Contributions to Zoology, 71 (1/2) 9-28 (2002)

SPB Academic Publishing bv, The Hague

and disc formation of the Orchestia Cleavage, gastrulation, germ amphipod cavimana (Crustacea, , )

Gerhard Scholtz & Carsten Wolff

Institut 10115 Humboldt-Universität zu Berlin, für Biologie/Vergleichende Zoologie, Philippstr. 13, Berlin,

Germany, e-mail: [email protected]

Keywords: cell lineage, cell migration, Arthropoda, phylogeny, evolution

Abstract Contents

Investigations ofamphipod embryonic developmenthave a long Introduction 9

tradition. of Material and methods 10 However, many aspects amphipodembryology are

still the of the controversial. These concern, among others, nature of Maintaining and embryos 10

the the of the the of Live observation 11 cleavage, origin germ disc, and mode

gastrulation. On the other hand, amphipods show the same Video recording 11

characteristic the and ofinvariant cell division pattern in germ Histology scanning electron microscopy 11 band as other malacostracans. Since amphipods seem to undergo Injections 11

a stereotyped pattern ofearly cleavage they are highly interesting Nomenclature 12

for our understanding ofthe evolution ofarthropod development. Results 12

In this The first four paper, we describe the cleavage patternof the amphipod cleavages 12

The and further Orchestia cavimana from the zygote to gastrulation fifth cleavage 14 and the formation of Mirror the germ disc using direct observation, images 17

The of scanning electron microscopy, histology, video recording, and formation the germ disc 17

lineage with follows Gastrulation tracing a vital dye. The early development 19 the mode of a total, radial, unequalcleavage with a determinate Discussion 22

stereotyped pattern. A small transient blastocoel is formed.The A secondary meroblastic radial cleavage despite

8-cell is stage characterisedby 4 micromeres and 4 macromeres. yolky eggs 22 One quadrant is smaller than the others. There are two kinds of Mirror images 24

e ggs that show The is the of the disc of a mirror handed image. 16-cell stage Origin germ by migrations macromere last regular stage after which the blastomeres divide highly derivatives 24 - The disc is formed the descendants Gastrulation in the asynchronously. germ by amphipods only “protostome” of the The other malacostracan macromeres and some micromere derivatives. 25

micromeres constitute the extra-embryonic region. Migration The embryonic development ofamphipods shows of macromere descendants is involved in disc formation germ many apomorphic characters 25 accompanied by the extrusion ofthe yolk. During this process Acknowledgements 26

some vitellophages are formed. The gastrulation sensu stricto References 27

is initiated by the micromere derivatives ofthe smallest quadrant at the anterior ofthe disc. A forming germ true blastopore occurs which involves an invagination and the immigration of cells. Introduction Our data help to correct erroneousinterpretations of former

students of We show that amphipod development. can many

chaiacters of The of amphipod embryonic developmentare apomorphic early development exhibits a great

supporting amphipodmonophyly. With the present investigation variety of modes with respect to virtually all as- we contribute to a complete understanding of the embryonic at level. We find all sorts cell pects every of cleavage lineage of from the formation amphipods egg to segment and modes from total to superficial (Anderson 1973; organogenesis. Scholtz 1997), different ways of gastrulation (Wey-

goldt 1979), numerous modes of germ band for-

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and of 8-cell mation segmentation processes (Scholtz 1997), originates from the micromeres the stage. and all kind of larval direct In and stages up to develop- contrast, Langenbeck (1898) Rappaport

ment (Anderson 1973). This diversity at the cellu- (1960) found that the macromeres of the 8-cell stage

rise to the disc cells. lar, morphological and ecological levels is also give germ Furthermore, some authors that the reflected at the genetic level where early pathways suggested cleavage pattern of

shows of are evolutionarily altered despite a similar result- amphipods aspects spiral cleavage (Sheader

& Shia which has been denied other ing or subsequent pattern (Abzhanov & Kaufman 1970) by re- searchers 1997; Wolff & Scholtz 1999; Jockusch et al. 2000; Davis & Patel 2002). (Scholtz 2002).

