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The molt cycle and its hormonal control in Rhithropanopeus harrisii larvae
DEVELOPMENTAL
BIOLOGY
74, 479-485 (1980)
BRIEF
NOTES
The Molt Cycle and Its Hormonal Control in Rhithropanopeus Larvae JOHN A. FREEMANANDJOHN Duke University
Marine
Received March
Laboratory,
Beaufort,
harrisii
D. COSTLOW North
Carolina
28516
12, 1979; accepted in rerised fornr June 22. 1979
Stages of the zoeal and megalopal molt cycles of Rhithropanopeus harrisii were characterized by the appearance of epidermal cells in the spines and antennae. Eyestalk removal of the beginning of the last zoeal instar slightly accelerated the molt cycle. Fourth-instar larvae which had undergone eyestalk ablation during the third instar progressed through the molt cycle significantly faster than did control larvae. Eyestalkless megalopae also demonstrated an enhanced molting rate. The results suggested that the larval eyestalks contain a factor (molt-inhibiting hormone) which plays a role in controlling the molting rate.
stalk removal during the zoeal period did not significantly affect the molting rate of the zoeal or megalopal stages (Costlow, 1966a). In Sesarma reticulaturn, eyestalk removal early in the zoeal period resulted in an enhanced molting rate in the megalopal, but not the zoeal, instars (Costlow, 196613). Thus, while some indication of hormonal control of molting in the megalopal stage was found, eyestalk removal appeared to have no effect on the zoeal instars. Since a zoeal instar has a short duration (several days), it is possible that the frequency of monitoring the molting rate in the earlier studies (on a daily basis) was not often enough to detect the subtle alterations in molt cycle stage durations which may have occurred following eyestalk removal. In the present study, the rate at which R. harrisii zoeae and megalopae passed through each molt cycle stage was observed in order to determine, in greater detail, the role of eyestalk factors in the control of the molting rate during larval development.
INTRODUCTION
In marine crabs the postembryonic development consists of several zoeal stages, in which slight external morphological alterations are expressed at each ecdysis, and one megalopal stage, which is intermediate in form between the zoea and adult. Metamorphosis is completed at the next ecdysis, when the adult form is attained. Although many studies on crab larvae have dealt with external morphological development and environmental growth parameters, few studies have attempted to explain the control of molting and metamorphosis. In adult crustaceans, molting is under the influence of two hormones: ecdysterone, which accelerates molting; and molt-inhibiting hormone (MIH), which retards molting (see Passano, 1960; Vernet, 1976). Previous efforts to demonstrate the presence of the latter hormone, MIH, in crab larvae have, however, produced equivocal results. Costlow (1963) found that molting was accelerated in Callinectes sapidus megalopae when the eyestalks (the source of MIH) were removed within the first 24 hr of the megalopal instar. Eyestalk removal after this time failed to accelerate the molting rate. In similar studies with Rithropanopeus harrisii larvae, it was found that eye-
*MATERIALS
AND
METHODS
Larvae of Rhithropanopeus harrisii were hatched and maintained in filtered seawater at 25 ppt and 21°C under a light 479 0012-1606/80/020479-07$C12.00/0 Copyright 0 1980 by Academic I’rrss, Int. All rights of reproductmn in any form wsrrvrd
480
DEVELOPMENTAL BIOLOGY
regime of 12:12 (L:D) throughout the study. Under these conditions survival of the larvae through the four zoeal and one megalopal stages approached 90%. The duration of the four zoeal stages was 16 days. The larvae were fed freshly hatched Artemia and the water was changed daily. When a zoea molted to the megalopa it was removed from the mass culture bowl and maintained individually in a small compartmented plastic box. Animals that were to have the eyestalks removed were placed in a small glass disk and covered with just enough water to keep them moist, in part to prevent movement. An iris scalpel was used to sever the eyestalk at the articulating membrane. Immediately after eyestalk removal the larva was placed in a small culture bowl with fresh seawater without food. After 6 hr all dead larvae were removed and Artemia were added. About 50% of the eyestalkless animals died within this period. In every case the eyestalks were extirpated within the first 12-16 hr of the instar, during molt cycle stages B and C. The beginning of each molt cycle was designated as 12:00 midnight. Almost no molting was observed before this time, or after 05:OO AM. Molt cycle stage determinations were made three or four times a day. The criteria for the molt cycle stages are defined in the Results. To eliminate confusion between the stages of the molt cycle and the stages (instars) of larval development, each instar of the zoeal period will be referred to by the letter Z followed by the instar number, i.e., Z-3 or Z-4, and the word “stage” will be used to designate a unit of the molt cycle. RESULTS Larval
