Regeneration of Walking Legs in the Fiddler Crab Uca pugilator

AMER. ZOOL., 33:348-356 (1993)

Regeneration of Walking Legs in theFiddler Crab Uca pugilator1

PENNY M. HOPKINS

Department of Zoology, University of Oklahoma, Norman, Oklahoma 73019

SYNOPSIS. Regeneration of walking legs in the fiddler crab Uca pugilatoris most efficient when it follows autotomy (the reflexive loss of a limb).Closure of the wound and would healing occur immediately followingautotomy and visible regeneration begins within a few days. Regenerationof the walking leg occurs in two distinct stages: The first stage, calledBasal Growth, involves mitosis and differentiation. The second stageinvolves primarily protein synthesis and water uptake and is called Proec-dysial Growth. Proecdysial Growth is, in part, under direct hormonalstimulation.

INTRODUCTION

Growth in arthropods is defined anddelimited by their rigid exoskeleton.Nowhere is this more obvious than in thecrustaceans where the chitinous exoskeletonis frequently reinforced with calcium salts.In order to grow, crustaceans must shed theold, hard exoskeleton and expand a newlysynthesized, soft exoskeleton. The result ofthis shedding (or ecdysis) and expansion isa larger animal. The growth pattern of acrustacean, therefore, is abrupt and episodicand resembles a rising staircase more thanthe standard "S-shaped" growth curve asso-ciated with many growth phenomena. Mostcrustaceans continue to molt and growthroughout the adult stages resulting in agrowth pattern that contains many "risers."Moreover, general body growth in crusta-ceans can be accompanied by another spe-cial growth associated with the regenerationof lost or damaged limbs.

Regeneration of limbs in crustaceans ismost efficient when it follows the involun-tary event called autotomy. Autotomy of aninjured limb was first described by Fred-ericq in 1882. Autotomy is a reflexiveresponse to injury that results in the castingoff of an injured limb at a predeterminedpoint proximal to the injury. Autotomy and

' From the Symposium on Hormonal Control ofGrowth and Reproduction in A rthropods of the Divisionof Comparative Endocrinology presented at the AnnualMeeting of the American Society of Zoologists, 27-30December 1991, at Atlanta, Georgia.

regeneration in crustaceans has captured theinterest and imagination of many scientistsbeginning with Aristotle and including suchluminaries as Charles Darwin (1854), andT. H. Morgan (1900, 1902, 1904). Forreviews see Skinner (1985) and Hopkins(1987).

MATERIALS AND METHODS

Most of the methods used in this paper(RIA, HPLC and in vitro limb bud incu-bations) have been described in detail else-where (Hopkins, 1982, 1986, 1989a). Limbbud proteins were extracted and precipi-tated overnight in ice cold 10% TCA. Fol-lowing centrifugation, the pellet wasextracted 4 times with 95% ethanol, twicewith acetone and once with ether. Total pro-tein was determined using a modified Lowrymethod (Markwell et ai, 1978). Other sam-ples were dissolved in sample buffer andheated for 5 min in a boiling water bath.These samples were applied to SDS-PAGEslab gels and separated in 1 dimension in10% acrylamide (25 ng of protein per lane).Standard molecular weight markers wereobtained from BioRad. Gels were fixed withacetic acid and methanol. Each lane was cutin half lengthwise and one half was extractedwith 30% H2O2 for counting. The other halfwas stained with silver stain (BioRad).

Entire coxae were removed prior to autot-omy and at various times following autot-omy. The coxa was fixed in 5% glutaral-dehyde for 4 hr and postfixed in OsO4 for2 hr. The entire coxa was embedded in gly-

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REGENERATION IN CRABS 349

col methacrylate. Thick sections (3-5were cut and stained with methylene blueand viewed with the light microscope. Thinsections (0.5 nm) were stained with leadcitrate and viewed with transmission elec-tron microscopy (Zeiss EM 9).

REGENERATION IN CRUSTACEANS

AutotomyThe best understood type of regeneration

in crustaceans is the regeneration that fol-lows autotomy. The autotomy reflexinvolves specialized autotomy muscles (seeMcVean, 1984) that insert on the proximaledge of the basioischiopodite (BI) segmentof each walking leg. The most crucial muscleto the autotomy reflex is a rotating levatormuscle which, when stimulated, switches thetension exerted by muscles that are nor-mally involved in walking so that the BI ispulled under and up against the edge of thecoxa. The pressure of the coxa upon the BIcauses the BI to rupture along a preformedline of weakened cuticle (Findley andMcVean, 1977).