In order to a better of This high degree of variation can be found even gain picture early amphi-

we several such within subgroups such as malacostracan pod development applied techniques

as direct observation of embryos, video re- crustaceans or hymenopteran insects (Grbic 2000; living

Scholtz 2000). cording, scanning electron microscopy (SEM), plas-

tic for and the vital Dil The present study describes the early cell lin- embedding histology, dye to cell In and disc as a marker study lineage. particular, we eage during cleavage, gastrulation germ address the in- following questions: what of cleav- formation of an amphipod crustacean. Although type total radial? age occurs, or superficial, spiral or vestigations on amphipod embryology started as

Where do the germ disc cells come from? When early as 1837 with Rathke’s report, there are still

are the axes of the determined? What kind controversial views and con- embryo many open questions In of gastrulation is found? a first we traced cerning the exact mode of amphipod embryonic paper, the fate of individual cells of the 8- and 16-cell development. Like all other malacostracans studied, & Here describe stages (Wolff Scholtz 2002). we amphipods undergo a distinct stereotyped pattern the cleavage pattern in detail, the formation of the of cell divisions from the formation of the post- germ disc, and the gastrulation process. naupliargerm band up to segmentation, neurogenesis, Our results confirm that the amphipod Orchestia limb bud formation, and mesodermal differentiation shows total cleavage with a highly stereotyped cell (Dohle & Scholtz 1988; Scholtz & Dohle 1996; lineage. The germ disc is formed by derivatives of Scholtz et al. 1994; Scholtz 1990,2000; Gerbcrding both micromeres and macromeres with the mac- & Scholtz 1999, 2001). However, most of the de- romeres giving rise to the largest part of the ecto- capod and peracarid studied with respect derm and to mesendodermal parts of the embryo in to their cell lineages the germ band undergo a whereas the micromeres contribute partly to the superficial cleavage that does not allow the tracing mesendodennand to the extra-embryonic ectoderm. of early cell lineages (if at all present) up to germ The cleavage is not spiral. Gastrulation takes place disc formation and gastrulation. This is different at the anterior end of the disc. germ in amphipods; there is some evidence from several The general pattern of early amphipod develop- studies that the early cleavage of amphipods shows ment is malacostracans and crusta- unique among a stereotyped pattern (e.g. van Beneden& Bessels ceans in general. It shows a numberof apomorphic 1869; Ulianin 1881; Langenbeck 1898; Heidecke characters related to cleavage, gastrulation, germ 1904; Rappaport 1960; Brcgazzi 1973; Meschen- band proliferation, and gene expression unifying moser 1987). However, in some respects these in- the different amphipod taxa. vestigations led to contradictory results. For instance,

La Valettc St. George (I860), Ulianin (1881) and

Mergault & Charniaux-Cotton (1973) claimed that Material and methods

the cleavage is not total but that the central yolk

mass remains undivided whereas authors such as Maintaining of the animals and embryos

Langenbeck (1898) and Percyaslawzewa (1888a)

suggested a clear total cleavage mode. Several Specimens of the terrestrial amphipod species Or- authors (e.g. Ulianin 1881; Bregazzi 1973; Mergault chestia cavimana were collected from beaches of

& Charniaux-Cotton 1973) stated that the germ disc the Tegeler See (Berlin) and from the river Weser

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close to the town ofElsfleth where they live in and Sony SSC-M370CE, analogous recorder: Panaso- feed detritus on (Rudolph 1995). The animals were The was one minute. nic). frequency picture per terrarium 18-20°C maintained in a at and fed with Later the recorded sequences were digitised. The

carrots and rolled oats. To receive in relevant eggs video films are put on the homepage of Contribu- the in stages a so-called praecopula (the male tions to Zoology thttp://www.uba.uva.nl/ctz). carries the female around for a while before copu-

lation) were caught and isolated. Usually after about

10-14 hours the animals and the female’s separate Histology and scanning electron microscopy ventral brood pouch (marsupium) contains eggs in

an early stage of cleavage. Females with in For eggs semi-thin sections the embryos were fixed for their where dazed in mineral marsupium carefully 30 min to 2 h in buffered 5% formaldehyde, Bouin’s

water C0 The were washed containing . eggs out solution or a mixture of acid and sublimate. 2 picric of the marsupium from anterior with a Pasteur The pi- egg envelopes were removed during fixation

pette. The were transferred salt dishes eggs to con- (if possible) to allow better penetration. After wash-

taining a saline which mimicks the in the in and liquid ing distilled water a dehydration in an etha-

marsupium saline saline = series the (isopod : NaCI-frog 1:2; nol embryos were transferred to the meta- [ saline: isopod 12 g NaCl, 1,6 g KC1, 1,6 CaCl medium g , crylate embedding 2 (Technovit) following 1,6 g NaHC0 1 I MgCl,, 0,2 g to aqua desk; NaCI- standard Serial semi-thin sections 3 protocols. (3pm) frog saline: 115 mM 2,5 mM KC1, 2,15 NaCl,' mM were produced with a Jung microtome. Toluidine-

