Investigation of hatching and early post-embryonic life of freshwater crayfish by in vitro culture,...

Investigation of Hatching and Early Post-EmbryonicLife of Freshwater Crayfish by In Vitro Culture,Behavioral Analysis, and Light and Electron Microscopy

Gunter Vogt

Zoological Institute and Museum, University of Greifswald, D-17487 Greifswald, Germany

ABSTRACT The late embryonic and early post-embry-onic life period of freshwater crayfish, which is the maintime period of organogenesis, is poorly investigatedbecause of the protective brooding behavior of crayfishmothers. A combination of in vitro culture, behavioralobservations, and microscopic investigations of organsinvolved in hatching, attachment, exploration of theenvironment, and searching and processing of foodyielded deeper insights in this important period of life.Experiments were performed with the robust partheno-genetic marbled crayfish. The following results wereobtained: (1) Marbled crayfish can be raised in simple invitro systems from 80% embryonic development to juve-nile Stage 4 with up to 100% survival; (2) Hatching isprepared by chemical weakening of the egg shell andcompleted by levering actions of the hatchling’s appen-dages; (3) The telson thread, a safety line that keeps thehatchling secured to the mother, is formed by secretionsfrom the telson and the detaching inner layer of the eggcase; (4) Molting Stage-1 juveniles are secured by ananal thread that results from delayed molting of thehindgut; (5) Active attachment of the hatchlings to thematernal pleopods with their 1st pereiopods is achievedby an innate fixed action pattern; (6) In vitro, juvenilesare motile from Stage 2 despite incomplete developmentof their balance controlling statocysts. Movement pat-tern and social behavior vary greatly among individuals;and (7) Feeding starts in Stage 3, when the mouthpartsand the gastric mill are fully developed. Onset of feedingis innate and does not require maternal contributions.In vitro culture of the isogenic marbled crayfish is rec-ommended for broader use in research because it ena-bles not only time and stage-specific sampling but alsoprecisely timed experimental manipulations. J. Morphol.269:790–811, 2008. � 2008 Wiley-Liss, Inc.

KEY WORDS: marbled crayfish; in vitro culture;hatching; development; digestion; sense organs

Freshwater crayfish differ from most other de-capod crustaceans by prolonged brood care (Anger,2001; Scholtz, 2002; Vogt and Tolley, 2004), whichincludes not only carrying of the developing eggsunder the mother’s abdomen but also carrying of theearly juvenile stages for weeks or even months(Andrews, 1907; Zehnder, 1934; Reynolds, 2002).Because of this hiding and protective broodingbehavior little is known on the biology of the earlylife stages of these freshwater macro-invertebrates.

The post-embryonic period of maternal care canbe subdivided into an attachment phase, in whichthe lecithotrophic juvenile stages are permanentlyhooked in pleopodal structures of their mother, anda sheltering period, in which the juveniles leave themother for feeding but stay on the pleopods for therest of the day. In the Astacidae, the attachmentphase comprises the first post-embryonic stage only(Andrews, 1907; Baumann, 1932; Bieber, 1940) butin the Cambaridae (Andrews, 1907; Price andPayne, 1984; Scholtz and Kawai, 2002; Vogt andTolley, 2004) and Parastacidae (Rudolph and Rios,1987; Sandeman and Sandeman, 1991; Hamr, 1992;Scholtz, 1995; Levi et al., 1999) the first and secondpost-embryonic stages. The sheltering period variesamong species (Gherardi, 2002; Reynolds, 2002)and appears to be prolonged under conditions oflimited space and scarcity of shelters (Andrews,1907; Levi et al., 1999; Vogt and Tolley, 2004). Inthe Cambaridae, the young usually remain on thepleopods until the end of juvenile Stage 3, in excep-tional cases until Stage 7 (Gherardi, 2002; Reyn-olds, 2002; Scholtz and Kawai, 2002; Vogt andTolley, 2004). The record holder of all crayfish is theparastacid Paranephrops zealandicus, which car-ries the eggs and juveniles for at least 15 months(Whitmore and Huryn, 1999).

Brooding females often do not feed and stay inshelters. Moreover, when disturbed they bend theirabdomen downwards to form a pouch, which sur-rounds the eggs or young almost completely, anddefend their brood with the chelipeds. These protec-tive and hiding behaviors make it difficult to inves-tigate the early life period of crayfish in situ, evenin captivity. Observations of the young are possiblefor short time intervals of a few minutes at best.These obstacles may be overcome by artificial incu-

Correspondence to: Gunter Vogt, Zoological Institute and Mu-seum, University of Greifswald, Johann-Sebastian-Bach-Strabe 11/12, D-17487 Greifswald, Germany. E-mail: [email protected]

Published online 25 April 2008 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10622

JOURNAL OF MORPHOLOGY 269:790–811 (2008)

� 2008 WILEY-LISS, INC.

bation of the eggs and in vitro culture of the earlyjuvenile stages. However, not many attempts havebeen undertaken to culture crayfish eggs and earlyjuvenile stages in vitro for research purposes, andmost of these studies only focused on the incubationof the eggs. The earlier literature on artificial incu-bation is summarized in Carral et al. (1988) andMatthews and Reynolds (1995). More recent dataare found in Perez et al. (1998, 1999), Henryon andPurvis (2000), Leonard et al. (2001), Nakata et al.(2004), and Melendre et al. (2006).

A major hindrance to a more widespread applica-tion of in vitro culture in the past could be relatedto the preferred use of European astacids as testorganisms. These species have a long developmentand delicate early life stages, and consequently,their culture requires time consuming efforts andsophisticated techniques (Matthews and Reynolds,1995). We have now tested the parthenogeneticMarmorkrebs or marbled crayfish, detected by us afew years ago (Scholtz et al., 2003), for its suitabil-ity to establish a simple laboratory culture systemfor eggs and early juvenile stages. This all-femalespecies, which is apparently a parthenogeneticstrain of the North American cambarid Procamba-rus alleni as revealed by analysis of the 16S hyper-variable region of the mitochondrial genome (KeithCrandall, personal communication), produces ge-netically identical offspring (Martin et al., 2007;Vogt et al., 2008), is highly resistant to handlingstress, and tolerates a broad range of experimentalconditions (Vogt and Tolley, 2004; Vogt et al., 2004;Seitz et al., 2005).

In an earlier paper we described some biologicalphenomena of the early life of the marbled crayfishby examination of eggs and juveniles taken frombrooding dams (Vogt and Tolley, 2004). Particularemphasis was given to the hatchlings, which wereinvestigated in depth by scanning electron micro-scopy (SEM). The embryonic stages and the earlydevelopment of the nervous system were recentlyinvestigated in detail by Seitz et al. (2005), Alwesand Scholtz (2006), and Vilpoux et al. (2006). Thepresent experiments, which combined in vitro cul-ture, behavioral observations, and microscopicanalyses, were performed to analyze the hatchingprocess, the formation of the telson thread and analthread, the attachment and motility of the depend-ent and independent juvenile stages, onset of feed-ing, and molting of the attached stages. In vitro cul-ture was expected to facilitate precise sampling ofany desired life stage and to enable behavioralobservations of any stage at any time. Onset offunctioning of the digestive tract and the senseorgans involved in exploration of the environmentand sensing and probing of food was expected to beidentified by the combination of behavioral observa-tion and microscopic investigation of the develop-ment of the respective organs.

MATERIALS AND METHODSRearing of crayfish and in vitro culture ofeggs and juveniles

The present experiments were performed with the partheno-genetic Marmorkrebs or marbled crayfish (Astacida, Cambari-dae) (Scholtz et al., 2003; Vogt et al., 2004), an aquarium ani-mal of unknown origin, that is genetically identical to the bisex-ually reproducing P. alleni (Faxon, 1884) from Florida (KeithCrandall, personal communication). In the following, the termin vivo applies to natural mother-offspring associations rearedunder aquarium conditions, and the term in vitro to progenyraised from the egg stage without maternal care in simplifiednet culture systems.

The eggs and juveniles used for in vitro culture and light andelectron microscopic investigations originated from dams thatwere communally reared in 60 3 30 3 30 cm aquaria filled at awater level of 15 cm. These aquaria were equipped with a layerof fine gravel and stones to provide shelter and to enablebreathing of atmospheric air. The crayfish were fed daily adlibitum with TetraWafer Mix pellets (Tetra, Melle, Germany).Water was completely changed once a week. The time point ofegg laying was determined by daily checks. Berried femaleswere individually kept in 30 3 20 3 15 cm aquaria. Photo-graphs of the mother-offspring associations were taken by plac-ing the brooding females in a photo-cuvette.

The eggs for artificial incubation were removed from thematernal pleopods at different stages of development (SD). SDis expressed as percentage of the total duration of development,i.e., the time point of egg removal divided by the time periodfrom oviposition to hatching x 100. A total of 165 eggs from 8dams with lengths of 3.9–6.6 cm (tip of rostrum to end of telson)was incubated in vitro and monitored until juvenile Stage 3, 4,or 5: 18 eggs of dam 1 from 88% SD, 18 eggs of dam 2 from74% SD, 36 eggs of dam 3 from 38% SD, 9 eggs of dam 4 from73% SD, 8 eggs of dam 5 from 83% SD, 9 eggs of dam 6 from65% SD, 17 eggs of dam 7 from 70% SD, and 50 eggs of dam 8from 56% SD.

