded sometime during the course of development. DISCUSSION
Precise staging of female prawns is essential in selection of females for experimental purposes, for example, biochemical or physiological analysis. Determination of developmental stages of the female giant freshwater prawns by external examination of the ovarian maturity is feasible and reproducible. The number of days of each developmental stage in laboratory-maintained females can be predicted. During the phase of somatic growth, ovarian development is latent. Initiation of ovarian development or vitellogenesis is apparent after the last regular molt. It takes only one molt cycle from the last regular molt to a premating molt to complete vitellogenesis of each batch of the oocytes. During vitellogenesis, the ovaries gradually develop from stage 1 to stage 3. Despite the cytoplasmic growth of the oocytes during vitellogenesis, the chromosomes are arrested at diplotene of the first meiotic prophase. Oocyte maturation is detected by the convoluted nuclear envelope (Merriam, I96 1) and by progress in the chromosomal stage in the ripe eggs 1 day prior to a premating molt. Germinal vesicles break down and the chromosomes enter the first meiotic metaphase in some ripe eggs at about the time of a premating molt. Most eggs are at this mature stage by 7 h after a premating molt. These mature eggs are fertilizable since eggs immediately spawned from unmated females are also
356
P. DAMRONGPHOL
ET AL.
at the first meiotic metaphase. Meiotic resumption occurs upon fertilization or activation (Damrongphol et al., 199 1). Mature oocytes in natantians arrested at first meiotic metaphase have been reported in Sicyonia ingentis (Clark et al., 1984) and Paluemon serratus (Cledon, 1986). Most females in the present study undergo the other round of ovarian growth after spawning, resulting in consecutive spawning. The rest procede to somatic growth after spawning. Therefore, the ovarian stage after spawning differs in these two groups. The former is apparently in the physiological condition ready for ovarian growth, and it retains a batch of oocytes which has begun vitellogenesis as evidenced by the presence of stage-l ovaries. On the other hand, the latter places stress on somatic growth and the ovarian development is latent. Thus, the ovarian stage is variable after spawning. The actual stage of the ovaries must be known if the stage of the berried female is determined with reference to embryonic development. Moreover, intermolt period producing ovarian growth is longer than that producing somatic growth. More specifically, stage C in the molt stages, which is a stage of tissue growth and accumulation of food reserve (Drach, 1939), is extended. During the phase of ovarian growth, the energy or food reserve normally utilized solely for somatic growth is also spent on ovarian growth. Consequently, the period of food intake is lengthened. Intermolt between two consecutive premating molts is longer in berried than in unberried females. A comparable observation has been reported in M. nobilli. Removal of the embryos from the brooding mother increases frequencies of spawning in the following molt (Pandian and Balasundaram, 1982). The presence of the embryos may physically interfere with molting, or extra energy may be required during brooding. Active production of secretions, which are involved in antimicrobial activity (Fisher, 1983) or in synthesis of the embryonic envelopes, as well as preening activity (Bauer, 1979) during the brooding period, may require extra energy expenditure. Removal of the embryos in the berried females seems to reduce the energy burden utilized in brooding so that ovarian growth is enhanced. Without ovarian growth, energy or food reserves seem to be adequate for both somatic growth and support for brooding, since the intermolt period between a premating molt and the next regular molt does not differ in berried or unberried females. The effect of mating on the spawning time is apparent in the present study. Mating does not induce spawning per se (Chow et al., 1982; O’Donovan et al., 1984) but a relatively predictable spawning time is found in mated females, whereas spawning time is delayed and variable in unmated females. The physical action of mating, chemical induction from the male or the presence of the spermatophore may somehow stimulate the final stage of ovarian activity i.e., ovulation, prior to spawning. These speculations are subject to further investigation.
SPAWNING CYCLE OF GIANT FRESHWATER PRAWNS IN THE LABORATORY
357
ACKNOWLEDGEMENTS
We thank K. Vejsanit and A. Duangkaew for their help with figures and typing. This work was supported by the office of the Science and Technology Developmental Board, Grant No. DSN 88A-l-05 1 I 7.