Na,HP0 mM mM *2H,0, 0,85 NaH,P0 *H,0, 1,8 blue was used for Some whole-mount 4 4 staining. CaCl 7 *H,0]). done of of preparations were eggs various stages.

In this case the fluorescent dye Bisbenzimide

(Hoechst) was used to stain nuclei. The stained eggs Live observation transferred into were glycerol and covered with a

cover slip. Micrographs were taken with a Zeiss For the observation of and we 1 with living eggs embryos Axiophot equipped a digital camera (Nikon used stereomicroscopes (Zeiss, Leica) equipped with Dl).

a camera and a Broods that The solutions cold-light source. were same for fixation as for histology

transferred to saline in salt dishes continu- used for were were scanning electron microscopy. After ously observed the had be turned the (although light to dehydration embryos were transferred into ac- off from time time dried to to avoid damage of the em- etone, at the critical point (Balzers Union) bryos). The progress of development was docu- and sputter coated (Balzers Union). Observations mented by drawings and photographs. were done with a Philips scanning electron micro-

scope.

Video recording

Injections The eggs were transferred in saline (see above) into

a s P e cially designed container with a The in the 8-cell glass (30 ml) eggs stage were fixed on mi- bottom made of a cover and on an inverted slides under small slip put croscopic cover slips that were microscope (Zeiss Telaval Fixation ofthe 31). eggs equipped with plasticine feet at the corners. The was done with a rubber on the cover could be the ring glued eggs brought in right position by care- s bp. The saline was with oxygenated an fully the cover With soft aquarium shifting slip. pressure on Pump through a filter (0.2 im) and the cover the changed every slip eggs were fixed for the injection. second The All day. light came from a cold source. of the 8- light macromeres cell stage were marked ermanent light destroys the so a switch was To eggs, subsequently. get suitable needles for the injec- integrated in the set which turned on the tion, diameter up light pipettes (Hilsberg, 1,0 mm, thick- 01 just one shot of the recording system (camera: ness 0,2 mm) were pulled (KOPF Puller 720). After

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this, the tips had to be sharpened (Bachofer). The Nomenclature

angle of the cutting edge varied between 20 and 30

the 2-cell degrees. The fluorescent marker was sucked into The blastomeres of stage cannot be rea-

the injection-needle. Oil (l,l'-dioctadecyl-3,3,3',3'- sonably labelled because the fate of the individual

tetramethyl-indocarbocyanin perchlorate, Molecular cells is unclear. At the 4-cell stage distinct differ-

Probes) was used as a vital marker. Dil is a lipo- ences between the quadrants are recognisable (Fig.

which binds the cell Viewed from ‘dorsal’ future micromere philic fluorescence-dye to mem- 1). (the

blastomere is named A brane. This guarantees that the dye is exclusively region), the smallest fol-

lowed and in a clockwise restricted to the daughter cells. A further advan- by B, C, D sequence.

tage is the kind of the application. The injection The 8-cell stage shows a characteristic pattern with

four micromeres and four As in needle does not have to penetrate the membrane of macromeres. the 4-cell the descendants of the smallest the marked cell. It is enough to penetrate both em- stage quad-

rant are labelled A (macromere) and a bryonic covers and to put down a drop of Dil on (micromere) the the other blastomeres are labelled in a the cell membrane. This drop diffuses over hole accordingly clockwise cell The done with sequence. Nomenclature for the 16-cell surface. cell marking was an

stage. Since the symmetry axis of the embryo runs inverse microscope equipped with a micromanipu-

lator through the A/C axis, the blastomeres are labelled (Leica DMIRB). Theopaque purple yolk makes their the axis of the according position to length the eggs non-transparent. Therefore, during Dil left and and anterior and cold embryo (I) right (r) (a) injection a light source was used to illuminate 1 6-cell posterior (p). After the stage the cleavage the eggs from the side. After the penetration of the products of equatorial cleavages are named d for two embryonic covers (chorion, vitelline membrane) blastomeres closer to the micromere pole and v for a drop of Dil was set on the membrane of single blastomeres closer to the macromere pole. cells. The diffusion was observed under blue fluo- rescence light. This wavelength causes only weak