Immediately proximal to the preformedfracture plane is a connective tissue mem-brane that spans the entire limb base anddivides the hemocoelic cavity between theBI and coxa. In Uca it is a double membranewith folded edges that surround the pedalnerve and blood vessels that pass from thebody to the leg (Fig. la). Upon autotomy,half of the membrane is torn away with theshed limb and half remains behind. Follow-ing autotomy the remaining membraneunfolds and acts like a valve: blood pressurein the body of the animal distends the mem-brane so that it expands outwardly fillingthe gap left by the autotomized limb. Thebulbous edges that once surrounded thepedal nerve swell to fill the hole left by thenerve and blood vessels. This type of imme-diate closure assures that there is very littleblood loss and minimal bacterial invasionwhen a limb is lost. Another advantage toautotomy is that relatively small amountsof tissue are damaged. No muscles extendacross the plane of fracture so there is nomuscle damage at the point of autotomy.The only tissue damage is to the nerve andblood vessels that pass from the body,

through the autotomy membrane and intothe limb (Fig. la). Because there is so littletissue damage, there is little phagocyticactivity following autotomy.

Blastema formation and basal growthAfter autotomy and the closure of the

breach by the autotomy membrane, thereis a rapid influx of granulocyte and blasto-cyte cells (Fig. 1 b). The granulocytes imme-diately degranulate and it appears that thecontents of the granules initiate the for-mation of a scab below the distended autot-omy membrane (Fig. lc). There is a contin-uous immigration of granulocytes andblastocytes into the coxal stump (Hopkinsand Mislan, 1986). These cells enter thestump by migrating up the severed pedalnerve or by moving up the coxal walls andout under the scab (Fig. Id). The cut end ofthe pedal nerve also becomes covered withgranulocytes and blastocytes. Moreover,there is an elongation and movement of theepidermal cells that line the sides of the coxaunder the scab.

Immigration of granulocytes, blastocytesand epidermal cells begins within 30 min-utes of autotomy and within the next fewhours, three distinct layers are evident inthe stump. The first layer is the degranulatedgranulocytes and blastocytes; the secondlayer is a loose assembledge of cells (Fig. lb)that eventually becomes filled with the blas-tema; and the third is a layer of blastocytesupon which the epidermal cells migrate.

A few days after autotomy, many of theimmigrant epidermal cells underlying thescab begin to divide mitotically (Emmel,1910). It is obvious that these cells makeup the new epidermis of the developing limbblastema for they begin to secrete a thincuticle below the scab (Adiyodi, 1972). Thefirst indication of the forming blastema isthis hollow epidermal (and cuticular) shell.By 5-6 days after autotomy, the shell is filledwith new cells. Some of the new cells areproduced mitotically from the epidermalcells but others are immigrant cells.

The origin of new muscle tissue is unclear.Some claim that muscle and other tissuearise metaplastically from local epidermalcells while others suggest that muscle tissuearises from immigrant blastocyte cells

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350 PENNY M. HOPKINS

FIG. 1. a. Light micrograph of autotomy membrane (AM) prior to autotomy. BV = blood vessel. PN = pedalnerve. HCC = hemocoelic cavity of coxa. HCBI = hemocoelic cavity of basioischiopodite. xl40. b. Lightmicrograph of coxal stump 30 min after autotomy. G = granulocyte. B = blastocyte. LC = loose assemblage ofcells. AM = autotomy membrane remnant. S = scab. C = cuticle of coxa. x450. c. Electron micrograph of coxalstump 30 min after autotomy. G = granulocyte. DG = degranulated granulocyte. B = blastocyte. S = scab. AM= autotomy membrane. Cr = chromatophore of migrating epidermal cell, x 2,400. d. Electron micrograph ofcoxal stump at point where autotomy membrane (AM) is attached to coxal cuticle (C) 1 hr after autotomy. G= granulocyte. E = elongated epidermal cell. S = scab, x 1,800.

(Emmel, 1910; Needham, 1952; Adiyodi,1972;Hoarau, 1973;Mittenthal, 1981). Theorigin of immigrant cells is also unclear.Some of these researchers suggest thatimmigrant cells are dedifferentiated cellsfrom various sources and others suggest thatthey are nondifferentiated reserve cells.