Molt Cycle
As a preliminary step in investigating the control of larval molting, the stages of the molt cycle were delineated using the morphological changes that occurred in the integument. The epidermis consists of a sin-
VOLUME 74.1980
gle layer of cells situated directly beneath a clear exoskeleton. These cells undergo size and shape changes throughout the molt cycle and are, thus, well suited for use in discerning the stages of the molt cycle. In addition, the epidermal events occur at approximately the same rate in all regions of the integument. As described previously for adult crustaceans (see Drach and Tchernigovtzeff, 1967; Skinner, 1962), the primary events which characterize the molt cycle stages include completion of the new exoskeleton during postmolt (stages A and B) and intermolt (stage C) and preparation for the next ecydsis (premolt, stage D). Separation of the epidermis from the old cuticle and secretion of the new exoskeleton are the primary events of premolt. Immediately following ecdysis to the fourth instar, the exoskeleton was very thin and the spines were flexible (stages A and B). The epidermis was enlarged and the cuticle was not completely expanded in some regions of the spines. As the larva approached intermolt (stage C!), the secretion of the endocuticle was completed, the spines became straight and rigid, and the epidermis decreased in size. The postmolt stages normally occupied the first 12-15 hr of each molt cycle. During stage C the epidermal cells regressed in size to a squamous shape and appeared as a thin line below the exoskeleton (Fig. 1). Stage C usually had a duration of 40-45 hr. The beginning of premolt (Stage Do) was signaled by an enlargement of the epidermis from a squamous to a cuboidal shape (Fig. 2). The increase in size occurred either in all the cells simultaneously or in patches of cells along the spine or appendage. Furthermore, a slight separation of the epiderma1 matrix from the cuticle, the initial stage of setagenesis, was seen at the base of the setae in the maxillipeds. Stage Do normally lasted about 15-20 hr. The increase in epidermal cell size was completed during stage D, and in many regions the cells were clearly separated from the cuticle. Stage D1
FIGS. 1-4. Appearance
of the epidermis along the proximal region of the dorsal spine of fourth-instar (zoea) during different stages of the molt cycle. c, cuticle; e. epidermts; nc, new cuticle. Fig. I. Stage C, x 750; Fig. 2, Stage Do, x 750; Fig. 3, Stage Dz, x 750; Fig. 4, Stage D,, x 3X
Rhithropanopeus harrisii
481
482
DEVELOPMENTAL BIOLOGY
normally lasted 20-30 hr. By the beginning of stage Dz the epidermis was retracted from the exoskeleton along the spines and antennae (Fig. 3). The new cuticle was visible and the epidermis began to acquire a pleated appearance due to the increased area of the new integument. During stage Da the epidermis reached its greatest retraction from the old exoskeleton (Fig. 4) and, in some regions, the degeneration of the old exoskeleton was evident. Stages Dz and Ds together accounted for the final 3040 hr of the molt cycle. Further subdivision of premolt into stage Dq, the brief period immediately preceding ecdysis, was not done. The criteria for the molt cycle of the megalopae were the same as those outlined for the zoeal instars. However, because the
VOLUME 74,198O
increased thickness and opacity of the cutitle in many regions of the integument hindered direct observation, only the epidermis and setae in the telson were used to determine the stages of the molt cycle. The molt cycle of the megalopal instar was more variable in duration than that of the zoeal instars and increased in length by 50%, compared to the fourth-instar duration. Stages Do and D1 lasted about 50 hr, while stages Dz and DS had a combined duration of about 40 hr. Effect of Eyestalk
Removal
In the first set of experiments the progression of the molt cycle of intact and eyestalkless fourth-instar zoeae was followed (Fig. 5). The eyestalkless larvae (Z4) reached the premolt stages DC,and Dz in
I 140
Zxea-
I
14il
IV
l t
-
12L
-
100
- 80
- 4u
- 20
-0
c
2-4
z-
Ecdysls
FIG. 5. Comparison of the period (hours) from ecdysis to molt cycle stages Do, D2, and the next ecdysis in fourth-intar zoeae. C, intact controls; Z-4, stage 4 zoeae with eyestalks removed during the first 12-16 hr of fourth instar; Z-3, stage 4 zoeae which had undergone eyestalk removal during the third instar. Each bar represents the mean -t 95% confidence limits of 15-30 larvae.