Incubation of the eggs and rearing of the juveniles was per-formed in 11 3 8 3 7 cm glass containers that were filled with150 ml water and equipped with a fine-meshed nylon net(pieces of a net curtain; Figs. 1H, 5C) for attachment. For cul-ture of the eggs, the water was first boiled to kill bacteria andfungi and then cooled to the culture temperature of 208C. Thisway we could avoid treatment with antibiotics. Water was notfiltered, aerated or changed until onset of feeding in juvenileStage 3. Evaporated water was replaced as necessary. Stockingdensity was usually 6 eggs or juveniles per container. The con-tainers were kept under dim light and slightly shaken twice aday to avoid sticking of the eggs to the glass. Water tempera-ture varied between 19.5 and 23.08C and dissolved oxygenbetween 2.5 and 4.6 mg/L. From Stage 3, the juveniles were feddaily ad libitum with TetraWafer Mix pellets unless otherwiseindicated. Excess food was siphoned out daily and water wascompletely changed once a week.

Behavioral observations and light andelectron microscopy

Eggs and juveniles, either bred in vivo or in vitro, were inves-tigated by a combination of behavioral analysis and microscopy.Behavioral analyses were applied with respect to hatching andattachment, motility, feeding and molting of juvenile Stages 1–5. Observations were made with the naked eye, through a cam-era with a macro-lens or under dissection microscopes. Docu-mentation was done with a Canon EOS D60 digital cameraequipped with a Tamron 90 mm lens (macro-photography) orWild M420 and Olympus SZH10 dissection microscopes (micro-photography). The Wild microscope was equipped with a con-ventional Leica camera and the Olympus microscope with anOlympus DP16 digital camera.

HATCHING AND BIOLOGY OF CRAYFISH JUVENILES 791

Journal of Morphology

Light microscopy (LM) was applied to search for glandsinvolved in hatching and to determine the developmental statusof the gastric mill and the pyloric filter in late embryos and

early juveniles. Late embryos and juvenile Stages 1–5 weretaken from both mother-offspring associations and in vitro cul-tures. They were fixed with Bouin’s fluid, dehydrated in ethanol

Fig. 1. Hatching and early postembryonic development of the marbled crayfish on the maternal pleopods (A–D) and in vitro (E–H). Macro-photography (A–D, H) and micro-photography (E–G). A: Cluster of hatchlings and late embryos (e) underneath thepleon. Note freshly hatched juvenile dangling on a telson thread (arrow), which keeps the newborn firmly roped to its egg case(arrowhead). B: Same hatchling like in A, bending upwards to attach with its chelipeds while secured by the telson thread (arrow).Arrowhead denotes hatchling successfully hooked in an egg case. C: Cluster of Stage-1 juveniles under the pleon, having close bodycontact with each other. Arrow denotes extensive yolk deposit. ec, egg case. D: Dense aggregation of Stage-2 juveniles (arrow) withinterspersed egg cases (arrowheads). E: First phase of hatching: sudden appearance of the cephalothorax. F: Final phase of hatch-ing: leaving the egg shell by levering movements of the appendages. G: Hatched juvenile in a side position on bottom of culturevessel, gripping its egg case with the cheliped (arrow). The telson (arrowhead) is attached to the inner face of the egg shell by anunexpanded telson thread. H: Stage-2 juveniles on net in typical resting position with distances between each other. Arrow denotesareola. Inset: Detail of areola with individual pigmentation pattern. Bars: 2 mm (A–D, H), and 500 lm (E–G).

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and embedded in paraplast, using tetrahydrofuran as interme-dium. Serial sections were taken with a Leica RM2125RTmicrotome, stained with Azan or Goldner’s stain, and examinedwith an Olympus BX60 light microscope equipped with anOlympus DP16 digital camera.Scanning electron microscopy (SEM) was applied to investi-

gate the structure of the telson thread in the hatchlings, thepereiopodal holding structures in the first five juvenile stages,and the developmental status of the mouthparts and senseorgans involved in exploration of the environment and sensingand probing of food. Samples for SEM, which were taken fromthe maternal pleopods and from in vitro culture, were fixed for2 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4),repeatedly washed in cacodylate buffer, and postfixed foranother 2 h with 1% osmium tetroxide. After washing in bufferand dehydration in a graded series of acetone (70–100%), thesamples were dried in a Bal-Tec CPD 030 critical point dryer,sputtered with gold, and examined with a Philips 505 scanningelectron microscope.Kodak Elitechrome 200 films were used for micro-photogra-

phy and Agfa APX 100 (7 3 5.4 cm) films for SEM. Picturestaken with the digital camera, the Olympus dissection micro-scope and the light microscope were directly loaded into thecomputer. The color slides obtained with the Wild dissectionmicroscope and the SEM positives were scanned before digi-tal processing. The background, scratches and small dirt par-ticles on the objects were retouched using Adobe PhotoshopCS.

Investigation of hatching and formation oftelson thread and anal thread

In order to determine the hatching rate and the survivalrate until juvenile Stage 5 in vitro cultured eggs and juve-niles were regularly inspected, counted and staged, with theexception of the offspring of dam 6, which were allowed todevelop without disturbances. The hatching process itself wasstudied in six in vitro incubated eggs, which were placedunder the dissection microscope for continuous observationfrom the time they had softened egg shells (indicates forth-coming hatching).The gripping behaviors of freshly hatched juveniles were

investigated in vivo and in vitro. Attachment of the newborn tothe maternal pleopods, first via telson thread and then addi-tionally by their 1st pereiopods, was observed through a camerawith macro-lens in dam 2, which was placed in a small observa-tion aquarium after onset of hatching in the clutch. In order toelicit the gripping reflexes observed in this mother-offspringassociation in isolated hatchlings, freshly hatched specimensfrom the net culture systems were lifted at their egg case witha forceps. The behavioral reactions of the lifted specimens werethen recorded either with the naked eye or under the dissectionmicroscope.Formation of the telson thread was reconstructed by serial

paraffin sections of late embryos, hatching specimens, andfreshly hatched juveniles and by SEM investigations of embryosat 95% SD, embryos ready to hatch, and freshly hatched juve-niles (n 5 3 per stage and treatment). The embryos and hatch-lings were first investigated in situ and documented by micro-photography. Thereafter, they were either sampled by removalof the egg case or carefully torn out from the split egg case witha forceps to artificially expand the telson thread. The hatchingspecimens were fixed during the hatching process, photo-graphed and then processed for LM.The anal thread was investigated under the dissection micro-

scope in Stage-1 exuviae (n 5 6) sampled from in vitro culturedspecimens and in freshly molted Stage-2 juveniles still linked totheir exuviae (n 5 6). In some of the later assemblages theholding capacity of the anal thread was tested by tearing with aforceps at the Stage-2 juvenile.

Investigation of attachment and motilityof the early juvenile stages, and developmentof sense organs involved in explorationof the environment

Motility of Stage-1 and Stage-2 juveniles was studied in vitroin four 11 3 8 3 7 cm containers, each equipped with a nylonnet. The 26 juveniles monitored were bred in the above contain-ers from eggs of dam 2 (2 containers, n 5 8 per container) anddam 6 (2 containers, n 5 5 per container). Individual positionson the net were documented during Stages 1 and 2 every day inthe morning and evening. Additionally, movements were contin-uously observed for 30 min each day.

The attachment mechanisms of the early juvenile stages wereinvestigated by placing the nets together with the juvenilesunder the dissection microscope, using in vitro-raised speci-mens. Attachment and crawling of Stages 3–5 were additionallystudied through a camera with macro-lens in a photo-cuvette,which included floating branches of the water plant Ceratophyl-lum demersum (Fig. 5D, E). The attachment structures of thejuveniles, the chelae of pereiopods 1–3 and the dactyls of pereio-pods 4 and 5 were examined in detail by SEM.

Since optical, olfactory, hydrodynamic, tactile, and geosensoryand balancing senses are essential for exploration of the envi-ronment we also investigated by SEM the development of thesesense organs in Stage-1 to Stage-5 juveniles, using at leastthree specimens or exuviae per stage. In particular, we ana-lyzed the compound eyes, the olfactory aesthetascs on the outerbranches of the 1st antennae, the receptor setae on the innerbranches of the 1st antennae, the geosensory statocysts on theprotopods of the 1st antennae, the hydrodynamic and tactile re-ceptor setae on the 2nd antennae, and the current-creating fanorgan, which is composed of the exopods of the maxillipeds 1–3.

Feeding experiments and investigationof organs involved in probing andprocessing of food

A series of feeding experiments was conducted to revealwhether feeding is innate or requires maternal contributionsand whether the early juvenile stages can feed on their eggcases and exuviae. TetraWafer Mix was used as food, as thesewafers were shown to attract crayfish rapidly and facilitatemonitoring of food uptake due to their intense color, whichis easily visible in the transparent intestinal tract of livingjuveniles.

The following experiments were performed. (1) Feeding ofnatural mother-offspring associations (dams 1 and 2), startingone day after the juveniles had molted into Stage 3. Fooduptake was monitored daily by gross inspection of the alimen-tary tract of juveniles shaken from the maternal pleopods. (2)Feeding of Stage-2 juveniles raised in vitro from the egg stage(n 5 12). (3) Feeding of in vitro-raised Stage-3 juveniles, start-ing feeding at the first day of this stage (n 5 12). (4) Feeding ofin vitro-raised Stage-3 juveniles with wafers plus maternalfeces, starting at the first day of this stage (n 5 12). (5) Feedingof in vitro-raised Stage-3 juveniles, starting at day 5 of thisstage (n 5 9). Egg shells and Stage-1 exuviae were not removedfrom the culture vessels. The in vitro-experiments 2–5 wereperformed in 11 3 8 3 7 cm containers (two containers perexperiment) with offspring of dam 2.