Whatever the source, the organization andregeneration of muscle tissue lags behindthat of the epidermis by 1 to 2 days. Thefirst appearance of the blastema is that of

an epidermal shell. This shell later fills withimmigrant cells and then muscle tissue.

Mitotic figures are first seen in the epi-dermis then in the internal cells. The mitoticgrowth of the blastema continues for severaldays. Cell immigration appears to stop oncemitosis begins. One side of the bud under-goes mitosis faster than the other and as aresult the bud curves in upon itself. Cutic-ular infoldings appear within the blastemaand divide it into segments. These ingrowths

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MOLT CYCLESUBSTAGES

Auto Emergence

DAYSFIG. 2. Diagrammatic representation of a regenerating limb bud. R-value calculation given in text. Molt CycleSubstages = Drach's Molt Cycle (1939). Stage C4 = anecdysis. DQ-D,^ = proecdysis. E = ecdysis. Auto = pointof autotomy of right third walking leg.

form the cuticular attachment sites for theflexor and extensor muscles that developlater.

Emergence (Fig. 2)As mitosis continues the blastema emerges

from the surface of the coxa and, in doingso, pushes aside the protective scab. At thispoint the blastema is called a papilla.

Basal growthMitosis continues after the emergence of

the papilla. Some tendons are evident butthe papilla is primarily an epidermal shellfilled with masses of undifferentiated cells.The period of growth after emergence iscalled Basal Growth (BG; see Fig. 2), andis a continuation of the mitosis and differ-entiation that began deep within the coxa.Once it has emerged from the coxa, the smallpapilla can be measured. The length of thepapilla (in mm) is divided by the width (inmm) of the carapace, multiplied x 100 andexpressed as an R-value (Fig. 2; Bliss, 1956).During BG, mitosis is also very evident inthe regenerating pedal nerve. The epidermal

cells begin to secrete droplets during BG andmyofibrils begin to appear within cells ofthe central mass of the bud. Mitosis is stillat its peak in all regions of the bud. Seg-mentation of the bud begins (Adiyodi, 1972).

Basal plateauA few days after emergence, mitosis slows

and then ceases. The epidermal cells appearto be less active and resume a more compactshape. After mitosis slows, muscles con-tinue to develop. The myofibrils occur inclusters and are surrounded by granular sar-coplasm. By an R-value of 7, functional syn-apses are present and a new autotomy mem-brane is formed (Govind et ai, 1973). Limbbuds at this stage are capable of an autotomyresponse (personal observation). The ces-sation of growth that follows BG is calledBasal Plateau (BP or Plateau of Anecdysis,Bliss and Boyer, 1964). BP occurs only dur-ing anecdysis (Drach's Molting Stage C4;Drach, 1939). Sometimes slight, sporadicspurts of growth occur during BP (these havebeen called Advancing Plateaus of Anec-dysis by Bliss and Boyer, 1964). During BP,

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FIG. 3. Total amount of protein (in /*g) extracted fromregenerating limb buds removed at various times fromcrabs with intact or ablated eyestalks. The solid curvedline above the bars is a diagrammatic representationof the Rj-value growth curve of a single regeneratingR3 walking leg from a crab with intact eyestalks. Thebars are placed at the point below the curve that cor-responds to the size of the bud when it was removedfor protein determination. Sample sizes are below barsin parentheses. Vertical lines above bars are standarderrors of the means.

some development of the bud continues.Segmentation of the bud is seen and cutic-uiar sculpting becomes evident. Chromat-ophores appear in the epidermis and thestriation of the muscles can be seen. Duringthis time, the small bud is enclosed in itscuticuiar sac and is protected by the coxaof the adjoining legs. The duration of BPcan be quite variable from crab to crab(Hopkins, 1982).

Proecdysial growthThe end of BP (and C4) is signalled by the

reinitiation of bud growth (Proecdysial

roD Eyestalks Intact

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FIG. 4. In vitro incorporation rates of l4C-leucine intoprotein by limb buds removed from crabs with intactor ablated eyestalks. Solid curved line above bars asin Figure 3. Bar placement, vertical lines and numbersin parentheses also as in Figure 3.