483
BRIEF NOTES
a shorter period of time than did the control animals. Both groups molted to the megalopal instar at about the same time. Fourthinstar larvae which had survived eyestalk removal in the third instar (Z-3, Fig. 5) reached stages Do and Dz in a much shorter period of time than did either the control larvae or the larvae which had been operated on at the beginning of the fourth instar. In addition, these animals molted to megalopae in a significantly shorter (P < 0.05) period of time than the control larvae. It is evident from these results that the effect of eyestalk removal on the molting rate of fourth-instar larvae was expressed to a much greater degree when the eyestalks were removed during the previous instar. The effect of eyestalk removal on molting in megalopae is shown in Fig. 6. When eyestalks were removed on the first day of the megalopal instar, the interval from ec-
dysis to the premolt stages, and to the next ecdysis, was decreased. Megalopae, which had molted from larvae operated on in the third or fourth instars (Z-3 and Z-4), advanced to stages Do and D2 in a considerably shorter period of time than did the intact control larvae. In fact several of the megalopae in the Z-3 and Z-4 groups molted to the first crab instar before the control larvae had entered premolt. These results indicate that, in the absence of molt-inhibiting hormone from the eyestalks, the molt cycle of the megalopa was accelerated. DISCUSSION
Based on the staging method used in this study, it was found that the zoeae of Rhithropanopeus harrisii undergo a diecdysic type of molt cycle. The diecdysic molt cycle is characterized by a relatively short intermolt period and a long premolt period (Knowles and Carlisle, 1956) and is most
1
Mega lapa
T L
f
A.
FIG. 6. Comparison of the period (hours) from ecdysis to molt cycle stages I>,,. Di. and the next ecdysis in megalopae. C, intact controls; M, megalopae with eyestalks removed during the first 1%hr of megalopal instar: Z-4 and Z-3, megalopae which had undergone eyestalk removal during the fourth or third nwal instars, respective1.v. Each bar represents the mean -C 95 “P confidence limits of 10-30 larvae.
484
DEVELOPMENTALBIOLOGY
often observed in rapidly molting crustaceans. Crustaceans which have a slower molting rate, such as adult crabs, usually have an anecdysic molt cycle, typified by a predominant intermolt period and a relatively short premolt period. In the R. harrisii megalopae observed in this study, it was found that the duration of stage C was approximately one-half of the molt cycle (compared to one-third in zoeae), which is indicative of an anecdysic molt cycle. Thus, it is possible that the transition from the rapidly molting zoea to the slowly molting adult in R. harrisii begins during the megalopal instar. Studies with larvae of several species of decapod crustaceans have demonstrated that the eyestalks of developing larvae contain the neuroendocrine centers which are capable of controlling several physiological processes (see Costlow, 1968; Jacques, 1975). The role played by the eyestalks in larval molting has been more difficult to ascertain. Eyestalk removal had no apparent abrupt effect of the molting rate of the larvae of two species of shrimp (Hubschman, 1963; Little, 1969), and produced variable results in several species of crabs (Costlow, 1963, 1966a, b). In the present research it was observed that the zoeal molt cycle was only slightly shortened when the eyestalks were removed at the beginning of the molt cycle. Since MIH may be present in the hemolymph, and thus controlling molting, only during postmolt, eyestalk removal may only shorten the initial stages of the molt cycle. Zoeae which had undergone eyestalk extirpation during the previous instar (Z-3) exhibited a decreased intermolt stage (C), as well as an enhanced molting rate. It is possible, therefore, that a sufficient quantity of MIH had been released during the first 12 hr of the fourth instar to have effectively regulated the intermolt duration after eyestalk removal. In this study, the greatest effect of eyestalk removal was observed in the megalopae. The molt cycle was significantly
VOLUME 74.1980