stimulation of Dil, and thus the dangers of dye fading Results and After damage to the egg were avoided. injec-

tion the into dish with saline eggs were put a petri Thefirst four cleavages (see above) and kept at 18-20°C in darkness. After

a defined period of development the marked eggs fertilized The oval shaped egg is about 500 pm long were put on a slide and fixed with a cover slip. The and 300 in diameter. It is rich of coloured pm purple observations were done with a fluorescence-micro-

marbled many of Polar blue yolk containing droplets lipid. scope (Zeiss Axiophotl) using light or green bodies differentiations or other cannot be seen, and light (strongest stimulation of Dil) and the results therefore no polarity can be recognised (Fig. 1A). were documented with analogous and digital cam-

The nucleus lies eccentrically in a stellate mass of eras (Zeiss C 80 (Agfa CT 100), Nikon Dl). The yolk-free protoplasm which has thin protrusions analogous slides were digitalized with a scanner

passing through the yolk forming a three-dimen- (EPSON Diascan) and tailored with Corel Draw to sional net. This plasmatic net is connected with a size. The retouched. the desired pictures were not thin layer of periplasm surrounding the yolk. If necessary, the embryos were dissected in buff- first first becomes The cleavage at visible as a ered 4% formaldehyde-solution and embedded in furrow on one side propagating around the egg. The DABCO-Glyccrol (25 mg DABCO [1,4 diazabi- cleavage is total and meridional, and with some in 1ml cydol-2,2,2-octane, Merck] PBS [8 g NaCl, variation the cleavage furrow is oriented obliquely 0,2 g KC1, 1,44 Na HP0 0,24 KH,P0 pH g g , 2 4, 4 to the long axis ofthe egg (Fig. IB). The nuclei of 7,2]to 9 ml Glycerol). the two daughter cells lie excentrically.

The second cleavage is again meridional. It leads

to a first distinct differentiation of cells which al-

lows to identify each cell with respect to its posi-

tion and size. The two blastomeres divide asym-

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Fig-1. Cleavage in Orchestia cavimana. Two A) living uncleaved eggs with the characteristic marbled purple yolk. B) The 2-cell sta ge (living The eggs). cleavage plane shows a certain variability. C) The 4-cell stage (unstained, living egg). The two small blastomeres A and C and the two blastomeres and is the smallest blastomere. large B D are recognisable. A The two large blastomeres touch each other in the median whereas the small blastomeres The 8-cell region are separated. D) stage(unstained, living egg). A tier of 4 micromeres (a-d) lies on top of4 The cells show radial A is the smallest macromeres (A-D). a arrangement. macromere, a the smallest nucromere. SEM E) of 16-cell The blastomeres The A is still picture a are = stage. radially arranged. quadrant the smallest, ch chrion. * Two living eggs the 5"’ cleavage. The white at the surface. The finished starting cytoplasm appears macromeres their division (right _ ogg, lateral view) whereas the micromeres (left “dorsal” view) are still in mitosis egg, (elongated white cytoplasm). The cells al and •>i lave not yet started their 5,h The 6"' cleavage (arrow). G) cleavage ofthe macromeres, lateral view. From left to right: the derivatives o Bp, Cl, and Cr are seen. Sister cells are connected by a line which indicates the spindle direction. This pattern is typical for the envatives of macromeres B, C and D. The derivatives ofA show a somewhat different pattern (see Figs. 8A, B) (compare Figs. 4C, "I The 7"' cleavage of the macromeres. As in the derivatives of and Cr be ' (G) Bp, Cl, can seen (compare Fig. 5). I) The 5"' cavage of the micromeres (“dorsal” All micromeres underwent their division ar view). except al, (arrow), ba, and da (compare Fig.