Growth in Fig. 2). This second period ofgrowth is invariably linked to the onset ofproecdysis. Proecdysis is the period thatprecedes ecdysis and has been divided intofive substages-D0 and D,^, (Drach, 1939).Proecdysial growth of regenerating limbbuds is limited to substage Do (Skinner,1962). In some limbs, BG may proceeddirectly into Do growth without any inter-vening basal plateau period. BP is, there-fore, not an obligatory stage in developmentbut rather a holding pattern between anec-dysial BG and proecdysial growth. BP isshortest in crabs that have been held in thelab for long periods of time and fed on aregular basis and also in eyestalkless crabs.The BP "holding period" is a time of accu-mulation of nutritional stores. The spurtsof limb bud growth observed during BP sug-gest that anecdysis is not a quiescent period,but rather a period of episodic activity.

Proecdysial growth of the limb bud is notdue to a resumption of mitosis but ratherto increases in the size of already existingcells (Adiyodi, 1972). The overall linearincrease in the bud can be three-fold and ismade possible by the flexibility of the cuticlesurrounding the bud.

The linear increase in size of the proec-dysial limb bud is a result of protein syn-thesis (Fig. 3, open bars). If eyestalks areablated (ESX), fewer total proteins are foundin the limb buds (Fig. 3, filled bars). Thisreduction is not due to a truncated regen-eration period, since intact and ESX crabsspend about the same amount of time inproecdysial stage D (Hopkins, 1982). Thereduced amounts of total protein are due tothe fact that eyestalkless crabs synthesizeprotein at a lower rate than do intact crabs(Fig. 4). The rate of protein synthesis in intactcrabs is greatest at the middle of proecdysisand is significantly reduced during that sametime in limb buds from ESX crabs. Thelinear increase in size of the limb buds fromESX crabs is due in a large extent toincreased water uptake (unpublished obser-vation).

Terminal plateauIn many brachyurans, there is a cessation

of linear bud growth prior to ecdysis that iscalled terminal plateau (TP, Bliss and Boyer,

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1964). TP is limited to Drach's substageD,^, (Fig. 2). During TP the rate of proteinsynthesis falls in limb buds from both intactand ESX crabs (Fig. 4).

The SDS-PAGE pattern of extractableproteins from TP limb buds differs fromthat of proteins from BG buds (Fig. 5, bars).The molecular weight pattern of proteinssynthesized in the limb buds changes as thelimb buds develop from BG (20 days priorto E) which is characterized by mitosis anddifferentiation to TP (5 days prior to E)which follows a period of intensive proteinsynthesis.

At ecdysis, the folded limb bud becomesfree of its cuticular sac. As the newly regen-erated bud fills with blood, it unfolds andstretches into a complete limb. The post-ecdysial limb is almost indistinguishablefrom its unregenerated neighbors.

HORMONAL CONTROL OF REGENERATION INCRUSTACEANS

In crustaceans levels of circulating ecdy-steroid hormones rise just prior to ecdysis.In Uca, there is more than one peak of RIA-active ecdysteroids in the blood duringproecdysis (Fig. 5, solid line, small dots).One peak occurs about 5 days prior to ecdy-sis (called Peak II in Hopkins, 1986) andappears to be primarily concerned with thesynthesis of a new exoskeleton (Hopkins,1989a). Another peak (also included as PeakII, Hopkins, 1986) occurs about 10 daysprior to ecdysis and coincides with the sep-aration of the old exoskeleton from theunderlying epidermis (Fig. 5). The epi-dermis of the entire crab as well as the epi-dermis of the TP limb bud are responsiveto these two peaks of ecdysteroids. Thesepeaks occur as limb bud growth is slowing(TP; D,^,) and are necessary for a successfulecdysis (Hopkins, 1983, 19896). It is con-cluded that the ecdysteroids of TP do notplay a role in proecdysial limb regeneration(see also, Hopkins, 1991).

A smaller peak (called Peak I in Hopkins,1986) of RIA-active ecdysteroids occurs atthe transition from anecdysial BP to proec-dysial growth (Fig. 5; 20 to 24 days prior toecdysis). This peak appears to be obligatoryfor the switch from basal growth to Dogrowth (Hopkins, 1989a). Similar peaks are

20

0 =350-70KQ =25-l5K

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FIG. 5. Comparison of protein composition of limbbuds, R-values and total circulating ecdysteroid hor-mones from eyestalk intact crabs. Bars represent meansof SDS-PAGE analysis of molecular weight distribu-tion of proteins (K = kilodaltons) extracted from R3buds removed 20 days prior to ecdysis and 5 days priorto ecdysis. Vertical lines on bars represent standarderrors of the means. Numbers below bars are days priorto ecdysis (E). Each bar is the mean of at least 8 limbbuds. Upper curved solid line with large dots is growthcurve of R3 limb bud. Lower solid line with small dotsis RIA-active ecdysteroids in crab blood on each of 25days prior to ecdysis. Each dot is the mean of at least15 animals. Standard error bars are omitted from thetwo curves for clarity.

also observed during anecdysis and may beresponsible for the spurts of growth calledAdvancing Plateaus of Anecdysis.