For assessment of the developmental status of the organsinvolved in probing and processing of food we investigated bySEM the mouthparts and the gustatory corrugated setae on thechelae of pereiopods 1–3 in juvenile Stages 1–5. Samples (exu-viae and complete specimens) were taken from both themother-offspring associations and the in vitro systems (n 5 3per stage and condition). The development of the digestive tractwas investigated by serial paraffin sectioning of late embryosand juvenile Stages 1–5 taken from the maternal pleopods andthe in vitro systems (n 5 3 per stage and condition).



Investigation of molting of thejuvenile stages

In order to document molting of the early juvenile stages indetail we sampled three Stage-2 juveniles in positions ready formolting (large gastroliths, juveniles tightly hooked into the net,no movements) and transferred them together with the netsinto a petri-dish to position them under the dissection micro-scope. Positioning was achieved without direct manipulation ofthe juvenile by bending of the net and fixing it with a needle.Such positioned juveniles were then uninterruptedly observedfor hours until molting. The lights of the microscope wereswitched on for short periods of time only to take the photo-graphs. Only during ecdysis the juveniles were continuouslyilluminated. Molting Stage-1 and Stage-3 to Stage-5 juvenileswere observed in the net culture systems by chance either withthe naked eye or under the dissection microscope.

RESULTSComparison of natural and in vitroconditions, viability of the eggs andhatching rate

Under natural (in vivo) conditions the developingeggs were carried in dense clusters on the maternalpleopods (Fig. 1A). They were regularly fanned andcleaned by the mothers. Decaying eggs, which wereorange to yellow instead of dark gray to brown, weremostly removed by the mother but occasionally theyremained for several days in the clutch. In the invitro systems there was no severe development offouling organisms on the eggs despite the absence ofmaternal cleaning, probably due to boiling of the cul-ture water and culture of the eggs in the dark.

The duration of embryonic development was verysimilar in vivo and in vitro as deduced from com-parison of eggs from the same mother incubated ei-ther on the maternal pleopods or in the net culturesystem. The range of embryonic development indams 1–8 was 21–26 days with an average of 24days. In vitro, clutch members often hatched within24 hours, e.g., in dams 4 and 6. In exceptionalcases, the hatching period spanned over 72 hours,e.g., in dam 2, but even then more than 50% of thejuveniles hatched within 24 hours.

Viability of the eggs cannot be determined accu-rately under in vivo conditions since the eggs can-not be counted exactly on the maternal pleopods(margin of error > 10%). However, differences in vi-ability were very obvious between dams 2 and 7.Both females carried more than 100 eggs after ovi-position but dam 2 produced 78 hatchlings and dam7 one hatchling only. In dams 1, 3, 4, 5 and 6 thenumbers of in vivo hatched Stage-1 juveniles were53, 63, 22, 58 and 34, respectively. Non-viable eggsdecayed in all phases of embryonic development.

Under in vitro conditions the hatching ratescould precisely be determined, referring to thenumber of eggs incubated at the start of the experi-ments. They differed greatly among the trials andreached 100% in the eggs derived from dams 2 and6. In dams 3, 4, 5 ,7 and 8 the hatching rates were16.7%, 77.8%, 75.0%, 5.9%, and 96%, respectively.

Non-viable eggs decayed in all stages of develop-ment like on the maternal pleopods. The mortalityrates of the early juvenile stages also differed con-siderably between clutches. In the undisturbed off-spring of dam 6 all hatchlings developed into Stage5. In dams 2, 4, 5, and 7 the survival rates fromhatching to Stage 4, the last life stage monitored,was 100%, 42.9%, 100% and 100%, respectively,although these juveniles were repeatedly measuredand photographed under the dissection microscope.In dams 3 and 8, Stage 3, the last stage monitored,was reached by 33.3% and 97.9% of the hatchlings,respectively.

Hatching and post-hatching behavior

In vivo, the hatchlings (5 Stage-1 juveniles) weresafeguarded by their telson thread during hatchingand in the hours thereafter (Fig. 1A). This structurekept the hatchlings roped to the egg case duringtheir attempts to attach with their chelipeds (5 1stpereiopods) on the pleopods of their mother (Fig.1B). Once firmly hooked, Stage-1 juveniles re-mained rather motionless in dense aggregations onthe maternal pleopods (Fig. 1C) and lived fromtheir extensive yolk reserves (Fig. 1C). The sameholds principally also for Stage-2 juveniles (Fig. 1D)but in these clusters there was much more move-ment. Stage-3 juveniles and often also Stage-4 andStage-5 juveniles aggregated on the maternal pleo-pods during prolonged resting periods but left themother regularly for feeding.

In vitro, the developing embryos could easily bemonitored through the transparent egg shell, whichmade it possible to sample late embryos at thedesired stages. The hatching process itself was ratheruniform with respect to time course and behavioralsequence. It was initiated by softening of the rigidegg shell several hours before eclosion. The elasticshell now could easily be dented by a forceps. In thisperiod there were no rotations of the embryo or flap-ping movements of the abdomen, which could haveexerted pressure on the shell, but there were slowrhythmic contractions and expansions of the yolksac. In earlier embryonic stages flapping of the abdo-men was occasionally seen but rotation or turning ofthe embryo was never observed.

Hatching itself lasted 10–12 min and startedwith rupture of the egg shell at the back of theembryo and sudden appearance of the dorsal thirdof the embryo (Fig. 1E), after which was a break ofapproximately five minutes. At that time, the sca-phognathites beat rapidly to propel water throughthe gill chambers. In the next three minutes, thesecond third of the embryo was slowly pushed outby stretching of the abdomen and the appendages.The last phase of hatching was characterized byintense movements of the long second antennaeand the pereiopods, which were used as levers topush the hatchling out of the egg (Fig. 1F). Freshly

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hatched juveniles appeared to be considerablylarger than the eggs (Fig. 1G).

The egg cases usually ruptured along the midlineof the dorsal cephalothorax. However, when thedorsal cephalothorax of the embryo was positioneddirectly underneath the egg stalk the egg shell rup-tured on the opposite side and the embryo then leftthe egg with the pereiopods first. Such hatchlingswere mostly not able to complete hatching and diedafter a few hours.

In vitro hatched juveniles remained directlyattached to the inner wall of the egg case with theirabdomen (Figs. 1G, 2B), which is in contrast tojuveniles hatched in vivo. These remained linked totheir egg case by a 1.5–2 mm-long telson thread(Fig. 2A) that was formed when the hatchlingdropped out from the egg towards the ground (Fig.1A). In vitro, no weight acted on the telson threadduring hatching, and consequently, it persisted asan unraveled tangle between the posterior marginof the telson and the inner side of the egg shell (Fig.2B). This linkage between telson and egg case occa-sionally survived from hatching until molting intoStage 2 but often ruptured earlier both in vivo andin vitro (Fig. 5B). When the in vivo situation wasmimicked in vitro immediately after hatching bytaking the egg shell with a forceps and lifting it, thetelson thread expanded completely. If the same pro-cedure was performed hours later the telson threaddid not stretch to its full length, indicating that theclotted material had already lost flexibility.

When in vitro hatched juveniles were taken at theegg shell with a forceps and lifted into a danglingposition they bent their abdomen and pushed theircephalothorax upwards while making searchingmovements with all appendages. The same behaviorwas observed in vivo in the hour following hatchingwhen the hatchlings tried to attach onto the mater-nal pleopods (Fig. 1A, B). If those hatchlings failed togrip suitable pleopodal structures such as empty eggcases or oosetae they stretched their body and againfell into a dangling position. After a resting period ofa minute or so the same procedure was repeateduntil they finally attained a firm hold (Fig. 1B, C). Invitro, bending and stretching of the body was alsorepeated, once or twice a minute. Even if laid back ina side position the hatchlings continued with thesebending and stretching movements, which wereaccompanied by searching movements of all appen-dages and snapping of the chelae, as observed underthe dissection microscope. This behavioral sequencewas shown in the hour after hatching by all of thehatchlings investigated but was not displayed 24hours later, even if the hatchlings were lifted into adangling position.

Formation of the telson thread

The telson thread of naturally hatched juvenilesextends from the distal end of the telson to the

inner face of the egg shell (Fig. 2A). Usually, it isheavily twisted as a result of passive rotations ofthe hatchling during the dangling phases. On thehatchling’s side the thread is attached to telsonspines (Fig. 2D, E) and on the egg’s side to the innerface of the egg case (Figs. 2A, 3G). The telson spinesare often surrounded by tiny filaments (Fig. 2E),which are apparently remnants of secretionsreleased from the tip of the spines (Fig. 2F).

Formation of the telson thread was investigatedin vitro in embryos of 95% development, embryosready to hatch, hatching specimens, and freshlyhatched juveniles. In embryos of 95% developmentthe pleon is bent forward on the ventral body sidelike in earlier stages and is almost completely cov-ered by the pereiopods. When such embryos wereremoved from the egg it became clear that the tel-son thread-linkage between the embryo and the eggshell was not yet established (Figs. 2C, 3A).

In embryos ready to hatch there was a drop oftransparent material visible between the pleon andthe inner layer of the egg shell. When such speci-mens were carefully pulled out from the egg andlifted by a forceps, a telson thread appeared whilethe transparent clot disappeared indicating thatthis material is used to form the thread or at least apart of it. Further careful lifting of the embryoresulted then in a large-area detachment of theinner layer of the egg shell and elongation of thethread, suggesting that this shell layer contributesto formation of the telson thread as well.