■Ticti ically producing a larger and a smaller descen- The cell A is 3A). the smallest blastomere (Figs. dant each do divide (Figs. 1C, 2A,B). They not 1C, 2B). Again the relative size ofthe blastomeres synchronously The division are and the show (Fig. 2A). planes cleavage planes some variation. There- Parallel. The resulting two descendants B the cell A not larger fore, is always unambiguously iden- and D meet at the top and the bottom of the egg, tifiable. Wlcie as the smaller blastomeres A and C have The third division is the first equatorial cleav- contact only in the centre ofthe (Figs. 1C, 2A,B, Each of the four egg age. blastomeres divides highly

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orientation. unequally with a clearly radial spindle

The divisions are not synchronous and show no

the smallest blastomere predictable sequence. Only

A always divides last. The result of the third cleavage

four micromeres labelled d and four are a, b, c, macromeres A, B, C, D (Figs. ID, 2C). The size relations of the quadrants at the four-cell stage are maintained during the third and subsequent cleav-

Since the blastomere of the ages (Figs. I D, 2C). A four-cell stage is the smallest, its descendants are also the smallest macromere A and the smallest micromere a of the eight-cell stage. During the 8- ccll stage the colour of the micromeres becomes lighter than the one of the macromeres. The small- est blastomere a is most advanced with respect to this. This phenomenon is caused by migration of the nuclei and the cytoplasm towards the periph-

of the micromeres ID and ery (compare Figs. IF).

The white colour of the cytoplasm is due to cal- cium concrements (Meschenmoser 1987). During the 8-cell a small blastocoel is formed 2. Six of the stage (Fig. Fig. stages same egg showing the sequence of

3B). early cleavages. A) Transition from the 2-cell stage to the 4-

cell meridional cleavage. The blastomeres A and and The fourth cleavage is again meridional leading stage; B,

C and D are sister cells. The cleavage furrow runs from the to a 16-cell stage with an upper tier of eight mi- the of The 4-cell 8- centre to margin the egg. B) stage. C) The cromeres and a lower tier of eight macromeres in cell stage. D) Transition from the 8-cell to the 16-cell stage. a radial IE, typical arrangement (Figs. 2F). Always Except for A, a all micromeres and macromeres have divided the one of large macromeres (B or D) starts divid- (B, b, C) or are in division (D, c, d). E) Transition from the 8-

cell the 16-cell All cells but micromere have finished ing. Its corresponding micromere mostly divides to stage. a their The The divisions. F) 16-cell stage. open space between next. Then a macromere divides, followed by its the micromeres allows a view into the blastocoel (see Fig. 3). sister micromere. Again, the sequence of divisions is not strictly determined. Only the smallest mac- romere A is always the last macromere to divide and the smallest micromere a is the last blastomere at all undergoing fourth cleavage (Figs. 2D-F). When it has finished its mitosis, the 16-cell stage is com- plete.

The and fifth cleavage further 3. Semi-thin sections Fig. through eggs duringearly cleavages,

Horizontal section the of 8-cell A) through macromeres an stage.

cell The second and third cleavage divisions leading to It is evident that all blastomeresare separated by membranes which is typical for total cleavage. The cytoplasm and the nuclei the 4- and 8-cell stages are already not synchro- lie in the centre of each cell. The arrow marks the A quadrant, This nous. tendency becomes much more distinct Transverse of 16-cell The B) section an egg after the stage. the fourth a 16-cell during cleavage. Nevertheless, to blastocoel. arrow points the still stage develops. However, this is the last “regu- lar” stage. This is due to several factors:

From the (i) 16-cell stage on mitoses of the mac- romeres are always in advance as compared to those

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sequence is not to be noticed. Each cell can start

this phase with the exception of the blastomeres

Ar and Al which always divide last. The cleavage

of the cells Ba, Bp, Cl, Cr, Da, Dp is equatorial

oriented and The lower unequal (Figs. IF, 4). po-

sitioned daughter cells Bav, Bpv, Civ, Crv, Dav,

and of Dpv are longitudinal shape and relatively

large, whereas the upper descendants Bad, Bpd,

Cld, Crd, Dad, and Dpd are smaller, round, and

lie around the equatorial area of the egg (Fig. 4).

of the The spindle directions single divisions are

determined. not strictly Their orientation depends

the and the the on space position of cell, which are

both due to earlier cleavages. In contrast to all the

Al and other macromeres, Ar divide more or less

equally (Fig. 4, see also Fig. 8A). The descendants

Alv and Aid and Arv and Ard show mostly a rhom-

boid arrangement, which is due to the inclined spin-

dle axes. Their nuclei and the white cytoplasm are

This now seen at the egg surface. stands in con-

the situation trast to in the other macromeres, where

the nuclei and the plasma reach the egg surface in

somewhat later stages.