REFERENCES Batter, R.T., 1979. Antifouling adaptations of marine shrimp (Decapoda: Caridea); gill cleaning mechanisms and grooming of brooded embryos. Zool. J. Linn. Sot., 65: 28 l-303. Chow, S., Ogasawara, Y. and Taki, Y., 1982. Male reproductive system and fertilization of the Palaemonid shrimp Macrobrachium rosenbergii. Bull. Jpn. Sot. Sci. Fish., 48: 177-l 83. Clark, W.H., Jr.. Yudin, AI., Griffin, F.J. and Shigekawa, K., 1984. The control of gamete activation and fertilization in the marine Penaeidae, Siqonia ingentia. In: W. Engels (Editor), Advances in Invertebrate Reproduction, 3. Elsevier, Amsterdam, pp. 459-472. Cledon, P. ,1986. Study on oocyte maturation and activation of the common prawn Palaemon serratus (Pennant): relationship between oocyte maturation and the molt cycle cytological aspects. Gamete Res., 13: 353-362. Damrongphol, P., Eangchuan, N. and Poolsanguan, B., 199 1. Chromosome behavior upon fertilization in eggs of the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Invert. Reprod. Devel., 19: 45-49. Drach, P., 1939. Mue et cycle d’intermue chez les crustaces decapodes. Ann. Inst. Oceanogr. Paris N.S., 19: 103-391. Fisher, W.S., 1983, Eggs of Palaemon macrodactylus. II. Association with aquatic bacteria. Biol. Bull., 164: 201-213. Ling. SW., 1969. The general biology and development of Macrobrachium rosenbergii (de Man ). FAO Fish. Rep., 57: 589-606. Merriam, R.W., 1961. Nuclear envelope structure during cell division in Chaetopterus eggs. Exp. Cell Res., 22: 93-107. O’Donovan, P., Abraham, M. and Cohen, D., 1984. The ovarian cycle during the intermoult in ovigerous Macrobrachium rosenbergii. Aquaculture, 36: 347-358. Pandian, T.J. and Balasundarum, C., 1982. Molting and spawning cycles in Macrobrachium nob&. Int. J. Invert. Reprod., 5: 21-30. Peebles, J.B., 1977. A rapid technique for molt staging in live Macrobrachium rosenbergii. Aquaculture, 12: 173- 180. Sagi, A. and Ra’anan, Z., 1985. Rapid identification of reproductive state and the receptive period of females in pond populations of Macrobrachium rosenbergii - a new technique. Aquaculture. 48: 361-367. Wickins, J.F. and Beard, T.W., 1974. Observations on the breeding and growth of the giant freshwater prawn Macrobrachium rosenbergii (de Man) in the laboratory. Aquaculture, 3: 159-174.
347
Spawning cycle and oocyte maturation in laboratory-maintained giant freshwater prawns (Macrobrachium rosenbergii) Praneet Damrongphol, Nittaya Eangchuan and Boonserm Poolsanguan Department of Bioloa, Faculty of Science, Mahidol University,Bangkok 10400, Thailand (Accepted 7 November
1990)
ABSTRACT Damrongphol, P., Eangchuan, N. and Poolsanguan, B., 199 1. Spawning cycle and oocyte maturation in laboratory-maintained giant freshwater prawns (Macrobrachium rosenbergii). Aquaculture, 95: 341-357. Ovarian development of laboratory-maintained female Macrobrachium rosenbergii was classified into four stages, stages 0, 1,2 and 3, based on external examination of coloration and the relative size of the ovaries to the size of the cephalothorax. Ovaries at each stage consisted of oocytes at various developmental stages. Mature ovaries had a high proportion of mature oocytes. Chromosomes of the oocytes were arrested at diplotene of the first meiotic prophase during vitellogenesis. An increase in the number of oocytes undergoing germinal vesicle breakdown and entering the first meiotic metaphase was found after a premating molt. The oocytes persisted at this stage even after spawning. The intermolt period producing ovarian growth lasted longer than that producing somatic growth. In some females the emphasis in development was on somatic growth after spawning, so that a regular molt followed. On the other hand, development of reproductively active females still emphasized ovarian growth after spawning and a premating molt followed, resulting in a consecutive spawning. Spawning in unmated females commenced much later than that in mated females; and the spawning time was relatively predictable in mated females.