shortened when eyestalk removal was done on the first day of the megalopal instar. As this effect was not observed in the zoeae (see Fig. 5,Z-4), it may be possible that the increased intermolt period (stage C) in the megalopa may be due to enhanced MIH secretion. Elimination of MIH early in the molt cycle would, thus, noticeably shorten the time from ecdysis to premolt. Larvae entering the megalopal period without eyestalks also showed a significant reduction in the time from ecdysis to premolt and the duration of the molt cycle, which is similar to the results obtained with the zoeae. Although these megalopae progressed through the molt cycle slightly faster than megalopae which had undergone eyestalk extirpation during the beginning of the megalopal period, the difference was not significant. The results of these experiments with both the zoeae and the megalopae clearly demonstrate that the larval eyestalks contain a factor which functions to control the molting rate during larval development. The apparent differences between the observations made in this study and those of earlier studies on R. harrisii larvae (Costlow, 1966a) could be explained by the different means employed to monitor the molting rate in animals following eyestalk removal. In the present study, the rate of progression of the larvae through each molt cycle stage was followed in addition to observing the total molt cycle duration, whereas, in the earlier study, only the total length of the zoeal period was measured. By increasing the frequency of the observations, a more accurate determination of the effect of eyestalk removal can be obtained. Another possible reason for the different findings may be the temperature at which the larvae were raised during the experimental period. Larvae in the earlier studies developed at 25°C while in thi:, study the animals were maintained at 21” C By increasing the temperature to 25”C, the length of the zoeal development is mark-
BRIEF NOTES
edly shortened (Costlow et al., 1966). This mav have the effect of eliminating. or obscuring, the differences in molting rates between control and eyestalkless animals. The authors wish to thank Mr. Terry L. West and Dr. David R. McClay for critically reviewing the manuscript. This work was supported by Contract No. NR-104-194 from the Office of Naval Research. REFERENCES COSTLOW, J. D., JR. (1963). The effect of eyestalk extirpation on metamorphosis of megalops of the blue crab, Callinectes supidus Rathbun. Gen. pomp. Endocrinol. 3, 120-130. COSTLOW, J. D., JR. (1966a). The effect of eyestalk extirpation on larval development of the mud crab, Rhithropanopeus harrisii (Gould). Gen. Comp. Endocrinol. 7, 255-274. COSTLOW, J. D., JR. (1966b). The effect of eyestalk extirpation on larval development of the crab, Sesarma reticulatum Say. In “Some Contemporary Studies in Marine Science” (H. Barnes, ed.), pp. 209-224. Appleton-Century-Crofts, New York. COSTLOW, J. D., JR. (1968). Metamorphosis in crustaceans. In “Metamorphosis: A Problem in Developmental Biology” (W. Etkin and L. I. Gilbert, eds.), pp. 3-41. Appleton-Century-Crofts, New York. COSTLOW, J. D., JR., BOOKHOUT, C. G., and MONROE, R. J. (1966). Studies on the larval development of
485
the crab, Rhithropanopeus harrisii (Gould) I. The effect of salinity and temperature on larval development. Physiol. Zool. 38,81-100. DRACH, P., and TCHERNIGOVTZEFF, C. (1967). Sur la methode de determination des stades d’intermue et son application generale aux crustaces. Vie MilLeu Ser A l&595-610. HUBSCHMAN, J. H. (1963). Development and function of neurosecretory sites in the eyestalks of larval Palaemonetes (Decapoda:Natantia). Biol. Bull. 125,96-113. JACQUES, F. (1975). Decouverte de la glande du sinus chez la larve de Squilla mantis (stade I) (Crustaces, Stomatopodes). Ultrastructure. C. R. Acad. Sci. Paris 280, 1575-1577. KNOWLES, F. G. W., and CARLISLE, D. B. (1956). Endocrine control in the Crustacea. Biol. Ret). 31, 396-473. LITTLE, G. (1969). The larval development of the shrimp, Palaemon macrodactylus Rathbun, reared in the laboratory, and the effect of eyestalk extirpation on development. Crustaceana 17,69-87. PASSANO, L. M. (1960). Molting and its control. In “Physiology of Crustacea” (T. H. Waterman, ed.), Vol. 1, pp. 473-536. Academic Press, New York. SKINNER, D. M. (1962). The structure and metabolism of a crustacean integumentary tissue during a molt cycle. Biol. Bull. 123, 635-647. VERNET, G. (1976). Don&es actuelles sur le determinisme de la mue chez les crustaces. Ann. Biol. 15. 115-188.
