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Reference: Biol. Bull 186: 241-246. (April, 1994) Regenerate Limb Bud Sufficient for Claw Reversal in Adult Snapping Shrimps GOVIND AND READ C. K. A. T. LifeSciences Division, Scarborough College, University ofToronto, 1265 Military Trail, Scarborough, Ontario, Canada MIC 1A4 Abstract. The paired, bilaterally asymmetric snapper than the remaining thoracic limbs and are bilaterally and pincer claws in the adult snapping shrimp Alpheus asymmetric, consistingofa pincerand snapperclaw. The heterochelis were simultaneously autotomized at the be- snapper is a much hypertrophied structure almost half ginning ofan intermolt, and the resulting growth ofthe thesizeoftheentire animal and isspecialized intoapow- limb buds was characterized into several stages. At the erful snapping tool; a hammer on the dactyl plunges into next molt the limb buds emerged as newly regenerated a matching socket on the pollex, resulting in a loud pop- clawsofthesame morphotype astheirpredecessors. Next, ping sound (hence the name snapping shrimps) and ajet the paired claws were autotomized sequentially, with the stream ofwater (Hazlett and Winn, 1966). The snapping second autotomy timed to different stages of limb bud behavior is used in agonistic encounters and also in growth at the first autotomy site. When the snapper is crushing the shells ofbivalves (McLaughlin, 1982). The autotomized and a limb bud varying from stages 1 to 5 contralateral pincerclaw issmallerand used primarily in is allowed to develop at this site before the pincer is re- burrowing and feeding (Read and Govind, 1991). moved, the pairedclaws regenerate in theirpreviouscon- Claw laterality, or handedness, is random in snapping figuration. Similarly, claw asymmetry is retained when shrimps, and the snapper appears with equal probability the pincer claw is removed first and an early limb bud on the right or left side ofthe animal. However, hand- (stage 1-2)isallowedtoform atthissitebeforethesnapper edness may be switched in adult shrimps. This happens is autotomized. However, claw asymmetry is reversed if when the snapper claw is removed at the beginning ofan an advanced limb bud (stage 3-5) is allowed to form at intermolt and in its place a new limb bud regenerates thepincersitebeforethe snapperclawisremoved. Under which at the next molt unfolds into a pincer claw, while these conditions a snapper regenerates at the pincer site the contralateral intact pincer claw is transformed into a and a pincer at the snapper site. Because the limb bud at snapperclaw(Przibram, 1901;Wilson, 1903). When only thispincersite regeneratesasa snapperratherthan apin- the pincer claw is removed, a new pincer regenerates in cer, claw transformation has occurred, with the stage 3- its place; when both claws are removed, the regenerates 5 limb bud substituting for an intact pincer. Therefore, appear in the same morphotype as their predecessors. the minimal requirement for pincer-to-snapper transfor- The latterprocedure ofremovingboth clawswithin an mation is a stage 3-5 limb bud. We postulate that the intermolt wasused in an original and imaginative manner newlytransformingsnapperclaw restrictsregeneration at by Darby (1934), who varied the time interval between the contralateral old snapper site to a pincer, thereby en- the two autotomies in the tropical shrimp Alpheus ar- suring that claw bilateral asymmetry is present, albeit re- millatus. These shrimps live offthe coast ofBahama in versed. ocean temperatures of 28-30C and have an intermolt period of 10.5 days, or 252 h. His findings may be sum- Introduction marizedasfollows. Intheexperimentinwhichthesnapper The first pair ofthoracic chelipeds, or claws, in adult claw is removed first and then the pincer claw, despite snapping shrimps ofthe Alpheid family are much larger varyingthetimeintervalbetweenthetwoautotomiesfrom 20-120 h, the paired claws regenerated in their previous Received 14 September 1993;accepted 14 December 1993. configuration. Autotomyofthepincerclawbeyond 120 h 241 242 C. K. GOVIND AND A. T. READ after autotomy ofthe snapper did not allow for a suffi- Materials and Methods cientlyadvanced limb bud to form on thepincersite,and a claw failed to regenerate at this site at the next molt. Adult snapping shrimps ofthe species Alpheus heter- Thus, sequential removal of first the snapper and then oc/ielis (Say) were collected from the tidal pools around the pincer within a single intermolt was similar to simul- Beaufort, North Carolina, and shipped to our laboratory taneousremovalofbothclaws,asboth proceduresresulted in Scarborough. Ontario. The animals were held in 25-1 in the regeneration ofpaired claws in their previous con- glass aquaria equipped with a bottom gravel filter and figuration (Wilson. 