INTRODUCTION
Molt stages in Mucrobruchiumrosenbergiican generally be determined by examination of internal setal development within the antenna1 scale (Peebles, 1977). Development in female prawns involves two physiological phases, somatic and ovarian growth. During the phase of somatic growth, regular molts occur intermittently. When the emphasis in development is more on ovarian growth, a so-called premating molt results; and spawning eventually follows (Ling, 1969). The period leading to a premating molt can be identified by the presence of large orange-colored ovarian mass and the receptiveness for mating (Sagi and Ra’anan, 1985 ). 0044-8486/91/$03.50
0 1991 -
Elsevier Science Publishers
B.V.
P. DAMRONGPHOL
348
ET AL.
Laboratory-kept female M. rosenbergii are able to develop eggs and spawn repeatedly (Wickins and Beard, 1974). The ovaries continue to develop in berried females (O’Donovan et al., 1984). The ovarian cycle in the berried female M. rosenbergii with reference to the developing embryos has been reported previously (O’Donovan et al., 1984). No study on direct determination of developmental stages of the female M. rosenbergii has been described. The precise developmental stage must be taken into account in selection of females for experimental analysis. The present report describes the external examination of ovarian maturity, molting and spawning cycles, and oocyte maturation in the laboratory-maintained female M. rosenbergii in order to establish a method for determination of developmental stages and to understand the reproductive cycle of the female prawns. MATERIALS
AND METHODS
Mature giant freshwater prawns (M. rosenbergii), 7-8 months of age which weighed about 30-35 g per individual, were purchased from prawn farms in Nakorn Pratom, Thailand. The prawns were acclimated in a large fiberglass tank containing half original water and half newly-added tap water at 2526°C for l-2 days. Each prawn was transferred to a separate chamber measuring 22 x 33 x 30 cm deep containing about 18 1 of recirculating tap water at 25-26°C. The chambers were aerated and exposed to luminescent light of a photoperiod of 12D: 12L. The prawns were fed daily on mussels. Half of the recirculating water was gradually replaced every 3 days. Developmental stages of the females were determined daily from December 1988 to January 1990. External features of the ovaries of each female were examined by viewing laterally and dorsally through the carapace. Moreover, the external appearance of the ovaries was examined dorsally through the muscular connection between the cephalothorax and the first abdominal segment while the female was held with the cephalothorax flexing downward. Coloration and the relative length of the ovaries to the length of the cephalothorax were recorded. Molt stages determined by setal development in the antenna1 scale (Peebles, 1977 ) were also examined. Mating was usually done between 6.00 and 12.00 h. A mature male was placed with a female which had newly completed a premating molt. Day and time of molting, mating and spawning were recorded. Ovaries at various developmental stages were dissected, fixed in Bouins solution, dehydrated and then embedded in paraffin. The sections were stained with hematoxylin-eosin for histological observations. Oocytes immediately spawned from unmated females, and ovaries from females about 1 day prior to, just after and 7 h after a premating molt were fixed in Camoy’s fixative for several days. The specimens were stained with 2% aceto-orcein for chromosome observations.