BIOLOGY
74, 479-485 (1980)
BRIEF
NOTES
The Molt Cycle and Its Hormonal Control in Rhithropanopeus Larvae JOHN A. FREEMANANDJOHN Duke University
Marine
Received March
Laboratory,
Beaufort,
harrisii
D. COSTLOW North
Carolina
28516
12, 1979; accepted in rerised fornr June 22. 1979
Stages of the zoeal and megalopal molt cycles of Rhithropanopeus harrisii were characterized by the appearance of epidermal cells in the spines and antennae. Eyestalk removal of the beginning of the last zoeal instar slightly accelerated the molt cycle. Fourth-instar larvae which had undergone eyestalk ablation during the third instar progressed through the molt cycle significantly faster than did control larvae. Eyestalkless megalopae also demonstrated an enhanced molting rate. The results suggested that the larval eyestalks contain a factor (molt-inhibiting hormone) which plays a role in controlling the molting rate.
stalk removal during the zoeal period did not significantly affect the molting rate of the zoeal or megalopal stages (Costlow, 1966a). In Sesarma reticulaturn, eyestalk removal early in the zoeal period resulted in an enhanced molting rate in the megalopal, but not the zoeal, instars (Costlow, 196613). Thus, while some indication of hormonal control of molting in the megalopal stage was found, eyestalk removal appeared to have no effect on the zoeal instars. Since a zoeal instar has a short duration (several days), it is possible that the frequency of monitoring the molting rate in the earlier studies (on a daily basis) was not often enough to detect the subtle alterations in molt cycle stage durations which may have occurred following eyestalk removal. In the present study, the rate at which R. harrisii zoeae and megalopae passed through each molt cycle stage was observed in order to determine, in greater detail, the role of eyestalk factors in the control of the molting rate during larval development.
INTRODUCTION
In marine crabs the postembryonic development consists of several zoeal stages, in which slight external morphological alterations are expressed at each ecdysis, and one megalopal stage, which is intermediate in form between the zoea and adult. Metamorphosis is completed at the next ecdysis, when the adult form is attained. Although many studies on crab larvae have dealt with external morphological development and environmental growth parameters, few studies have attempted to explain the control of molting and metamorphosis. In adult crustaceans, molting is under the influence of two hormones: ecdysterone, which accelerates molting; and molt-inhibiting hormone (MIH), which retards molting (see Passano, 1960; Vernet, 1976). Previous efforts to demonstrate the presence of the latter hormone, MIH, in crab larvae have, however, produced equivocal results. Costlow (1963) found that molting was accelerated in Callinectes sapidus megalopae when the eyestalks (the source of MIH) were removed within the first 24 hr of the megalopal instar. Eyestalk removal after this time failed to accelerate the molting rate. In similar studies with Rithropanopeus harrisii larvae, it was found that eye-
*MATERIALS
AND
METHODS
Larvae of Rhithropanopeus harrisii were hatched and maintained in filtered seawater at 25 ppt and 21°C under a light 479 0012-1606/80/020479-07$C12.00/0 Copyright 0 1980 by Academic I’rrss, Int. All rights of reproductmn in any form wsrrvrd
480
DEVELOPMENTAL BIOLOGY
regime of 12:12 (L:D) throughout the study. Under these conditions survival of the larvae through the four zoeal and one megalopal stages approached 90%. The duration of the four zoeal stages was 16 days. The larvae were fed freshly hatched Artemia and the water was changed daily. When a zoea molted to the megalopa it was removed from the mass culture bowl and maintained individually in a small compartmented plastic box. Animals that were to have the eyestalks removed were placed in a small glass disk and covered with just enough water to keep them moist, in part to prevent movement. An iris scalpel was used to sever the eyestalk at the articulating membrane. Immediately after eyestalk removal the larva was placed in a small culture bowl with fresh seawater without food. After 6 hr all dead larvae were removed and Artemia were added. About 50% of the eyestalkless animals died within this period. In every case the eyestalks were extirpated within the first 12-16 hr of the instar, during molt cycle stages B and C. The beginning of each molt cycle was designated as 12:00 midnight. Almost no molting was observed before this time, or after 05:OO AM. Molt cycle stage determinations were made three or four times a day. The criteria for the molt cycle stages are defined in the Results. To eliminate confusion between the stages of the molt cycle and the stages (instars) of larval development, each instar of the zoeal period will be referred to by the letter Z followed by the instar number, i.e., Z-3 or Z-4, and the word “stage” will be used to designate a unit of the molt cycle. RESULTS Larval