1903; Govind etui. 1986). partitioned into 12 compartments with fiberglass screens In anotherseriesofexperiments Darby (1934) removed (Govind ela/., 1986). Theaquariawerefilledwithartificial first the pincerclaw, soon aftera molt, and then at varying seawater that was kept at room temperature (22C). A time intervals the snapper. If the snapper claw was re- specially prepared diet ablended mash ofchicken livers moved up to 29 h afterpincerautotomy. the paired claws and hearts, carrots and commercial cereal was fed to regenerated in theiroriginal configuration. Laterremoval theanimalsdaily, and occasionally live food wasprovided ofthe snapper claw led increasingly to a reversal ofclaw in the form of Tubifex worms. The shrimps were sexed asymmetry; i.e., a pincer regenerated in place ofthe pris- on arrival in the laboratory and their molt history during tine snapper claw and a snapper regenerated in place of captivity was recorded. thepristine pincerclaw. Thus,when thesnapperclawwas Shrimps ofboth sexes were used, and only those with removed 33 h after pincer autotomy. reversal of asym- well-differentiated pincerand snapperclawswere selected. metry was seen in 50% ofthe animals; removal 40 h and These animals were allowed to molt twice before being 72 h afterpincerautotomy produced reversal in 67% and used; this ensured that the claws were pristine, because 100% oftheanimals respectively. In theseexperiments in earlierstudies (Read and Govind, 1991) have shown that which thepairedclawsareautotomized sequentially, those at least three intermolts are required for a newly regen- withtheshortertime interval between pincerandsnapper erated limb to be fully differentiated. These claws are re- autotomy are equivalent to removing both claws at the garded as pristine in the present report (Fig. 1A). In our same time, because claw asymmetry is retained in the laboratory, the intermolt period was between 14 and previousconfiguration, whereasthosewith thelongertime 26 days (an average of 18.2 days) at 22C. intervals are equivalent to removing the snapper alone, Removal ofaclawwasaccomplishedbygently pinching because asymmetry is reversed. During these longertime the limb near the base ofthe merus, thereby inducingthe intervals, what transpires that leads to reversal? animal to autotomize the claw at a preformed fracture One ofthe events that transpires after a claw has been plane. The following experiments were performed using athuattotdeovmeilzoepdsidsutrhiengfotrhmeatiinotne,rmaotltthainsdsietem,eorfgeaslaismba nbeuwd thi(s1p)roAceddauyreo.r two after a molt, the paired claws were clawat the next molt. Because reversal ofclawasymmetry removed within minutes ofeach other; this is regarded as involves transformation ofan existing pincer claw into a snapper, we considered the possibility that a limb bud may serve in place ofan intact pincer as a suitable target for transformation. This would explain Darby's (1934) findings that claw asymmetry reverses at the longer time intervals between pincer and snapper autotomy, because a limb bud has had time to form at the pincer site. To test this hypothesis, we monitored the development of limb buds at the autotomy sites when both claws are removed simultaneously, producing a chart for limb bud growth. Using this growth chart, we repeated Darby's experiments but, rather than removing the second claw based on the time elapsed after removing the first claw, we removed the second claw at different stages of limb bud growth at the first autotomy site. We find that the presenceofasufficiently advanced limbbud at the pincer pincer pincer snapper site when the snapper is autotomized leads to reversal of claw asymmetry, thereby explaining Darby's results. Ad- Figure1. Representativepristine(A)andnewlyregenerated(B)pincer dtihteiomnailnlyi,mouumr rreesqulutisrdeemmeonntstfroartpei,ncfoerr-tthoe-fsinrastppteirmet,rtahnast- aasicnztdee,riswsnhtaiecprpepealrsuntcghleearwnsoenwslhiytoswrdieangcegtnyletrh(aeatrehrdyopwoe)n.retsPrroaiprshetiisneiedmicsllnaarawpsipnaersreizwaeisatynhmdmteshtuebrscithcaanri--n formation is a limb bud. tially smaller. Scale 3 mm; magnification X5.5. SHRIMP CI.AW ASYMMETRY 243 simultaneous autotomy. The regenerating limb buds at SNAPPER PINCER both sites were monitored daily and sketches were made that were subsequently categorized into a series ofdevel- opmental stages. The regenerating limb buds were also measured, in blind observations, under a dissecting mi- croscopewith acalibrated eyepiece. Toreducethechance ofdamaging the verydelicate limbbuds by repeated han- dling, measurementsweremadeat 3-4day intervals. Even so, 1 1 ofthe 27 animals suffered damage to the buds and were excluded from the study. To compensate for differ- ences in animal size, limb bud measurements were ex- pressed in terms of a regeneration (R) value where R 45 = (limb bud length/carapace length) X 100 (Bliss. 1960). 