Investigation by SEM revealed that the embryosmolt in the very final period of embryonic develop-ment (Fig. 3B), uncovering recurved spines at thechelae of the pereiopods. Although the telson threadappears approximately at the same period of time itis apparently not a part of the exuvia because theold exoskeleton is shed in fragments (Fig. 3B, C)and not as a whole like in later life stages. Inembryos ready to hatch the telson thread seemed toterminate at the anterior margin of the telsonrather than at its end (Fig. 3B, D), the telson stillbeing covered by a cup-like remnant of the old cuti-cle (Fig. 3D).

Investigation of telson threads of both in vivohatched juveniles and in vitro hatched specimenstorn out from their egg cases with a forcepsrevealed that this structure is composed of twoclearly distinguishable parts. The section adjacentto the telson, having a length of 200–400 lm,appeared translucent under the dissection micro-scope and rather homogeneous using SEM (Fig. 3C,F). The section adjacent to the egg case, in contrast,which is usually much longer (ca. 1–1.5 mm), wasopaque under the dissection microscope and hetero-geneous under the SEM (Figs. 2A, 3C, E).

In order to identify the site of synthesis of thesecretion, which forms the proximal part of the analthread, we serial sectioned the telsons of lateembryos and hatchlings. None of these stages dis-



Fig. 2. Formation of the telson thread in the marbled crayfish. Micro-photography (A–C), SEM (D–F) and LM, Azan staining(G–L). A: Naturally hatched Stage-1 juvenile taken from the maternal pleopods. The telson thread (arrowhead) is completelyexpanded and is continuous with the inner layer (arrow) of the everted egg shell. B: In vitro hatched Stage-1 juvenile. The telsonthread is unexpanded (arrow), and keeps the telson linked to the inner face of the egg case. C: Late embryo with egg case. Arrowdenotes posterior margin of telson lacking a telson thread. D–F: Telson thread of naturally-hatched specimens. D: Attachment oftelson thread (tt) to spines (arrow) at the posterior margin of the telson (t); ventral view. E: Filamentous material (arrows) aroundtelson spines and telson thread. F: Top view of telson spine with attached filament (arrow). G–I: Telson of late embryos with weakegg shell. G: Cross section of telson showing numerous vacuolar epithelial cells (arrows). Arrowhead denotes detached cuticle. h,hindgut; u, uropod anlagen. H: Epithelial cells with vacuoles (arrow) in detail. Arrowhead, musculature. I: Anlagen of telson spines(arrow). J–L: Unexpanded telson thread of in vitro-hatched specimen. J: Spine-producing cell group at posterior end of telson andtelson thread. Note differences in structure and color between telson thread and partly detached cuticle (c). Arrow denotes filamen-tous material between cell cluster and tip of spine (arrowhead). K: Section through unexpanded part of telson thread. L: Telsonthread material around tip of pereiopod 5. Bars: 500 lm (A-C), 50 lm (G), 30 lm (D, H, K, L), 20 lm (J), 10 lm (E, I), and 3 lm(F).

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played glandular structures in the interior of thetelson. However, using cytological criteria we wereable to identify two potential sites of synthesis ofsecretory materials, the epithelial cells of the telson

posterior to the anus (Fig. 2G, H) and groups ofcells at the very end of the telson (Fig. 2I, J). Inembryos ready to hatch, the epithelial cells of thetelson included large vacuoles (Fig. 2G, H), which

Fig. 3. Formation of the telson thread in the marbled crayfish, continued. SEM.A: Pleon (p) and telson (t) of late embryo lacking ter-minal spines and a telson thread.B–D: Embryo ready to hatch, torn out from its egg case by a forceps.B: Specimen with well developedtelson thread (tt) and exuvial fragments (arrowheads). C: Telson thread of same specimen from a different angle. The proximal part ofthe thread is composed of a homogeneous mass (arrowhead) that is linked to the partly detached inner layer (arrow) of the egg case (ec).D: Side view of isolated pleon showing emergence of the telson thread (arrow) between pleomere 5 (p5) and telson. The telson is stillcovered by a cup-like part of the old cuticle. E–G: In vitro-hatched juvenile, lifted by a forceps to artificially expand the telson thread.E: Specimen with partly expanded thread. The thread is composed of a rather homogeneous proximal section (arrowhead) and a hetero-geneous distal section (arrow). F: Structural details of proximal (ps) and distal (ds) sections of the thread.G: Continuity of telson threadwith partly detached inner layer of egg case (arrow). Bars: 200 lm (B, E, G), and 100 lm (A, C, D, F).



was neither observed in earlier embryonic stagesnor in later life stages. These cells may produce asecretion that might accumulate underneath theunshed cuticle of the telson. A further secretion isapparently synthesized by the posterior cellsgroups, which also produce the terminal spines(Fig. 2I). This secretion is thought to be releasedthrough the spines. Sections through the unex-panded telson threads of specimens fixed duringhatching revealed that the thread differed from thecuticle of the hatchling with respect to structureand staining properties (Fig. 2J, K). The tips of theposterior appendages were often stuck on this ma-terial (Fig. 2L).

Formation of the anal thread

During molting of Stage-1 juveniles, the emerg-ing Stage-2 juveniles were again secured by a safetyline (Fig. 4A, B) but this so-called anal thread iscompletely different from the telson thread withrespect to origin and structure. It consists of the cu-ticular lining of the hindgut of the Stage-1 juvenile(Fig. 4C, D) and is produced by delayed molting ofthe hindgut. Separate Stage-1 exuviae with analthreads or Stage-1 exuviae connected to Stage-2juveniles by anal threads are difficult to sample invivo because the exuviae are usually torn intopieces by crawling movements of the Stage-2 juve-

niles. In vitro, in contrast, both can easily beobserved, sampled or manipulated with a forceps.

The anal thread of the marbled crayfish has alength of ca. 2 mm when completely pulled out fromthe intestine of a Stage-2 juvenile (Fig. 4C). On theexuvial side, the thread ends at the anus and is con-tinuous with the cuticle of the telson (Fig. 4C, inset).At its distal end it was often seen to have a bulb-likeextension (Fig. 4C, D), which may contribute to itsholding capacity. The anal thread can easily hold theweight of a Stage-2 juvenile in the water and resistmoderate tearing forces, as tested by lifting experi-ments with a forceps. The strength of the anal threadconnection was highest when its greater part wasstill in the intestine of the Stage-2 juvenile. In ournet culture system, this structure kept the emergingStage-2 juvenile passively connected to the net viathe Stage-1 exuvia, which remained hooked in thenet with the 1st pereiopods. The anal thread connec-tions were eventually disconnected by crawling andflapping movements of the Stage-2 juveniles. Usually,this process lasted several hours.

Attachment and motility of early juveniles

In vivo it is very difficult to study how the juve-niles attach to the maternal pleopods and howmotile they are on the pleopods. Comparison of digi-tal photos taken during short observation intervals

Fig. 4. Anal thread of in vivo-raised specimens of the marbled crayfish. Macro-photography. A: Stage-2 juvenile (ju2) linked toits Stage-1 exuvia (ex1) by an anal thread (arrow). B: Detail of anal thread connection. C: Stage-1 exuvia showing an anal threadin total length (arrow). Inset: attachment site (arrowhead) of anal thread. D: Detail of anal thread with terminal extension (arrow).Bars: 1 mm (A), 500 lm (C), and 300 lm (B, D).

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suggest that Stage-1 juveniles are usually station-ary and do not change position. Stage-3 juvenilesand higher stages, in contrast, often crawl aroundand change position. Sometimes, their grip to thepleopods seems quite firm but at other times theyare easily shaken off from the mother. The pictureis less clear for Stage-2 juveniles. Generally, therewas much more movement in clutches of Stage-2juveniles than of Stage-1 juveniles but this move-ment seems to reflect turnings of the bodies aroundfixed attachment sites rather than positionalchanges.

To overcome the limitations of such in vivo obser-vations we have examined attachment and motilityof Stage-1 to Stage-5 juveniles in vitro by using ny-lon nets as artificial substrates to hold on. In blankculture vessels Stage-1 juveniles were unable towalk or swim but were capable of moving shorterdistances in a side position by flapping of their ab-domen or levering movements of their 2nd anten-nae and pereiopods. When hatched on a net orplaced on a net after hatching they hooked in eitherin upright (Fig. 5A), horizontal or hanging position.Sometimes it took hours until the juveniles were

Fig. 5. Attachment behaviorand holding structures in earlyjuvenile stages of the marbledcrayfish. Micro-photography (A–C), macro-photography (D–E)and SEM (F-K). A: Stage-1 juve-nile in typical attachment pos-ture on nylon net. Inset: Onlythe cheliped (arrowhead) ishooked in the net. B: Stage-1 ju-venile adhering to its egg shellwith the chelipeds (arrow). Theother pereiopods are free. C:Stage-2 juvenile gripping thenet with all pereiopods. Arrowdenotes cheliped tightly hookedin the net. Inset: Hooked-in che-liped in detail. D: Stage-3 juve-nile crawling on Ceratophyllumdemersum. The branches of theplant are grasped with all per-eiopods. E: Resting Stage-3 ju-venile keeping hold with pereio-pods 2 and 3 only. F: SEM-image of cheliped chela ofStage-1 juvenile with stronglyrecurved terminal hooks(arrow). G: Cheliped of Stage-2juvenile with less recurvedhooks (arrow). Arrowhead de-notes gustatory corrugatedsetae. H: Third pereiopod ofStage-2 juvenile with stronglycurved hooks. I: Fourth pereio-pod of Stage-2 juvenile withstrongly curved hook (arrow). J:Cheliped of Stage-3 juvenilewith moderately curved hooks.K: Cheliped of Stage-5 juvenilewith slightly curved hooks.Bars: 1 mm (D, E), 500 lm (A-C), 40 lm (F, G, K), and 20 lm(H–J).



firmly attached to the net, in extreme cases morethan 24 hours.