Molt Cycle
As a preliminary step in investigating the control of larval molting, the stages of the molt cycle were delineated using the morphological changes that occurred in the integument. The epidermis consists of a sin-
VOLUME 74.1980
gle layer of cells situated directly beneath a clear exoskeleton. These cells undergo size and shape changes throughout the molt cycle and are, thus, well suited for use in discerning the stages of the molt cycle. In addition, the epidermal events occur at approximately the same rate in all regions of the integument. As described previously for adult crustaceans (see Drach and Tchernigovtzeff, 1967; Skinner, 1962), the primary events which characterize the molt cycle stages include completion of the new exoskeleton during postmolt (stages A and B) and intermolt (stage C) and preparation for the next ecydsis (premolt, stage D). Separation of the epidermis from the old cuticle and secretion of the new exoskeleton are the primary events of premolt. Immediately following ecdysis to the fourth instar, the exoskeleton was very thin and the spines were flexible (stages A and B). The epidermis was enlarged and the cuticle was not completely expanded in some regions of the spines. As the larva approached intermolt (stage C!), the secretion of the endocuticle was completed, the spines became straight and rigid, and the epidermis decreased in size. The postmolt stages normally occupied the first 12-15 hr of each molt cycle. During stage C the epidermal cells regressed in size to a squamous shape and appeared as a thin line below the exoskeleton (Fig. 1). Stage C usually had a duration of 40-45 hr. The beginning of premolt (Stage Do) was signaled by an enlargement of the epidermis from a squamous to a cuboidal shape (Fig. 2). The increase in size occurred either in all the cells simultaneously or in patches of cells along the spine or appendage. Furthermore, a slight separation of the epiderma1 matrix from the cuticle, the initial stage of setagenesis, was seen at the base of the setae in the maxillipeds. Stage Do normally lasted about 15-20 hr. The increase in epidermal cell size was completed during stage D, and in many regions the cells were clearly separated from the cuticle. Stage D1
FIGS. 1-4. Appearance
of the epidermis along the proximal region of the dorsal spine of fourth-instar (zoea) during different stages of the molt cycle. c, cuticle; e. epidermts; nc, new cuticle. Fig. I. Stage C, x 750; Fig. 2, Stage Do, x 750; Fig. 3, Stage Dz, x 750; Fig. 4, Stage D,, x 3X
Rhithropanopeus harrisii
481
482
DEVELOPMENTAL BIOLOGY
normally lasted 20-30 hr. By the beginning of stage Dz the epidermis was retracted from the exoskeleton along the spines and antennae (Fig. 3). The new cuticle was visible and the epidermis began to acquire a pleated appearance due to the increased area of the new integument. During stage Da the epidermis reached its greatest retraction from the old exoskeleton (Fig. 4) and, in some regions, the degeneration of the old exoskeleton was evident. Stages Dz and Ds together accounted for the final 3040 hr of the molt cycle. Further subdivision of premolt into stage Dq, the brief period immediately preceding ecdysis, was not done. The criteria for the molt cycle of the megalopae were the same as those outlined for the zoeal instars. However, because the
VOLUME 74,198O
increased thickness and opacity of the cutitle in many regions of the integument hindered direct observation, only the epidermis and setae in the telson were used to determine the stages of the molt cycle. The molt cycle of the megalopal instar was more variable in duration than that of the zoeal instars and increased in length by 50%, compared to the fourth-instar duration. Stages Do and D1 lasted about 50 hr, while stages Dz and DS had a combined duration of about 40 hr. Effect of Eyestalk
Removal
In the first set of experiments the progression of the molt cycle of intact and eyestalkless fourth-instar zoeae was followed (Fig. 5). The eyestalkless larvae (Z4) reached the premolt stages DC,and Dz in
I 140
Zxea-
I
14il
IV
l t
-
12L
-
100
- 80
- 4u
- 20
-0
c
2-4
z-
Ecdysls
FIG. 5. Comparison of the period (hours) from ecdysis to molt cycle stages Do, D2, and the next ecdysis in fourth-intar zoeae. C, intact controls; Z-4, stage 4 zoeae with eyestalks removed during the first 12-16 hr of fourth instar; Z-3, stage 4 zoeae which had undergone eyestalk removal during the third instar. Each bar represents the mean -t 95% confidence limits of 15-30 larvae.