SPAWNING CYCLE OF GIANT FRESHWATER PRAWNS IN THE LABORATORY
349
The chi-squared test was used to evaluate statistical significance for the data of Tables 2 and 4. Student’s t-test was used to evaluate statistical significance for the data of Tables 3 and 5. RESULTS
I. Determination offemale developmental stages by ovarian development It was found that, in female M. rosenbergii, a pair of ovaries were fused at the anterior ends and were situated dorsally to the hepatopancreas. Immature ovaries were tiny transparent masses without coloration or with faint orange color overlain by two dark pigmented stripes. They could not be seen externally through the carapace, but they were visible dorsally at the connection between the cephalothorax and the first abdominal segment if the female was held with the cephalothorax bending downward. Developing ovaries became noticeable externally. They were bright orange and enlarged. The anterior ends extended toward the base of the last spine of the rostrum, and the posterior ends penetrated between the muscular masses in the first abdominal segment. Mature ovaries were bright orange and very large. The anterior ends reached the base of the second or third last spine of the rostrum; the posterior ends penetrated further. Ovarian development was then classified into four stages, stages 0, 1,2 and 3, based on coloration and the relative size of the ovaries to the size of the cephalothorax (see Fig. 1 and Table 1) . No seasonal variation in the ovarian development was observed. Similar percentages of females exhibited spawning each month (Table 2). Ovaries during the period leading to a regular molt were immature and were designated stage 0. Ovaries during the period leading to the last regular molt, the molt preceded a premating molt, were still immature but became faint orange and were designated stage 1. After stage 1, ovarian development progressed rapidly. It took an average of 18.5 + 10.9 s.d. days to enter stage 2 (Table 3 ), a stage in which developing ovarian masses began to be visible externally through the carapace. At the end of stage 2, an average of 9.6 + 4.0 s.d. days later (Table 3)) the anterior ends of the ovaries reached the base of TABLE 1 Coloration and the relative size ofthe ovaries in different developmental
stages
Stage
State of ovary
Coloration
Relative size
0 1 3
Immature Immature Developing
Clear, transparent Faint orange Bright orange
3
Mature
Bright orange
Small Small Large, extend anteriorly to the base of the last spine of the rostrum Very large, extend anteriorly to the base of the second or third last spine of the rostrum
350
Fig. 1. Diagram
P. DAMRONGPHOL ET AL.
0
1
2
3
showing the external
features of the ovaries in four different ovarian develop-
mental stages. TABLE 2 Percentages of laboratory-maintained to January 1990)
females which exhibited spawning each month (December
1988
Month
Year
Percentage of females exhibited spawning
No. of females exhibited spawning
Total no. of females
December January February March April May June July August September October November December January
1988 1989 1989 1989 1989 1989 1989 1989 1989 1989 1989 1989 1989 1990
50.0 40.0 45.1 33.3 44.8 30.8 51.5 41.9 32.0 36.0 52.5 41.7 40.0 45.0
11 10 16 8 13 8 17 18 8 9 21 10 14 9
22 25 35 24 29 26 33 43 25 25 40 24 35 20
the last spine of the rostrum. The ovaries grew furthur during stage 3. By the time of a premating molt, an average of 12.12 4.1 s-d. days later (Table 3 ), the ovaries were fully mature and marked the end of stage 3. The anterior ends of the ovaries reached the base of the second or the third last spine of
SPAWNING CYCLE OF GIANT FRESHWATER PRAWNS IN THE LABORATORY
351
TABLE 3 Intermolt periods between different types of molt Type of intermolt
dk s.d. (days ) Regular molt to regular molt Regular molt to last regular molt Last regular molt to premating molt Stage 1 to stage 2 Stage 2 to stage 3 Stage 3 to premating molt Premating molt to premating molt Berried female Unberried female Premating molt to regular molt Berried female Unberried female
No. of observations
Intermolt period Range (days)
30.9t
4.1
25-67
8
28.2f
5.3
21-43
5
9.8 10.9 4.0 4.1
27-56 8-42 3-22 7-22
14 12 37 61
35.2 k 10.2 38.0f 9.7 28.7& 8.2
21-59 28-59 21-59
34 24 10
26.82 27.0& 26.3+
22-31 22-3 1 26-27
10 7 3
38.7t 18.5& 9.6? 12.li
2.7 3.2 0.5