There is a drop in total circulating ecdy-steroids (called "Valley" in Hopkins, 1986)18 to 10 days prior to ecdysis (Fig. 5).Because this drop in ecdysteroids coincideswith the period of fastest limb bud growth(Do), it was first thought that proecdysiallimb bud growth might be independent ofecdysteroid control.

The total ecdysteroid content of the bloodof Uca can be separated by HPLC into fourmajor components (Hopkins, 19896, 1991).These include ecdysone (E), 20-hydroxyec-dysone (20HE), as well as 25-deoxyecdy-sone (25DE) and Ponasterone A (PA). Theratios and levels of these four componentsvary considerably with the molting stage ofthe crab (Hopkins, 1991).

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I4C-LEUCINE INCORPORATION DISTRIBUTION

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• = CONTROLS V2 = ADDED CX =ADDEDFIG. 6. The effects of ecdysone (a) and 20-hydroxyecdysone (/3) on the distribution of l4C-leucine incorporationin vitro into various molecular size classes of proteins. Limb buds used in incubations were removed duringearly proecdysis (PE = D?) and terminal plateau (TP = Dw). Open bars = controls. Cross hatched bars =ecdysone added (25 pg/^1 incubation media). Stippled bars = 20-hydroxyecdysone added (50 pg/^l incubationmedia). Each bar is the mean of at least 5 limb buds.

When the four major ecdysteroid metab-olites in the blood of Uca at the various moltstages were separated by HPLC, quantifiedby RIA, then analyzed by means of regres-sion analysis against rates of protein syn-thesis at the same molt stages, only levelsof circulating E were highly correlated withprotein synthesis (Hopkins, 1991). Despitethe fact that overall levels of ecdysteroidsdrop during Do there is actually an increasein circulating E and 20HE levels at that time(Hopkins, 1991).

In vitro limb buds from crabs with intacteyestalks respond to added E and 20HE byincreased incorporation of l4C-leucine intoextractable protein (Figs. 6, 7; also Hopkins,1989a). These data show that E (and also20HE) induce the synthesis of specific pro-teins during Do (PE buds) in limb buds fromcrabs with intact eyestalks. These data alsosupport the conclusion that ecdysteroids donot stimulate protein synthesis during D,^,(TP buds).

It has been known for many years that

eyestalk removal results in the early onsetof proecdysis. I discussed earlier that eye-stalk removal reduces the ability of the limbbud to synthesize protein and results in limbbuds with less total protein than limb budsfrom intact crabs. Does an eyestalk factorparticipate in protein synthesis or does eye-stalk removal cause sufficient disruptions inecdysteroid metabolism that it indirectlyaffects limb bud protein synthesis? I havepreviously shown (Hopkins, 1991) that eye-stalk removal results in striking changes inamounts and ratios of the four majormetabolites in the blood of Uca. Eyestalk-less (ESX) crabs have more total circulatingecdysteroids but the ratios of steroids in theblood always favor 25DE and PA. The lev-els of E and 20HE are fairly high immedi-ately following ESX but fall to low levelsthereafter (Hopkins, 1991). A reduction incirculating ecdysone can account for someof the reduction of protein synthesis seen inESX crabs. In Figure 7 we see that addedecdysone is able to stimulate in vitro protein

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FIG. 7. The effects of ecdysone (25 pg//il incubation media) and 20-hydroxyecdysone (20HE; 50 pg/jil incubationmedia) on the rate of in vitro l4C-leucine incorporation into proteins by limb buds removed at various stagesof the molt cycle from eyestalk intact and eyestalk ablated crabs. Group 1 = limb buds with R3 values of 9-11(early Do). 2 = R3v = 13-15 (Do). 3 = R3v = 19-20 (late Do). 4 = Terminal plateau (D,^). Sample sizes givenbelow bars in parentheses. Each bar is the mean and the vertical lines are standard errors of the means.

synthesis only in limb buds removed fromESX crabs in early proecdysis but not inlimb buds removed later in proecdysis.Moreover, in ESX crabs, 20HE has aninhibitory effect on protein synthesis. Theseinteresting differences may reflect the vari-ations observed in the circulating ecdyste-roids in ESX crabs.