1 2 3 (2) Within one ortwodays followinga molt, the snap- LJMB BUD STAGES per claw was removed and the regenerating limb bud at this site was monitored daily. Next, the pincer claw was Figure 2. Stages in the regeneration ofa limbbud at the siteofan removed at varying stages oflimb bud growth at the old autotomized daw in adult snapping shrimps. The development ofthe snapper site. Of the 52 animals subjected to these se- (l1i-m6b)bsuedp.araalttehosutagghesabcaosnetdinounouesxtperroncaelssl,ahnadsmabrekesn;caattesgtoagreiz6edthientolismibx quential autotomies, 38 successfully regenerated paired buds are differentiated into snapper and pincer types (see text for de- claws at the next molt. scription ofeach stage). (3) The experimental protocol was similar to that in experiment 2. except that the pincer claw was removed firstandatvariousstagesofitslimbbudgrowth the snap- claws results in the regeneration of the paired claws in per claw was removed. Twenty-four of 36 animals suc- their previous configuration, these studies did not report cessfully regenerated paired claws at the next molt. on the growth ofthe limb buds. We repeated this experiment by autotomizing the paired claws within minutes Results ofeach other, one ortwodaysaftera shrimp had molted, Stages ojlimb bud regeneration and monitoringlimbbudgrowth in theensuingintermolt. Following autotomy of a claw, a new limb gradually Limb bud growth was qualitatively similar on the two regeneratesatthebase. Thelimbbudthat formsiscovered sidesand followedthecriteria listedabove. Moreover, the byatough flexible cuticularcoat that persists throughout regenerate limb buds ofthe two sideswere also similar in theintermolt and isdiscarded onlyatthe molt. Limbbud length throughout theintermolt (Fig. 3). At specifictimes tfhoartmaetniloanrgbeesgiinnstoimamnedaipiactaellyblaassatesmmaal(lstpaagpeil2l)a ((sFitga.ge2)1.) wduoruilndgbtehesliignhttelrymomlotr.eeiatdhvearnctehed,sbnuatpptehreroerwpaisncneorcbound- The blastema elongates and acquires a club-like appear- sistent pattern and frequently both buds were equal in ance distally. In the next stage (stage 3), a longitudinal size. When these limb buds unfolded as claws at the next furrow appears in the distal tip, marking the beginning molt (Fig. IB), they were much smaller than their pre- ofsegmentation by dividing this region into the putative decessors (Fig. 1A) but otherwise similar in morphotype. dactyl and propus segment. This is followed by the In all 16 ofthe 27 animals that successfully regenerated appearance of a series of transverse furrows along the paired claws, the previous asymmetric configuration was length ofthe limb bud (stage4). By stage 5, segmentation retained (Fig. 4A). iscomplete and the limb has acquired typical pincer-like Snapper autotomyfollowed bypincerautotomy proportions. Further differentiation into a snapper-type claw is marked by the characteristic appearance of a In this experiment, the snapper claw was autotomized plungeronthedactyl anda matchingsocketonthepollex one or two days after the shrimp had molted, and the (stage 6) and, in the case of a pincer-type claw, the pincer claw was then autotomized at different stages of appearance ofa fringe ofhair on the propus and dactyl limbbud regeneration on thesnapperside. Thus, thepin- ofmale shrimps. These six stages represent the majorex- cerwasremovedateach stageoflimbbudgrowthclassified ternal landmarks in the regeneration ofa claw and were as stages 1 to 5. Ofthe 33 animals in which the pincer used as markers in the present study. claw was autotomized at different limb bud stages, 24 animals successfully regenerated both claws. In all cases, Simultaneous mapper andpincer autotomv the paired claws mimicked their previous configuration. Although earlierexperiments (Darby, 1934; Govind et In a few additional trials, the pincer claw was also re- al., 1986)had shown that simultaneousautotomvofboth moved when the limb bud at the snappersitewasat stage 244 C. K. GOVIND AND A. T. READ The results ofthis series ofexperiments may be sum- marized as follows(Fig. 4C): removal ofthe snapperclaw 60 RNCER when a limb bud ofstage 1-2 is present at the pincer site SNAPPER results in retention ofclaw asymmetry at the next molt 50 in 50% ormoreoftheexperimental animals, but removal ofthesnapperclawwhenalimbbudofstage 3-5 ispresent at the pincer site results in reversal ofclaw asymmetry in S 40 all ofthe experimental animals. 