Attachment was mainly done with the 1st pereio-pods (Fig. 5A, B), the chelae of which are equippedwith strongly recurved and sharp terminal hooks(Fig. 5F). The 2nd and 3rd pereiopods also havechelae with recurved hooks but these are generallysmaller than those of the chelipeds. The 4th and5th pereiopods are non-chelate and have dactylswith recurved or strongly curved terminal spikes.Pereiopods 2–5 were sometimes used to assist thegripping process but often hung free once the hatch-ling was firmly attached with its chelipeds (Fig.5B). Heavily disturbed Stage-1 juveniles were prin-cipally able to unhook and to move slowly byrepeated gripping and release of the net. Normally,however, they remained in an attached position forthree to four days until molting into juvenile Stage2. If the hatchlings had no access to a substratesuitable to cling to they hooked their 1st pereiopodsinto their own egg shell (Figs. 1G, 5B) and re-mained in that position until molting.

Stage-2 juveniles also used mainly their 1st per-eiopods to adhere to the nylon net but attachmentwas regularly supported by the chelae of pereiopods2 and 3 and the dactyls of pereiopods 4 and 5 (Fig.5C). The terminal hooks of their chelipeds are lessprominent than in Stage 1 but are still considerablyrecurved (Fig. 5G). The hooks of pereiopods 2 and 3(Fig. 5H) and the dactyls of pereiopods 4 and 5 (Fig.5I) are strongly curved or even slightly recurvedand are thus also suitable for use as hooks. There-fore, when firmly attached Stage-2 juveniles triedto detach from the net they normally needed sev-eral attempts, resembling cats trying to unhooktheir claws from a carpet.

Instead of hooking in, Stage-3 juveniles grippedappropriate structures with the chelae of their per-eiopods 1–3 to hold on. Due to their larger size thechelae of the 1st pereiopods certainly exert thetightest grip, and therefore, they were usually usedduring climbing (Fig. 5D). At rest, however, thebody was often held by the other pereiopods alone(Fig. 5E). The cheliped chelae of Stage-3 juvenilesare also equipped with prominent terminal spikes(Fig. 5J) but these are less curved than in earlierstages and are therefore less suitable for hooking.The attachment behaviors and also the holdingstructures of Stage-4 and Stage-5 juveniles are sim-ilar to those of Stage-3 juveniles but the terminalspikes of their chelipeds are straighter (Fig. 5K).

On the maternal pleopods, Stage-1 and Stage-2juveniles were generally found in dense aggrega-tions with close body contact to each other (Fig. 1Cand D). In the in vitro systems, these life stagessometimes aggregated as well, but sometimes theydispersed and preferred to be solitary. Stage-1 juve-niles were occasionally seen to move away fromeach other over a distance of a few millimeters to acentimeter, particularly after they had struggled

with one another. Stage-2 juveniles, which canactively move over greater distances, used to dis-perse across the entire available space (Fig. 1H).Since the juveniles are individually identified bytheir pigmentation pattern, for instance that of theareola of the carapace (Fig. 1H, inset), their move-ment patterns could be tracked individually.

In vitro, resting Stage-2 juveniles were oftenfound on top of the net in a sitting position (Fig.1H) with their abdomen bent under the cephalo-thorax like their siblings on the maternal pleopods(Fig. 1D) but were also present on the underside ofthe net in a hanging position. Upon disturbance,advanced Stage 2 juveniles fled backwards by tailflipping as seen in later juvenile stages, which wasnot the case on the maternal pleopods. Motility ofStage-2 juveniles was generally higher towards theend of this life stage than at the beginning. Laterjuvenile stages either rested on the maternal pleo-pods in dense aggregations or roamed around indi-vidually or in smaller groups. They could easilyattach to the vertical glass walls to rest or to crawlupwards in order to breathe atmospheric air at thewater surface. Stage-3 juveniles and later stagesmostly fled by rapid backwards or, even more fre-quently, upwards swimming.

Development of sense organs involved inexploration of the environment

The optic, hydrodynamic, tactile, olfactory, andgeo-sensory and balancing sense organs, which areessential for exploration of the environment, werenot developed in Stage-1 juveniles. The eyes al-ready were considerably pigmented in late embry-onic stages but their typical compartmentation intoommatidia only was finished in Stage 2, as shownby SEM and paraffin sectioning (Fig. 6B, C). Thegeo-sensory statocysts, which are located in the ba-sal article of the first antenna, appeared first inStage-1 juveniles but were only structurally com-plete in Stage 3 (Fig. 6D-G). In Stage-1 juvenilesthe statocyst caves were already present but setaewere generally lacking. In Stage-2 juveniles geo-sensory hairs were present in considerable amountsbut there was no statolith and the entrance of thestatocyst was not yet covered by setae. From Stage3 the entrance of the statocyst was protected bysetae (Fig. 6D) and the sensory setae were linked toa statolith composed of debris (Fig. 6E, F). In lifestages cultured on gravel the statoliths alsoincluded tiny sand particles. The sensory hairs ofthe juveniles had the same morphology like in theadults (Fig. 6F, G). These results match differencesrecorded with respect to locomotion of Stage-2 andStage-3 juveniles: crawling Stage-2 juveniles (Fig.6A) often fell on their side or back, whereas Stage-3juveniles displayed controlled walking movementslike those of the adults.

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The hydrodynamic and tactile receptor setae onthe 2nd antennae (‘‘antennae’’) (Fig. 7A) and on theinner branches of the 1st antennae (‘‘antennules’’)(Fig. 7F), which include feathered setae and smoothsetae with terminal pores (Fig. 7B, C, G, H),appeared first in Stage-2 juveniles. These receptorsetae, which differed in detail among the two anten-nal types, displayed the same structural featureslike in later stages but were less numerous. Thesame holds for the olfactory aesthetascs on theouter branches of the first antennae (Fig. 7D, E)and their accompanying setae (Fig. 7E), whichappeared first in Stage-2 juveniles as well.

The fan organ, which serves to propel odor mole-cules from the surroundings of the crayfish to itsfrontal sense organs, is structurally complete fromStage 2 (Fig. 7J). It is composed of the exopods of

the maxillipeds 1–3 (Fig. 7I), which have long feath-ered setae at their tips forming a fan-like structure(Fig. 7J, K).

Onset of feeding and development of organsinvolved in probing and processing of food

Under in vivo conditions Stage-1 and Stage-2juveniles were non-feeding, thriving on their yolkreserves. Onset of feeding started in Stage 3. In themother-offspring associations Stage-3 juvenilesbegan to ingest the food in the first day of feedingalthough they had still extensive yolk reserves attheir disposal. Successful food uptake was indicatedby the presence of intensely colored food particlesin the stomach and the intestine, which could easilybe seen through the transparent integument. In the

Fig. 6. Motility of early juvenile stages of the marbled crayfish and development of optical and geo-sensory sense organs. Micro-photography (A) and SEM (B–G). A: In vitro-raised Stage-2 juvenile in motion. B–C: Compound eyes. B: Eye of Stage-1 juvenile.The typical subdivision into ommatidia is not yet visible. C: Eye of Stage-2 juvenile with well developed rectangular corneas(arrow), reflecting completion of ommatidia formation. D–G: Statocysts. D: Entrance of statocyst of Stage-3 juvenile covered withbranched setae. E: Statolith (sl) and sensory hairs (arrow) in Stage-5 juvenile. F: Attachment of sensory hair (sh) to statolith bylateral setules (arrow) in Stage-3 juvenile. G: Bases of sensory hairs in Stage-5 juvenile. Bars: 1 mm (A), 50 lm (B, C), 30 lm (D),10 lm (E), 5 lm (G), and 2 lm (F).



in vitro systems, the flakes were approached by theStage-3 juveniles within minutes after offeringfood, in some cases even within seconds. One hourlater all juveniles had food in their stomach, andanother hour later the intestines of most specimenswere filled. After three hours, feces strings werefound.

Under natural conditions, early Stage-3 juvenilesmight use the exuviae or the egg cases as first food

as these structures usually persist on the maternalpleopods during the non-feeding Stages 1 and 2(Fig. 1D) but disappear thereafter. In vitro, Stage-3juveniles were seen to eat their Stage-1 and Stage-2exuviae and also their egg cases if they were not fedduring the first days of Stage 3. When they werefed early in Stage 3, the exuviae and egg cases weresometimes eaten and sometimes not. The egg cases,in particular, often remained.

Fig. 7. Development of olfactory and hydrodynamic sense organs and current-creating fan organ in the marbled crayfish. SEM.A–C: Second antennae (‘‘antennae’’). A: Terminal part of 2nd antenna of Stage-2 juvenile with sensory setae (arrow). B: Hydrody-namic (arrowhead) and tactile (arrow) setae on 2nd antenna. C: Tip of hydrodynamic seta showing beak-like terminal structurewith pore (arrow). D–H: First antennae (‘‘antennules’’). D: First antenna of Stage-2 juvenile with olfactory aesthetascs (arrow-heads) on outer branch (ob) and feathered setae (arrow) on inner branch (ib). E: Olfactory aesthetascs (arrow) in Stage-3 juvenile.Arrowhead denotes smooth accompanying seta. F: Terminal part of inner branch of 1st antenna of Stage-2 juvenile with smooth(arrowhead) and feathered (arrow) setae. G: Feathered seta in detail. H: Tip of smooth seta with terminal pore (arrowhead). I–K:Fan organs. I: Fan organ of Stage-5 juvenile, being composed of the exopods (arrowheads) of the maxillipeds 1–3. The exopod ofmaxilliped 3 (m3) is broken off. a2, 2nd antenna; ca, carapace, gc, gill chamber. J: Side view of terminal setae of the exopod of max-illiped 3 in Stage-2 juvenile. K: Top view of terminal setae in Stage-3 juvenile. Bars: 200 lm (I), 100 lm (D, K), 30 lm (A, E, F, J),10 lm (B, G), and 2 lm (C, H).