the rostrum. After spawning, the ovaries returned either to stage 0 or 1 depending on the type of the successive molt (see section III). Histological observations of the horizontal sections of the ovaries passing at the levels of the oviducts revealed that ovaries at each stage contained oocytes at various developmental stages. Ovaries in stage 0 were predominately filled with immature oocytes in early meiotic prophase and in previtellogenesis (Fig. 2a). Ovaries in stage 1 contained mainly oocytes in primary vitellogenesis in the central area and oocytes in secondary vitellogenesis at the periphery (Fig. 2b). Developing ovaries in stage 2 were occupied by many oocytes in secondary vitellogenesis (Fig. 2~). Mature ovaries in stage 3 were full of ripe eggs, but groups of immature oocytes were distributed around the central area of each ovary (Fig. 2d). II. Oocyte maturation Immature oocytes in previtellogenesis, about 40 pm diameter, were at the stage of pachytene of the first meiotic prophase (Fig. 3a). Oocytes in primary vitellogenesis, about 130 pm in diameter, were at the stage of diplotene (Fig. 3b). Oocytes in secondary vitellogenesis were still arrested at this stage. Most ripe eggs 1 day prior to a premating molt were advanced to the stage of diakinesis; the chromosomes were enclosed in a convoluted nucleoplasm (Fig. 3~). After a premating molt, some ripe eggs had undergone germinal vesicle
Fig. 2a. Horizontal section of the ovaries in stage 0. Each ovary contains predominately immature oocytes in early meiotic prophase (arrow) and in previtellogenesis. Scale bar= 200 pm. Fig. 2b. Horizontal section of the ovaries in stage 1. Most oocytes are in primary vitellogenesis; oocytes in secondary vitellogenesis (white arrow) are found at the periphery of each ovary. Scale bar = 200 /*m. Fig. 2c. Horizontal section of the ovaries in stage 2. Oocytes in secondary vitellogenesis are predominant; groups of oocytes in previtellogenesis and in primary vitellogenesis (arrow) are found in the central area of each ovary. Scale bar= 200 pm. Fig. 2d. Horizontal section of the ovaries in stage 3. Most oocytes are ripe and groups of immature oocytes (arrow ) are found in the central area of each ovary. Scale bar = 200 ,um.
SPAWNING
CYCLE
OF GIANT
FRESHWATER
PRAWNS
353
IN THE LABORATORY
c
e-'
,.
“,
Fig. 3a. Oocyte in previtellogenesis at the stage of pachytene. Scale bar = 10 pm. Fig. 3b. Oocyte in primary vitellogenesis at the stage of diplotene. Scale bar=20pm. Fig. 3c. Chromosomes at the stage of diakinesis in the ripe egg found 1 day prior to a premating molt. Scale bar= 20 pm. Fig. 3d. Chromosomes at the first meiotic metaphase in the mature egg found after a premating molt. Scale bar= 20 pm. Fig. 3e. Chromosomes at the first meiotic metaphase in the egg immediately spawned from the unmated female. Scale bar = IOpm.
breakdown and entered the first meiotic metaphase (Fig. 3d). About 7 h after a premating molt, most ripe eggs were at this mature chromosomal stage. Eggs immediately spawned from unmated females were also at the first meiotic metaphase (Fig. 3e). III. Molting and spawning cycles Various intermolt periods in the female giant freshwater prawns were listed in Table 3. The intermolt period between regular molts, which places stress
354
P. DAMRONGPHOL
ET AL.
on somatic growth, took 30.9 ? 4.1 s.d. days, whereas the intermolt period between the last regular molt and a premating molt, which emphasized ovarian growth, lasted 38.759.8 s.d. days. Moreover, the intermolt period between consecutive premating molts in berried females was about 10 days longer than that in unberried females. After spawning, the ovaries of reproductively active females returned to stage 1 and, thus, a premating molt followed, resulting in consecutive spawning. However, for some females, the ovaries were in stage 0 after spawning. Therefore, a regular molt followed. The general scheme of molting cycles is depicted in Fig. 4A. Relation of time span in the molt stages as described by Peebles ( 1977) and the ovarian stages described in the present report is shown in Fig. 4B. Most premating molts (51.4% of observations) and spawning (52.9% of A
B MOLT STAGES
ABC
DA
D ABC
D ABC
111 1111 1111 OVARIAN
STAGES
0
1
0
0
40
20
60
11 23
PM
100 80 TIME IN DAYS
Fig. 4. A. Scheme of molting cycles. B. Relation of time span in the ovarian and the molt stages. M, regular molt; ML, the last regular molt; PM, premating molt. TABLE 4 Percentages of observations riods of the day Event
Premating molt: Spawning: Mated female Unmated female
showing occurrence of a premating molt and spawning at different pe-
Percentage of observation
No. of observations
0.00-6.00 h
6.00-l 2.00 h
12.00-18.00 h
18.00-24.00 h
11.4 3.8 3.5 5.9
14.3 10.6 4.6 41.2
22.9 32.1 29.9 47.0
51.4 52.9 62.0 5.9
35 104 87 17
SPAWNING CYCLE OF GIANT FRESHWATER PRAWNS IN THE LABORATORY
355
TABLE 5 Hour intervals between a premating molt and spawning, and between mating and spawning State of female
Hour interval (2ks.d.)