483
BRIEF NOTES
a shorter period of time than did the control animals. Both groups molted to the megalopal instar at about the same time. Fourthinstar larvae which had survived eyestalk removal in the third instar (Z-3, Fig. 5) reached stages Do and Dz in a much shorter period of time than did either the control larvae or the larvae which had been operated on at the beginning of the fourth instar. In addition, these animals molted to megalopae in a significantly shorter (P < 0.05) period of time than the control larvae. It is evident from these results that the effect of eyestalk removal on the molting rate of fourth-instar larvae was expressed to a much greater degree when the eyestalks were removed during the previous instar. The effect of eyestalk removal on molting in megalopae is shown in Fig. 6. When eyestalks were removed on the first day of the megalopal instar, the interval from ec-
dysis to the premolt stages, and to the next ecdysis, was decreased. Megalopae, which had molted from larvae operated on in the third or fourth instars (Z-3 and Z-4), advanced to stages Do and D2 in a considerably shorter period of time than did the intact control larvae. In fact several of the megalopae in the Z-3 and Z-4 groups molted to the first crab instar before the control larvae had entered premolt. These results indicate that, in the absence of molt-inhibiting hormone from the eyestalks, the molt cycle of the megalopa was accelerated. DISCUSSION
Based on the staging method used in this study, it was found that the zoeae of Rhithropanopeus harrisii undergo a diecdysic type of molt cycle. The diecdysic molt cycle is characterized by a relatively short intermolt period and a long premolt period (Knowles and Carlisle, 1956) and is most
1
Mega lapa
T L
f
A.
FIG. 6. Comparison of the period (hours) from ecdysis to molt cycle stages I>,,. Di. and the next ecdysis in megalopae. C, intact controls; M, megalopae with eyestalks removed during the first 1%hr of megalopal instar: Z-4 and Z-3, megalopae which had undergone eyestalk removal during the fourth or third nwal instars, respective1.v. Each bar represents the mean -C 95 “P confidence limits of 10-30 larvae.
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DEVELOPMENTALBIOLOGY
often observed in rapidly molting crustaceans. Crustaceans which have a slower molting rate, such as adult crabs, usually have an anecdysic molt cycle, typified by a predominant intermolt period and a relatively short premolt period. In the R. harrisii megalopae observed in this study, it was found that the duration of stage C was approximately one-half of the molt cycle (compared to one-third in zoeae), which is indicative of an anecdysic molt cycle. Thus, it is possible that the transition from the rapidly molting zoea to the slowly molting adult in R. harrisii begins during the megalopal instar. Studies with larvae of several species of decapod crustaceans have demonstrated that the eyestalks of developing larvae contain the neuroendocrine centers which are capable of controlling several physiological processes (see Costlow, 1968; Jacques, 1975). The role played by the eyestalks in larval molting has been more difficult to ascertain. Eyestalk removal had no apparent abrupt effect of the molting rate of the larvae of two species of shrimp (Hubschman, 1963; Little, 1969), and produced variable results in several species of crabs (Costlow, 1963, 1966a, b). In the present research it was observed that the zoeal molt cycle was only slightly shortened when the eyestalks were removed at the beginning of the molt cycle. Since MIH may be present in the hemolymph, and thus controlling molting, only during postmolt, eyestalk removal may only shorten the initial stages of the molt cycle. Zoeae which had undergone eyestalk extirpation during the previous instar (Z-3) exhibited a decreased intermolt stage (C), as well as an enhanced molting rate. It is possible, therefore, that a sufficient quantity of MIH had been released during the first 12 hr of the fourth instar to have effectively regulated the intermolt duration after eyestalk removal. In this study, the greatest effect of eyestalk removal was observed in the megalopae. The molt cycle was significantly