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In vitro culture also revealed that maternal assis-tance is not necessary for onset of feeding, as artifi-cially raised Stage-3 juveniles started feeding in theabsence of a mother, indicating that searching offood and proper use of the mouthparts are innate.However, when such juveniles could choosebetween pellet food and maternal feces they alwayspreferred the feces and left the pellets unscathed.In vivo, juveniles were also regularly seen to feedon the maternal feces.

Stage-2 juveniles, which were non-feeding in themother-offspring associations, often approached theflakes in the in vitro systems and examined themfor longer periods of time but never ingested them.SEM investigations revealed that the main organs

necessary for locating, sensing and probing offood—the eyes, the fan organ, the olfactory aesthe-tascs and the gustatory corrugated setae on pereio-pods 1–3 (Fig. 8A, B)—are already developed in thislife stage. In Stage-1 juveniles, these organs areeither not developed at all, i.e., the aesthetascsand corrugated setae, or only partly developed, i.e.,the eyes and the fan organ.

The mouthparts are already present in Stage-1juveniles but are not yet equipped with sensory andmasticatory structures (Fig. 8C). Stage-2 juveniles,in contrast, have some setae on their mouthpartsand also teeth-like structures on the mandibles andthe cristae dentatae of the 3rd maxillipeds (Fig. 8D)but only from Stage 3 do the mouthparts seem to

Fig. 8. Development of organs involved in probing and processing of food in the marbled crayfish. SEM. A,B: Gustatory corru-gated setae. A: Corrugated setae (arrowhead) on chela of 2nd pereiopod of Stage-2 juvenile. Arrow denotes another type of sensoryhair. B: Higher magnification of corrugated setae (cs) in Stage-2 juvenile showing typical corrugations (arrow) and terminal pore(arrowhead). C–G: Mouthparts. C: Ventral aspect of mouthparts of Stage-1 juvenile extending from mandibular palp (mp) to 3rdmaxilliped (m3). Note general absence of setae. D: Mouthparts of Stage-2 juvenile characterized by presence of some setae (arrow)and moderately developed masticatory structures (arrowhead). E: Inner margins of mouthparts of Stage-3 juvenile with high diver-sity of sensory and masticatory structures. m, mandible; m1, 1st maxilliped; ma1, 1st maxilla; ma2, 2nd maxilla. F: Crista dentataof 3rd maxilliped of Stage-3 juvenile with prominent teeth. G: Sensory hair with terminal pore on 2nd maxilla of Stage-5 juvenile.Bars: 200 lm (D), 100 lm (C), 30 lm (E, F), 10 lm (A), 2 lm (B), and 1 lm (G).



have the same set of sensory and masticatory struc-tures like in later stages (Fig. 8E-G).

The digestive tract of Stage-1 juveniles is domi-nated by a huge yolk sac (Figs. 1C, 9F) that is con-nected to the posterior part of the stomach, themidgut and the hindgut. Yolk material was nor-mally not observed in the stomach, the hepato-pancreatic tubules or the hindgut (Fig. 9F) but wasregularly found in the midgut, which, in this lifestage, has broad luminal continuity with the yolksac. In late Stage-1 juveniles, the gastric mill is al-ready present. It is composed of primordia of the

median and lateral teeth but these are not yet scle-rotized (Fig. 9A). The pyloric filters, in contrast, arefully developed and include ca. 9 filter tubescovered by setae on each body side (Fig. 9B). Thesesecondary filters and also the primary filters onthe bottom of the cardiac stomach are formedduring Stage 1. Freshly hatched specimens haveprominent gastroliths (Fig. 9C) in ventrolateralpockets of the cardiac stomach. These calciumdeposits are dissolved in the hours after hatchingand are re-established before molting into juvenileStage 2.

Fig. 9. Development ofstomach (A–E) and hepatopan-creas (F–H) in the marbledcrayfish (LM, Azan staining).A: Gastric mill primordium inStage-1 juvenile composed ofmedian tooth (mt) and lateralteeth (lt). B: Pyloric filter inStage-1 juvenile with welldeveloped filter tubes coveredby filter setae (arrow). C:Partly resolved gastrolith(arrow) in stomach pocket ofhatchling. D: Gastric mill ofStage-2 juvenile with moder-ately sclerotized (brown) me-dian and lateral teeth. The cu-ticular teeth are detached fromtheir epithelia. E: Gastric millof Stage-3 juvenile with mark-edly sclerotized teeth. F: Crosssection of posterior part ofcephalothorax of Stage-1 juve-nile showing yolk sac (ys),midgut (m), and hepatopan-creas tubules (asterisks). g,ganglion; gc, gill chamber. G:Cross-section of hepatopancreastubule showing R-cells (r) withlipid globules (arrowhead), F-cells (f), and a developing B-cell(arrow). H: Cross-section ofhepatopancreas tubule of feed-ing Stage-3 juvenile displayingwell-developed B-cells (b). Noteabsence of yolk from the stom-ach (A–E) and the hepatopan-creas tubules (F–H). Bars: 200lm (F), 40 lm (A, D, E), and 30lm (B, C, G, H).

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The hepatopancreas of Stage-1 juveniles is com-posed of several tubules (Fig. 9F), which includethe typical cell types of this organ (Vogt, 2002), R-cells (absorption and storage of nutrients), F-cells(synthesis of digestive enzymes) and B-cells (prob-ably synthesis of fat emulsifiers) (Fig. 9G). The R-cells already include considerable amounts of lipiddroplets, which is in contrast to the situation in lateembryos. F-cells are easily detectable by their baso-philic staining, which reflects proliferation of therER. The B-cells are inconspicuous in this life stageand only have small central vacuoles (Fig. 9G). Thestructural features of F-and B-cells indicate thatthey are not yet active, suggesting that the hepato-pancreas is not involved in catabolism of the yolk.

In Stage-2 juveniles the teeth of the gastric millare more prominent and slightly sclerotized (Fig.9D). The pyloric filters include now approximately13 filter tubes per body side, and the hepatopan-creas is considerably enlarged as well. The R-cellsinclude numerous lipid globules but the F-cells andB-cells still show no structural signs of functioning.Like in Stage-1 juveniles, yolk is normally notfound in the stomach or the hepatopancreas. How-ever, some yolk is usually present in the anteriordorsal caecum, which runs from the yolk sac to themidgut-hindgut junction. Feces strings were notproduced in this life stage but during heavy stressthe juveniles sometimes released small portions oforange to brownish yolk material via the hindgut.

In Stage-3 juveniles the teeth of the gastric millare further sclerotized (Fig. 9E) and the pyloric fil-ters are further enlarged. The hepatopancreas isalso markedly enlarged. The B-cells are now veryprominent and display large central vacuoles (Fig.9H). F-cells are also more prominent and, in con-trast to earlier stages, include numerous prominentGolgi-bodies, indicating synthesis of digestiveenzymes. These structural features, which are simi-lar to those of the adult hepatopancreas, indicatenormal functioning of the organ.

Molting of early juvenile stages

The net culture system proved to be particularlysuitable to investigate molting of the attached juve-nile Stages 1 and 2 in detail. Stage-1 juveniles werepermanently attached to the net in a fixed positionand thus could easily be observed. Stage-2 juvenileshooked firmly into the net hours before molting andremained in that position until ecdysis. Forthcom-ing molting was indicated in both stages by the for-mation of relatively large, bluish gastroliths in thelateral walls of the cardiac stomach. These were dif-ficult to see through the dorsal or lateral parts ofthe cephalothorax because of the large yolk depos-its, but were easily detectable from the front.

Figure 10 illustrates molting of a Stage-2 juve-nile, which may serve as an example for all early

juvenile stages. The specimen depicted was foundfirmly attached to the net with all pereiopodsapproximately two hours before molting (Fig. 10A).It attracted our attention because it showed nomovements and did not react to slight touches witha forceps. Its abdomen was slightly curved andthe 1st and 2nd antennae as well as the 3rd maxilli-peds were horizontally extended (Fig. 10A). Theonly remarkable action during that immobile periodwas the release of a long opaque string fromthe intestine, which occurred half an hour beforeecdysis.

Ecdysis itself lasted less than 30 s. It started withbending of the abdomen and downward movementsof the antennae followed by upward movements ofthe cephalothorax. The cephalothoracic cuticle wasthen shed and turned down anteriorly (Fig. 10B).At the same time the pleopods were pulled out ofthe old exoskeleton. In the next phase, the cephalo-thorax became free by more intense upward move-ments of the entire body (Fig. 10C), and subse-quently, the long 2nd antennae and the pereiopodswere pulled out (Fig. 10D). Finally, the juvenile leftthe exuvia by flapping of the abdomen and swim-ming movements of the pereiopods and pleopods(Fig. 10E) and sank to the bottom where it startedwalking almost immediately (Fig. 10F). The exuviaremained firmly attached to the net (Fig. 10F).Interestingly, the freshly molted Stage-3 juvenileshowed no tendency to hide or rest as one wouldexpect.

Molting of Stage-1 and Stage-3 to Stage-5 juve-niles was in many aspects similar to molting instage 2 but differed in others. Molting Stage-1 juve-niles, for instance, remained attached to their exu-via by the anal thread, as described earlier. Stage-3to Stage-5 juveniles molted either on the net, underthe net or in the free area of the culture vessels inside position. In any case, their exuviae floatedfreely after ecdysis indicating that these stages didnot firmly attach to the net for molting. Freshlymolted Stage-3 to Stage-5 juveniles were motile im-mediately after hatching, roamed around and wereable to escape by intense tail-flipping. The juvenileStages 1–5 molted at any time during the day or atnight.