The mitoses of the fifth in cleavage the mac-

romeres are mostly finished by the time the mi-

4. Fig- Three of the the of start to stage same egg showing sequences cromeres divide. Sometimes the small macro- the 5"’ th and 6 th cleavage ofthe macromeres and of the 5 cleavage Al and Ar meres divide synchronously with the °1 the micromeres. A)The eight micromeres ofthe 16-cell stage micromeres starting the fifth cleavage. As in the are still undivided. The elongated cytoplasm (except in al and

ar a defined of mitoses cannot )’ lh macromeres, sequence however, indicates the beginning ofthe next (5 ) cleavage. The be in the 5"' of the found micromeres. Often divide al- cleavage macromeres is almost finished. Only they the blastomeres Al and Ar have not finished their mitosis. B) A most synchronously, with the exception of the blas- slightly advanced to that of stagecompared (A). Mostmicromeres tomeres ar da al, and ba, which show a retarded

underwent ,h lh the 5 C) The of the 6 cleavage. beginning cleavage further development (Figs. 11, 4). As in the mac- "i the macromeres. The show upper macromere derivatives a the orientations meiidional romeres, spindle are more or less cleavage whereas the lower derivatives undergo an

equatorial ,h to the one of the cleavage (compare Figs. 1, 5). The 5 cleavage of perpendicular previous cleavage. die micromeres is still The smallest al incomplete. The derivatives of a and the micromeres, and ar, undergo their

adjacent cells ba and da show a delayed development(see Fig. mitoses last. The spindle axes of this division are

perpendicular to the one of the previous mitosis.

the Furthermore, spindles are slightly inclined to- °fthe micromeres - with increasing tendency (Figs. wards the interior of the egg. Consequently, the * 4)- During germ disc formatiort the macromeres cells of al and daughter ar are in a lower position tic about three divisions ahead, (ii) The cells Ar somewhat sunken into the yolk. This is the begin- Tnd Al, the smallest macromeres, and ar and al, of the ning blastopore formation in the area of the the smallest micromeres, and their cells adjacent gastrulation centre. The blastomeres adjacent to al show retarded mitoses further in develop- and along ar, the cells ba and da, show a retarded develop- ment (Fig. 4). ment in this stage. Their daughter cells also become Once the 16-cell stage is complete, the mac- involved in the early gastrulation process. Flowever, romeres are next to divide. Again a determined these do show phenomena not an identical pattern

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in all The of these mitoses is eggs. sequence ap-

parently not fixed. During the sixth cleavage of the

macromeres the retarded micromeres sink deeper

into the yolk. Thereby they undergo a change of

their shape. The nuclei and the yolk-free cytoplasm

are now superficial and the yolk of each cell is shifted

towards also the centre of the egg (Figs. II, see

the cells columnar Figs. 7, 8). Thus, assume a ap- pearance.

The sixth cleavage ofthe macromere region starts

before the micromeres have finished their fifth cleav-

First the smaller cells Cld, age. Bad, Bpd, Crd,

Dpd, and Dad divide almost synchronously into

Bada and Badp, Bpda and Bpdp, Crdr and Crdl,

Cldr and Cldl, Dpda and Dpdp, Dada and Dadp

(Figs. IG, 4C). All these mitoses are equal. The

spindle axes are again perpendicular to those of

the previous divisions. This means it is in principle

a meridional cleavage. Flowever, as in the last cleav-

be ages many deviations can found caused by ear-

lier positional changes of single blastomeres. These

deviations of direction be to 90°. spindle can up Fig. 5. Schematic representation of the cleavage pattern ofthe these there exist about 10 to 13 During processes macromeres B,C, I) lateral (view). A shows a somewhat diffe-

micromeres. The divisions of the is retarded subsequent larger rent pattern and its development (see text). The

th ventralmost cell from the behaves like a stem macromere derivatives Bav, Bpv, Crv, Civ, Dpv, 5 cleavage on

cell with divisions in dorsal direction. The dorsal and the asymmetric and Dav are again unequal spindle direc- follow the oriented cells typical sequence of perpendicularly in last tion is vertical as their cleavage (equatorial cleavage planes. The left number indicates the cleavage stage, cleavage) (Figs. IG, 4C). The upper derivatives the right number represents the number ofmacromeres at that