No. of observations
Premating moltSpawning Mated female: Productive eggs Unproductive eggs Unmated female:
19.8k5.8 19.9+51. 20.6 t 7.4 40.0+ 5.4
Hour interval (8+ s.d.)
No. of observations
Mating-Spawning
30 20 10 5
9.6 * 5.4 8.5k4.1 14.2f7.3
87 70 17
observations) occurred during 18.00-24.00 h (Table 4). Spawning time in the mated female was relatively predictable; it was 19.8? 5.8 s.d. h after a premating molt (Table 5 ), while in unmated females, spawning time was relatively variable and delayed to 40.0 2 5.4 s.d. h after a premating molt (Table 5 ). Eggs spawned from unmated females were usually discarded within l-2 days. Most of the eggs spawned from mated females were productive, i.e., they developed to hatching. The rest, the unproductive eggs, were found to be discarded sometime during the course of development. DISCUSSION
Precise staging of female prawns is essential in selection of females for experimental purposes, for example, biochemical or physiological analysis. Determination of developmental stages of the female giant freshwater prawns by external examination of the ovarian maturity is feasible and reproducible. The number of days of each developmental stage in laboratory-maintained females can be predicted. During the phase of somatic growth, ovarian development is latent. Initiation of ovarian development or vitellogenesis is apparent after the last regular molt. It takes only one molt cycle from the last regular molt to a premating molt to complete vitellogenesis of each batch of the oocytes. During vitellogenesis, the ovaries gradually develop from stage 1 to stage 3. Despite the cytoplasmic growth of the oocytes during vitellogenesis, the chromosomes are arrested at diplotene of the first meiotic prophase. Oocyte maturation is detected by the convoluted nuclear envelope (Merriam, I96 1) and by progress in the chromosomal stage in the ripe eggs 1 day prior to a premating molt. Germinal vesicles break down and the chromosomes enter the first meiotic metaphase in some ripe eggs at about the time of a premating molt. Most eggs are at this mature stage by 7 h after a premating molt. These mature eggs are fertilizable since eggs immediately spawned from unmated females are also
356
P. DAMRONGPHOL
ET AL.
at the first meiotic metaphase. Meiotic resumption occurs upon fertilization or activation (Damrongphol et al., 199 1). Mature oocytes in natantians arrested at first meiotic metaphase have been reported in Sicyonia ingentis (Clark et al., 1984) and Paluemon serratus (Cledon, 1986). Most females in the present study undergo the other round of ovarian growth after spawning, resulting in consecutive spawning. The rest procede to somatic growth after spawning. Therefore, the ovarian stage after spawning differs in these two groups. The former is apparently in the physiological condition ready for ovarian growth, and it retains a batch of oocytes which has begun vitellogenesis as evidenced by the presence of stage-l ovaries. On the other hand, the latter places stress on somatic growth and the ovarian development is latent. Thus, the ovarian stage is variable after spawning. The actual stage of the ovaries must be known if the stage of the berried female is determined with reference to embryonic development. Moreover, intermolt period producing ovarian growth is longer than that producing somatic growth. More specifically, stage C in the molt stages, which is a stage of tissue growth and accumulation of food reserve (Drach, 1939), is extended. During the phase of ovarian growth, the energy or food reserve normally utilized solely for somatic growth is also spent on ovarian growth. Consequently, the period of food intake is lengthened. Intermolt between two consecutive premating molts is longer in berried than in unberried females. A comparable observation has been reported in M. nobilli. Removal of the embryos from the brooding mother increases frequencies of spawning in the following molt (Pandian and Balasundaram, 1982). The presence of the embryos may physically interfere with molting, or extra energy may be required during brooding. Active production of secretions, which are involved in antimicrobial activity (Fisher, 1983) or in synthesis of the embryonic envelopes, as well as preening activity (Bauer, 1979) during the brooding period, may require extra energy expenditure. Removal of the embryos in the berried females seems to reduce the energy burden utilized in brooding so that ovarian growth is enhanced. Without ovarian growth, energy or food reserves seem to be adequate for both somatic growth and support for brooding, since the intermolt period between a premating molt and the next regular molt does not differ in berried or unberried females. The effect of mating on the spawning time is apparent in the present study. Mating does not induce spawning per se (Chow et al., 1982; O’Donovan et al., 1984) but a relatively predictable spawning time is found in mated females, whereas spawning time is delayed and variable in unmated females. The physical action of mating, chemical induction from the male or the presence of the spermatophore may somehow stimulate the final stage of ovarian activity i.e., ovulation, prior to spawning. These speculations are subject to further investigation.