DISCUSSION

It was the aim of this work to investigate the lateembryonic and early post-embryonic life period offreshwater crayfish in depth by combining in vitroculture with behavioral analysis and microscopicinvestigations of hatching, attachment structures,digestive organs and major sense organs. The par-thenogenetic marbled crayfish, which was chosenas experimental animal due to its hardy and unde-manding nature, turned out to be particularly suit-able for this purpose.



Viability of the eggs and development of thefirst post-embryonic life stages

The viability of the eggs of crayfish can only pre-cisely be determined in vitro since exact counting ofa clutch is impossible without completely removingthe ova from the pleopods. Our experiments re-vealed that the eggs of the marbled crayfish can becultured in simple in vitro systems with low mortal-ity at least from 50% embryonic development. The

survival rate of the eggs is apparently dependenton the time of stripping from the maternal pleo-pods, as can be concluded from survival rates of16.7% and 100% in eggs removed at 38% and 65%development, respectively. In vivo the survival rateof the eggs may exceed an estimated 80% underfavorable conditions but can also be a few percentonly or even zero, as is sometimes the case in first-time spawners or water temperatures higher than258C (Seitz et al., 2005). A further source of egg loss

Fig. 10. Molting of Stage-2 juvenile of the marbled crayfish in vitro. Micro-photography. A: Stage-2 juvenile 30 min before ecdy-sis, being largely immobile and tightly hooked into the net. B: Beginning of ecdysis by bending of the abdomen, upward movementof the cephalothorax and shedding of the carapace that is turned down anteriorly (arrow). C, D: Withdrawal of the appendagesfrom the old exoskeleton by intense upward movements. E: Leaving of the exuvia. F: Freshly molted Stage-3 juvenile crawling onbottom of petri-dish. The exuvia is still firmly attached to the net. Bars: 1 mm.

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in vivo is heavy disturbance by aquarists or compe-tition among crayfish, particularly when sheltersare rare (Gherardi, 2002; Reynolds, 2002).

The developmental speed of the embryos and thenon-feeding Stage-1 and Stage-2 juveniles from thesame mother was roughly similar under in vivo andin vitro conditions, indicating that in captivitymaternal contributions are not required to success-fully complete embryogenesis and the first post-em-bryonic life stages. Consequently, the role of mater-nal care in this period is mainly to be seen in pro-tection. Mortality in the first post-embryonic lifestages was negligibly low in our in vitro experi-ments. In two of eight trials the survival rate waseven 100% from stripping of the eggs to juvenileStage 4, suggesting that in vitro culture of themarbled crayfish is a suitable means to producehigh numbers of vital crayfish juveniles for re-search purposes.

Hatching

Eggs ready for hatching are recognized by thepresence of a fully developed embryo (Seitz et al.,2005; Alwes and Scholtz, 2006) and a weakened eggshell. Softening of the rigid egg case starts approxi-mately a day prior to eclosion and is probablycaused by a hatching enzyme, which in the noblecrayfish Astacus astacus was found to be expressedin high quantities in the pre-hatching period (Geierand Zwilling, 1998). This hatching enzyme is amember of the astacins, a zinc-metalloprotease fam-ily, which also includes the eponymous crayfish di-gestive enzyme astacin (Mohrlen et al., 2001; Vogt,2002). An increase in elasticity and a decrease inthickness of the egg shell in the hours before eclo-sion were also observed in Procambarus clarkii(Suko, 1961; Davis, 1981).

Hatching of the marbled crayfish in vitro isachieved by a stereotyped sequence of behavioralpatterns, which differ in some aspects from pub-lished concepts on hatching in crayfish (summar-ized in Vogt and Tolley, 2004). For instance, therewas no flapping of the abdomen or rotation of theembryo prior to hatching, which were earlierassumed to provide the mechanical power for eclo-sion. Our data rather indicate that rupture of theegg case and sudden appearance of the dorsal partof the hatchling is either caused by a strong inter-nal pressure built up by water uptake into theembryo, or by pushing with the pereiopods, or by acombination of both mechanisms. The first idea issupported by the larger size of freshly hatchedembryos compared to the egg diameter. Waterinflux into the egg and then into the embryo may bepromoted by the described structural alterations ofthe egg case prior to hatching. The second idea issupported by general splitting of the egg case alongthe backside of the cephalothorax of the embryo.Hatching is generally completed by stretching and

levering movements of the appendages. Mortalitiesduring hatching were only rarely observed andoccurred mainly when the juveniles hatched withthe pereiopods first.

Our in vitro experiments also revealed thathatching in crayfish does not require maternal as-sistance, for instance by exerting pressure onto theeggs by bending of the abdomen as hypothesized byBieber (1940). Hatching in crayfish also does notrequire a chemical maternal signal as is the casein cirriped crustaceans, where hatching is stimu-lated by a metabolite of the eicosapentaenoic acidsecreted into the mantle cavity (Crisp et al., 1991).This signal synchronizes hatching with a suitableplanktonic environment.

In vivo-hatched juveniles dangling from thematernal pleopods and in vitro-hatched juvenileslifted at their egg cases with a forceps bent theirbodies upwards in an almost 1808 arc and fell backagain into a dangling position if they failed to gripstructures to hold onto. During these movementsthey were secured by the telson thread. Sincethis behavioral pattern is stereotypically repeatedit must be an innate reflex. The accompanyingsearching movements of all appendages and snap-ping of the chelae even continued when in vitro-hatched juveniles were laid back in a horizontalposition suggesting that this behavior is a fixedaction pattern, which serves for attaching onto thematernal pleopods.

Origin of the telson thread

Safeguarding of hatching by a telson thread is anautapomorphy of freshwater crayfish (Scholtz,2002; Vogt and Tolley, 2004). The telson thread actslike a safety line, keeping the hatchling secured toits mother and preventing it from being dislodgedby the water current. The origin of this thread hasbeen controversially discussed for more than a cen-tury. It was believed to originate from an exuviashed during hatching or from a secretion of telsonglands or by a combination of both mechanisms(reviewed by Andrews, 1907; Bieber, 1940; Scholtz,1995; Scholtz and Kawai, 2002; Vogt and Tolley,2004).

Our investigations revealed that the telsonthread of the marbled crayfish is composed of twocomponents, a proximal part originating from secre-tions of the telson and a distal part originating fromthe detaching inner layer of the egg shell. Therewere apparently no exuvial parts involved in forma-tion of the thread, although the hatchlings moltedshortly before hatching. However, unlike in laterstages, this exoskeleton was shed in small frag-ments unsuitable to form a telson thread. Ourinvestigations also revealed that the telson becomesattached to the inner layer of the egg shell only inthe very final phase of embryonic development. Atelson thread is only formed if mechanical forces



are exerted, which is the case under natural condi-tions when the hatchlings drop out of the egg. Suchmechanical forces convert the secretion drop andthe inner layer of the egg case into a string-likestructure.

Motility and attachment of theearly juveniles

The in vitro culture system also proved very suit-able to gather information on the motility patternand the attachment mechanisms of the early juve-nile stages. Stage-1 juveniles usually hooked into asuitable substrate and remained in that positionalmost motionless until molting into Stage 2. Inthis life stage, all organs necessary for orientationand sensing of environmental signals, i.e., the eyes(Hafner et al., 2003), statocysts (Takumida andYajin, 1996; Vogt, 2002), olfactory aesthetascs (San-deman and Sandeman, 2003), tactile and hydrody-namic receptor setae on the 1st and 2nd antennae(Zeil et al., 1985; Sandeman, 1989; Breithauptet al., 1995; Basil and Sandeman, 2000) and thecurrent-creating fan organ (Breithaupt, 2001) wereeither partly developed or not developed at all,making an independent life in nature impossible.However, Stage-1 juveniles have enough yolkreserves at their disposal to survive in a predator-free in vitro system. By avoiding crawling move-ments, which are possible to a certain degree, theyreduce energy consumption to a minimum.

Striking differences in the movement patternsbetween in vivo and in vitro conditions were notedfor Stage-2 juveniles, the first life stage with ratherwell developed external sense organs. In vitro,Stage-2 juveniles often moved around and changedposition, whereas on the maternal pleopods theyseemed to remain stationary. Walking and swim-ming, however, was less coordinated and elegantthan in later stages, probably because of the incom-plete development of the telson (Vogt et al., 2004)and the statocysts. In vitro, Stage-2 juveniles oftendispersed over the entire available space, and a con-siderable percentage of them preferred to be soli-tary when hooking into the net, which is in sharpcontrast to the aggregating behavior of their sib-lings on the maternal pleopods. In mother-offspringassociations, the aggregating behavior may be pro-moted by a juvenile-attracting pheromone releasedby the brooding female (Little, 1976).

The development of firm attachment mechanismsis of particular importance for Stage-1 and Stage-2juveniles, as these permanently adhering life stagesare often heavily shaken by fanning movements ofthe maternal pleopods. Attachment is still impor-tant in the first independent stages, as these stagesboth rest on the maternal pleopods and crawl inwater plants for foraging. Our in vitro investiga-tions revealed two principal and stage-specificattachment mechanisms, one based on hooking and

one based on clipping. The first mechanism is real-ized in Stage-1 and Stage-2 juveniles and the sec-ond in the later stages.