30 Discussion 20 When the paired claws were removed sequentially within an intermolt, they regenerated in their previous 10 configuration ifthe snapper was autotomized first and a 24 limb bud allowed to form at this site before the pincer was autotomized, providing there is enough time to re- 6 8 10 12 14 16 NUMBER OF DAYS generate a claw at the second site. This is the same as when the paired claws are autotomized simultaneously, Figure 3. Percent regeneration ofsnapperand pincerlimb buds in the limb buds on the two sides regenerate at equivalent adult shrimps following simultaneousautotomy ofboth claws. Percent regenerationwasobtainedasfollows: (limbbud length/carapacelength) rates, and claw asymmetry is retained. X 100. Atotal of 128 limbbudswere measured from 16 animals;each The paired claws also regenerate in theirpreviouscon- datapoint representsan averageof4-5 measurements. figuration when the pincer is removed first and an early limb bud (stage 1-2) isallowed to form beforethe snapper is removed in 50% or more ofthe animals. This is equiv- 6 and already differentiated into a pincer claw. In these alent to removing both claws at the same time. However, animals, a pincer appeared at the old snapper site, but a paired claws regenerate in the reversed configuration in claw failed to form at the old pincer site because there 100% ofthe animals when the pincer is removed and a was not enough time for limb regeneration. more advanced limb bud (stage 3-5) is allowed to form Thus, allowing a limb bud to develop to an advanced at this site before the snapper is removed. This is equiv- stageat the snappersite before the pincer isremoved leads alent to removingthe snapperin the presence ofan intact to the regeneration ofthe paired claws in their previous pincer, in which case the existing pincer claw transforms configuration (Fig. 4B). In effect, this is similar to simul- into a snapper at the next molt and a new pincer regen- taneous autotomy of the paired claws, where the limb eratesat the old snapper site. A stage 3-5 limb bud at the buds form at equivalent rates on the two sides and claw pincersitethereforeactslikean intact, fully formed pincer asymmetry is retained as described above. claw in that they both transform into a snapper claw in response to removal ofthe contralateral snapperclaw. In Pincerautotomyfollowed by snapper autotomy other words, a stage 3-5 limb bud is a suitable target for In this experiment, the snapper claw was autotomized transformation. at different stages oflimb bud regeneration on the pincer Stage 3 limb buds are characterized by a longitudinal side following autotomy ofthe pincer claw. At the next furrow markingthebeginningofdivision ofthetwo most molt the morphotype of the newly regenerated paired distalsegments, thedactylandpropus. Themoreproximal claws was assessed in terms of retention or reversal of segments carpus, merus, and basi-ischium are delin- claw asymmetry. The results (Table I) show that with in- eated in stage 4 buds, and segmentation iscomplete with creasingtimeintervalbetween removalofthepairedclaws, final delineation ofthe dactyl and propus in stage 5 limb and hence increasing limb bud development, claw asym- buds. Although the most advanced stage 4 and 5 limb metrywasreversed. In otherwords, asthe limb bud stage buds resemble an intact pincer limb in possessing all of advances, the proportion ofclaw reversal increases until the segments, stage 3 budswithjust the beginning ofseg- 100% reversal is reached at stage 3-5 limb buds. mentation least resemble an intact limb; yetastage 3 bud Removal of the snapper claw when the limb bud is may be signaled into developing as a snapper. It would already sufficiently advanced to bediscernible asa pincer appear that the transforming signal released with auto- type (stage 6) does not result in transformation; the limb tomyofthesnapperclawcan influencethedifferentiation bud at the pincer site unfolds as a pincer claw, whereas of tissue, not only in intact pincer claws, but also in a the snapper site lacks a newly regenerated limb or limb developinglimbbudatthepincersite. Thus, forexample, bud because ofinsufficient time for regeneration. the intact pincer claw has a closer muscle whose mixture molt molt Snapper& pincer autotomy: Regenerate: pincer B molt molt Pristine: Snapper autotomy followed by pincer autotomy: Regenerate: snapper stage 1-5 limb bud molt molt Pristine: Pincer autotomy followed by snapper autotomy: Regenerate: stage 1-2 limb bud snapper pincer 3-5 stage limb bud Figure 4. Pictorial representation ofthe configuration ofthe paired, asymmetric pincer and snapper claws ofadult snappingshrimps in the following experiments. (A) Pincerand snapperclaws autotomized simultaneously;regenerateclawsappearedinthepristineconfiguration.(B)Thesnapperclawwasautotomized and. at different limb hud stagesat thissite, the contralateral pincerclaw wasautotomized: the regenerate clawsappeared in the pristine configuration. (C) The pincerclawwasautotomized and, at different stages intheformationofalimbbudatthissite,thesnapperclawwasautotomized;theregenerateclawsappeared inthepristineconfigurationwithstage 1-2budsandinthereversedconfigurationwithstage3-5limbbuds. 245 246 C. K. GOVIND AND A. T. READ Table I Configuration o)clawasymmetry, whetherretainedin theprevious configuration orreversed,followingantinomyolfirst thepincerclaw andthen thesnapperclawduringasingleinlermoll inadultsnapping shrimps Asymmetry