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shortened when eyestalk removal was done on the first day of the megalopal instar. As this effect was not observed in the zoeae (see Fig. 5,Z-4), it may be possible that the increased intermolt period (stage C) in the megalopa may be due to enhanced MIH secretion. Elimination of MIH early in the molt cycle would, thus, noticeably shorten the time from ecdysis to premolt. Larvae entering the megalopal period without eyestalks also showed a significant reduction in the time from ecdysis to premolt and the duration of the molt cycle, which is similar to the results obtained with the zoeae. Although these megalopae progressed through the molt cycle slightly faster than megalopae which had undergone eyestalk extirpation during the beginning of the megalopal period, the difference was not significant. The results of these experiments with both the zoeae and the megalopae clearly demonstrate that the larval eyestalks contain a factor which functions to control the molting rate during larval development. The apparent differences between the observations made in this study and those of earlier studies on R. harrisii larvae (Costlow, 1966a) could be explained by the different means employed to monitor the molting rate in animals following eyestalk removal. In the present study, the rate of progression of the larvae through each molt cycle stage was followed in addition to observing the total molt cycle duration, whereas, in the earlier study, only the total length of the zoeal period was measured. By increasing the frequency of the observations, a more accurate determination of the effect of eyestalk removal can be obtained. Another possible reason for the different findings may be the temperature at which the larvae were raised during the experimental period. Larvae in the earlier studies developed at 25°C while in thi:, study the animals were maintained at 21” C By increasing the temperature to 25”C, the length of the zoeal development is mark-
BRIEF NOTES
edly shortened (Costlow et al., 1966). This mav have the effect of eliminating. or obscuring, the differences in molting rates between control and eyestalkless animals. The authors wish to thank Mr. Terry L. West and Dr. David R. McClay for critically reviewing the manuscript. This work was supported by Contract No. NR-104-194 from the Office of Naval Research. REFERENCES COSTLOW, J. D., JR. (1963). The effect of eyestalk extirpation on metamorphosis of megalops of the blue crab, Callinectes supidus Rathbun. Gen. pomp. Endocrinol. 3, 120-130. COSTLOW, J. D., JR. (1966a). The effect of eyestalk extirpation on larval development of the mud crab, Rhithropanopeus harrisii (Gould). Gen. Comp. Endocrinol. 7, 255-274. COSTLOW, J. D., JR. (1966b). The effect of eyestalk extirpation on larval development of the crab, Sesarma reticulatum Say. In “Some Contemporary Studies in Marine Science” (H. Barnes, ed.), pp. 209-224. Appleton-Century-Crofts, New York. COSTLOW, J. D., JR. (1968). Metamorphosis in crustaceans. In “Metamorphosis: A Problem in Developmental Biology” (W. Etkin and L. I. Gilbert, eds.), pp. 3-41. Appleton-Century-Crofts, New York. COSTLOW, J. D., JR., BOOKHOUT, C. G., and MONROE, R. J. (1966). Studies on the larval development of
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the crab, Rhithropanopeus harrisii (Gould) I. The effect of salinity and temperature on larval development. Physiol. Zool. 38,81-100. DRACH, P., and TCHERNIGOVTZEFF, C. (1967). Sur la methode de determination des stades d’intermue et son application generale aux crustaces. Vie MilLeu Ser A l&595-610. HUBSCHMAN, J. H. (1963). Development and function of neurosecretory sites in the eyestalks of larval Palaemonetes (Decapoda:Natantia). Biol. Bull. 125,96-113. JACQUES, F. (1975). Decouverte de la glande du sinus chez la larve de Squilla mantis (stade I) (Crustaces, Stomatopodes). Ultrastructure. C. R. Acad. Sci. Paris 280, 1575-1577. KNOWLES, F. G. W., and CARLISLE, D. B. (1956). Endocrine control in the Crustacea. Biol. Ret). 31, 396-473. LITTLE, G. (1969). The larval development of the shrimp, Palaemon macrodactylus Rathbun, reared in the laboratory, and the effect of eyestalk extirpation on development. Crustaceana 17,69-87. PASSANO, L. M. (1960). Molting and its control. In “Physiology of Crustacea” (T. H. Waterman, ed.), Vol. 1, pp. 473-536. Academic Press, New York. SKINNER, D. M. (1962). The structure and metabolism of a crustacean integumentary tissue during a molt cycle. Biol. Bull. 123, 635-647. VERNET, G. (1976). Don&es actuelles sur le determinisme de la mue chez les crustaces. Ann. Biol. 15. 115-188.