Stage-1 juveniles attach almost exclusively withtheir prominent 1st pereiopods that are equippedwith sharp recurved hooks. This kind of attachmentis very firm, and consequently, loss of juveniles israre. Stage-1 juveniles are mostly able to unhookbut usually they avoid doing so once firmlyattached. In Stage-2 juveniles, hooking with the 1stpereiopods is still the predominant principle ofattachment but the other pereiopods are regularlyused as well to strengthen the contact. Principally,Stage-2 juveniles can attach and detach more easilythan Stage-1 juveniles. Stage-3 juveniles and laterstages grip appropriate structures with the chelaeof their pereiopods 1–3 rather than hooking. Thisclipping mode of attachment certainly requiresmuch more energy than the hooking principle ofthe earlier stages since pressure must be exertedpermanently to hold the grip.

Stage-3 juveniles and later stages were regularlyseen to crawl along the lateral walls of the culturevessels to the water surface to breathe atmosphericair. For this purpose they located one of the gillchambers in a lateral position at the water surfaceand propelled a mixture of water and air throughthe gills. Attachment of the pereiopods to thesmooth surface of the aquarium is certainly notaccomplished by the terminal hooks of the pereio-pods but perhaps by the corrugations of the corru-gated setae, which in situ are oriented towards thesubstrate. Breathing of atmospheric air is also typi-cal of later life stages. Berried and brooding femaleseven fan their eggs or juveniles in atmospheric airif they have access to the water surface. Therefore,the marbled crayfish can easily be reared withoutaeration by placing in the center of the aquariumappropriate facilities for climbing such as stones.This culture technique eliminates the risk ofescapes from the aquaria because crayfish normallyescape along the aeration tubes.

Onset of foraging and feeding

Onset of foraging and feeding in Stage-3 juvenilesis innate and does not require maternal contribu-tions as revealed by the in vitro experiments. Themajor sense organs involved in locating and probingof food, the eyes, the aesthetascs on the 1st anten-nae (Giri and Dunham, 1999; Kraus-Epley andMoore, 2002) and the gustatory fringed setae onpereiopods 1–3 (Altner et al., 1983; Vogt, 2002) al-ready are well developed in Stage 2, suggestingthat foraging could principally start in this stage.However, when brought together with food, Stage-2juveniles refused to feed although they examinedthe food intensively. Refusal to internalize the foodmay be related to incomplete development of themouthparts, which only show the structural fea-

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tures of adult crayfish (Vogt, 2002; Garm, 2004) inStage 3.

Stage-2 juveniles already possess sclerotizedteeth in their stomach suitable for macrophagousfeeding, which is in contrast to the situation inStage-1 juveniles. The pyloric filters already arewell developed in Stage 1, suggesting that the stom-ach of Stage-2 juveniles should in principle be readyfor feeding. In the Australian parastacid Cheraxquadricarinatus the teeth of the gastric mill wereonly sclerotized from Stage 3 (Loya-Javellana et al.,1994). Onset of digestion of food not only requireswell-developed teeth in the stomach but also func-tioning of the hepatopancreas, the site of synthesisof the digestive enzymes and major organ of absorp-tion of nutrients in crayfish (Vogt et al., 1989; Vogt,1993, 1994, 2002; Mohrlen et al., 2001). In the mar-bled crayfish, all hepatopancreatic cell types, thenutrient absorbing R-cells, the enzyme producingF-cells and the B-cells, which are thought to syn-thesize the fat emulsifiers, are discernable in juve-nile Stage 1 but only the R-cells seem to be func-tional. During Stage 1 they accumulate increasingamounts of lipid droplets, probably by absorptionfrom the hemolymph, reflecting a gradual transferof energy storage from the yolk sac to the hepato-pancreas.

Our data suggest that the yolk is exclusively cat-abolized by the epithelium of the yolk sac, particu-larly by the large cells of its dorsal lining. Althoughthe yolk sac has luminal connections with the poste-rior part of the stomach and the midgut, and indi-rectly also with the hepatopancreas tubules, theseorgans are apparently not involved in yolk digestionas hypothesized by Fioroni (1969) because their epi-thelial cells appear inactive in Stage-1 and Stage-2juveniles. In early Stage-3 juveniles, yolk normallywas not found in the stomach-midgut-hepatopan-creas system, suggesting that yolk digestion canoccur in parallel with digestion of food, which wouldsupport the above idea. In P. clarkii, a speciesclosely related to the marbled crayfish (Scholtzet al., 2003), Hammer et al. (2000) measured anincrease of the specific activities of three digestiveenzymes (a-amylase, trypsin and non-specific ester-ase) before food was internalized for the first time,indicating that the molecular digestive competenceis attained shortly before onset of feeding in Stage3. This fits our cytological findings that F-cells andB-cells are functional from Stage 3 only.

It has repeatedly been hypothesized that the firstfood of crayfish juveniles might be their exuviae oregg cases (Andrews, 1907), because these structuresusually disappear from the maternal pleopods aftermolting of the Stage-2 juveniles. Our in vitroexperiments revealed that Stage-3 juveniles inprinciple are able to eat their exuviae and egg casesbut if other food sources are available they oftenleave the more rigid egg cases untouched. Undernatural conditions the egg shells may either be

eaten or be mechanically removed by the intensemovements of the Stage-3 juveniles on the pleopods.A unique means to get rid of the empty egg cases isknown from the crab Sesarma hematocheir, wherethey are chemically stripped off by release of anovigerous setal stripping substance (Saigusa andIwasaki, 1999).

When Stage-3 juveniles of the marbled crayfishwere allowed to choose between pellet food andmaternal feces they always preferred the feces.This may simply be due to the weaker consistenceof the feces but coprophagy could also be a means tointernalize maternal microbial symbionts (Micke-niene, 1999). However, we have not observed bettergrowth and development in juveniles raised withmaternal feces compared to siblings raised withoutmaternal feces.

Molting of early juveniles

Molting of early juveniles, to my knowledge, hasnot been documented in detail as yet, neither incrayfish nor in other decapods. The first post-em-bryonic stages of the marbled crayfish leave the oldexoskeleton in the same way as the older life stages,namely through a dorsal slit between the cephalo-thorax and abdomen. They also build up gastrolithsbefore molting. Differences in molting in adoles-cents and adults concern the time of molting andthe movement patterns before and after ecdysis.

In vitro, the juvenile Stages 1–5 molted at anytime of the 24-hour cycle, which is in contrast to theadolescents and adults that generally molted dur-ing the day when their conspecifics were in thedens. Stage-2 juveniles were immobile for hoursbefore molting, needed a few seconds for ecdysisand were immediately motile thereafter. Adoles-cents and adults, in contrast, moved around almostuntil ecdysis, needed one to two minutes for ecdysis,and stayed up to half an hour at the site of moltingto limber up their appendages, test escape reflexes,and refill their statocysts with sand particles.

A very special aspect of molting in Stage-1 juve-niles is the delayed shedding of the hindgut, whichproduces the anal thread. In vitro tests revealedthat this curious structure was firm enough to keepthe emerging Stage-2 juveniles linked to the mater-nal pleopods via the attached Stage-1 exuvia, sug-gesting that in the wild the anal thread is a suitablemeans to protect freshly molted Stage-2 juvenilesfrom being washed away. An anal thread has beenfound in representatives of the Cambaridae andParastacidae but is lacking in the Astacidae andapparently also in the the cambarid genus Cambar-oides (Scholtz and Kawai, 2002).

Concluding remarks and perspectives

The combination of in vitro culture, behavioralanalysis and microscopic investigation of late



embryos and early post-embryonic stages of theparthenogenetic Marmorkrebs proved particularlysuitable to gain deeper insights into obscure lifephenomena of freshwater crayfish. In vitro cultureof the marbled crayfish, which can be accomplishedin very simple culture systems with low mortality,appears to be especially promising for research onorganogenesis in crayfish, as developmental eventscan be precisely correlated with time and can beexperimentally manipulated. Our data indicatethat the digestive tract and most sense organs onlybecome functional during the first post-embryoniclife stages.

The combination of in vitro culture, behavioralanalysis and microscopic investigation is also suita-ble to distinguish between innate and learnedbehaviors and to examine the variability of inbornbehaviors among isogenic batch mates. In vitroculture of genetically identical marbled crayfish isfurther expected to facilitate investigation of theestablishment of self-reinforcing circuits involvingaggression, growth and endocrine feedback. Mean-while, in vitro culture of the Marmorkrebs wasshown to also have considerable potential as an in-vertebrate embryo-toxicity test system (Vogt, 2007),using 12-well micro-plates for culture.

Finally, I would like to stress that culture of thisunique crayfish is recommended for research only,which can be performed under secure laboratoryconditions, but not for aquaculture due to its poten-tial ecological threat discussed in an earlier paper(Vogt et al., 2004).

ACKNOWLEDGMENTS

The author thanks Ute Bieberstein for takingcare of the marbled crayfish stem population, Clau-dia Kempendorf (Department of Zoology, Universityof Heidelberg) for technical assistance in scanningelectron microscopy, Erika Becker (Zoological Insti-tute and Museum, University of Greifswald) forpreparation of the serial sections, Gisela Adam(Department of Zoology, University of Heidelberg)for photo-technical assistance, Volker Storch(Department of Zoology, University of Heidelberg),Werner Franke (German Cancer Research Center,Heidelberg) and Gerd Alberti (Zoological Instituteand Museum, University of Greifswald) for supportwith technical equipment. Many thanks also to twoanonymous referees and Frederick W. Harrison forvaluable comments and linguistic corrections.

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Investigation of hatching and early post-embryonic life of freshwater crayfish by in vitro culture,...

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Transcript of Investigation of hatching and early post-embryonic life of freshwater crayfish by in vitro culture,...