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Adiyodi, R. G. 1972. Wound healing and regenera-tion in the crab, Paralelphusa hydrodromus. Int.Rev. Cytol. 32:257-289.

Bliss, D. E. 1956. Neurosecretion and the control ofgrowth in a decapod crustacean. In K.. G. Wing-strand (ed.), Bertil Hanstrom, Zoological Papersin Honour of His Sixty-fifth Birthday, Nov. 20,1956, pp. 56-75. Zool. Inst., Lund, Sweden.

Bliss, D. E. and J. R. Boyer. 1964. Environmentalregulation of growth in the decapod crustacean,Gecarcinus lateralis. Gen. Comp. Endocrinol. 4:15-41.

Darwin, C. 1854. A monograph on the sub-class Cir-ripedia. Ray Society, London.

Drach, P. 1939. Mue et cycle d'intermue chez les

Crustaces Decapodes. Ann. Inst. Oceanogr. Mona-co 19:103-391.

Emmel, V. E. 1910. A study of the differentiation oftissues in the regenerating crustacean limb. Am.J. Anat. 10:109-156.

Findlay, I. and A. McVean. 1977. The nervous con-trol of limb autotomy in the hermit crab, Pagurusbernhardus (L) and the role of the cuticular stressdetector, CSD. J. Exp. Biol. 70:93-104.

Fredericq, L. 1882. Amputation des pattes parmouvement reflexe chez le crab. Arch. Biol. 3:235-240.

Govind, C. K., H. L. Atwood, and F. Lang. 1973.Synaptic differentiation in a regenerating crab limbmuscle. Proc. Nat. Acad. Sci. U.S.A. 70:822-826.

Hoarau, F. 1973. Comportement de l'hypoderme etprogression de la differentiation au cours de laregeneration d'un pereiopode chez l'isopode ter-restre Helleria brevicornis ebner. Ann. Embryol.Morphol. 6:125-135.

Hopkins, P. M. 1982. Growth and regeneration pat-terns in the fiddler crab, Uca pugilator. Biol. Bull.163:301-319.

Hopkins, P.M. 1983. Patterns of serum ecdysteroidsduring induced and uninduced proecdysis in thefiddler crab, Uca pugilator. Gen. Comp. Endocri-nol. 52:350-356.

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Hopkins, P. M. 1986. Ecdysteroid titers and Y-organactivity during late anecdysis and proecdysis inthe fiddler crab, Uca pugilator. Gen. Comp. Endo-crinol. 63:362-373.

Hopkins, P.M. 1987. Control of regeneration in crus-taceans. In H. Laufer and R. G. H. Downes (eds.),Endocrinology of selected invertebrate types, pp.327-340. A. R. Liss, Inc., New York.

Hopkins, P. M. 1989a. Ecdysteroids and regenera-tion in the fiddler crab, Uca pugilator. J. Exp. Zool.252:293-299.

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Hopkins, P. M. 1991. Hormonal control of the moltcycle in the fiddler crab, Uca pugilator. Amer. Zool.(In press.)

Hopkins, P. M. and T. Mislan. 1986. Wound healingin an autotomized limb: An electron and lightmicroscopic study of the regenerating limb of Ucapugilator. Amer. Zool. 26:60A.

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brane and lipoprotein samples. Anal. Biochem.87:206-210.

McVean, A. 1984. Autotomy. In D. E. Bliss (ed.),The biology of Crustacea, pp. 107-132. AcademicPress, New York.

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Morgan, T. H. 1900. Further experiments on theregeneration of the appendages of the hermit crab.Anat. Anz. 17:1-9.

Morgan, T. H. 1902. The reflexes connected withautotomy in the hermit crab. Am. J. Physiol. 6:278-282.

Morgan, T. H. 1904. Notes on regeneration. Biol.Bull. 6:159-172.

Needham, A. E. 1952. Regeneration and wound-healing. Barnes and Noble, New York.

Skinner, D. M. 1962. The structure and metabolismof a crustacean integumentary tissue during a moltcycle. Biol. Bull. 123:635-647.

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Regeneration of Walking Legs in the Fiddler Crab Uca pugilator