SPAWNING CYCLE OF GIANT FRESHWATER PRAWNS IN THE LABORATORY
357
ACKNOWLEDGEMENTS
We thank K. Vejsanit and A. Duangkaew for their help with figures and typing. This work was supported by the office of the Science and Technology Developmental Board, Grant No. DSN 88A-l-05 1 I 7.
REFERENCES Batter, R.T., 1979. Antifouling adaptations of marine shrimp (Decapoda: Caridea); gill cleaning mechanisms and grooming of brooded embryos. Zool. J. Linn. Sot., 65: 28 l-303. Chow, S., Ogasawara, Y. and Taki, Y., 1982. Male reproductive system and fertilization of the Palaemonid shrimp Macrobrachium rosenbergii. Bull. Jpn. Sot. Sci. Fish., 48: 177-l 83. Clark, W.H., Jr.. Yudin, AI., Griffin, F.J. and Shigekawa, K., 1984. The control of gamete activation and fertilization in the marine Penaeidae, Siqonia ingentia. In: W. Engels (Editor), Advances in Invertebrate Reproduction, 3. Elsevier, Amsterdam, pp. 459-472. Cledon, P. ,1986. Study on oocyte maturation and activation of the common prawn Palaemon serratus (Pennant): relationship between oocyte maturation and the molt cycle cytological aspects. Gamete Res., 13: 353-362. Damrongphol, P., Eangchuan, N. and Poolsanguan, B., 199 1. Chromosome behavior upon fertilization in eggs of the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Invert. Reprod. Devel., 19: 45-49. Drach, P., 1939. Mue et cycle d’intermue chez les crustaces decapodes. Ann. Inst. Oceanogr. Paris N.S., 19: 103-391. Fisher, W.S., 1983, Eggs of Palaemon macrodactylus. II. Association with aquatic bacteria. Biol. Bull., 164: 201-213. Ling. SW., 1969. The general biology and development of Macrobrachium rosenbergii (de Man ). FAO Fish. Rep., 57: 589-606. Merriam, R.W., 1961. Nuclear envelope structure during cell division in Chaetopterus eggs. Exp. Cell Res., 22: 93-107. O’Donovan, P., Abraham, M. and Cohen, D., 1984. The ovarian cycle during the intermoult in ovigerous Macrobrachium rosenbergii. Aquaculture, 36: 347-358. Pandian, T.J. and Balasundarum, C., 1982. Molting and spawning cycles in Macrobrachium nob&. Int. J. Invert. Reprod., 5: 21-30. Peebles, J.B., 1977. A rapid technique for molt staging in live Macrobrachium rosenbergii. Aquaculture, 12: 173- 180. Sagi, A. and Ra’anan, Z., 1985. Rapid identification of reproductive state and the receptive period of females in pond populations of Macrobrachium rosenbergii - a new technique. Aquaculture. 48: 361-367. Wickins, J.F. and Beard, T.W., 1974. Observations on the breeding and growth of the giant freshwater prawn Macrobrachium rosenbergii (de Man) in the laboratory. Aquaculture, 3: 159-174.