Bavd, Crvd, Clvd, and Davd are Bpvd, Dpvd, stage. The latter is idealisedsince the mitoses ofthe macromeres

again roundly shaped and smaller than their lower are not synchronous (in particular, those of A) but it gives an

estimate of the cell numbers. sister cells Bavv, Bpvv, Crvv, Clvv, Dpvv, and

Daw which are elongated (Fig. 1H). As in the

previous cleavages, the descendants of the blas-

the tomeres A divide much later. Beside their retarda- 16-cell stage onwards. This mode of division

tion the mode of their mitoses is different. Arv and is best described as teloblastic with the large basal

Ard, Alv and Aid divide equally again, and the blastomeres showing the asymmetric unidirectional

arrangement of the daughter cells does not show division sequence which is typical forteloblast stem

the typical pattern that is found in the other mac- cells. The smaller macromere derivatives, on the

romere derivatives. other hand, always alternate their division plane

in 90° This mode The sixth cleavage the micromere area starts about (Fig. 5). is kept up to eighth

before the A of has this an is made group macromeres undergone cleavage. Beyond stage analysis

its sixth the cell cleavage. Again, no predictable pattern in impossible by migration during formation

the be of the disc. The mitoses of the seventh cleav- sequence can seen (data not shown). germ

The seventh in the in the smaller cleavage of macromere descendants age macromere area again begin

follows close the same mode as described for the sixth cells to the micromere descendants (Fig. 1H).

cleavage. The basal macromere derivatives divide They are synchronous in a larger region. As before,

unequally with an equatorial division plane. Thus all these events show some variations concerning

their they keep up spindle orientation starting from the sequence, the spindle direction and the area where

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the cleavage starts. The larger ventral blastomeres in eggs every single brood is about 1/1. This phe-

lag a little bit behind with their mitoses. do nomenon can be stated They up to relatively late embry-

not show a predictable As in sequence either. all onic stages as is indicated by the inclination of the earlier the divisions of the A stages group are re- band in relation to the axis of the germ long egg. tarded and the cleavage pattern is different. All

mitoses are and the equal spindles are more or less

vertically oriented. It was observed in The the living eggs formation of germ disc that the during seventh cleavage a few cells of the

macromere region to towards the begin immigrate The cleavage divisions of the macromeres have the centre of the egg probably forming vitellophages result that the macromere derivatives have the similar (data not Whether shown). these events show a size and appearance as the micromere derivatives

reproducible could not be clarified. Nei- pattern (Figs. 7A, 8B). Furthermore, a differentiation takes ther a defined number nor a specific area was iden- place mainly concerning the A region, the smallest tified. These fill small vitellophages out the blas- Both the quadrant. macromeres Al, Ar as well as tocoel do cavity. They obviously not undergo fur- the micromeres d and their a, b, descendants, re- ther mitoses and their nuclei degenerate during spectively differ from their neighbour cells with formation of the germinal disc. In sections of these regard to their position, their morphology and the stages traces only of can be found in the and cytoplasm retardation orientation of their mitoses. As yolk (data not shown). described above, the with nuclei'together the cyto- plasm migrate relatively early (after the 16-cell

towards the stage) egg surface whereas the yolk of Mirror images these cells is transferred towards the egg centre. The derivatives of A are characterised by a round There are two kinds of a mirror of the white which eggs showing sym- margin cytoplasm stands in con-

metry. This can be 4 recognised from the to 8-cell trast to the stellate or amoeboid shape of the cyto- stage on. For if example, one looks on the in the other macromere upper plasm descendants. Together side of at eggs the 16-cell there is to be seen with these of the cell stage changes shape a retardation that the cells and Al in of the divisions ar, al, Ar, point one case occurs. During the eighth cleavage towards the left direction in the other towards the division of the other macromeres they differenti- right (Fig. The relation 6). of these two types of ate in a comparable The nuclei and way. the cyto- plasm of the macromere derivatives migrate towards

the become egg periphery, they superficial (Fig. 9). The external of the shape blastomeres changes from spherically to flat. This coincides with the

of the separation yolk from the cytoplasm of each cell. The of process yolk extrusion leads to central

yolk compartments which are surrounded by mem- branes. This phenomenon is comparable to what

has been described for other crustaceans as so-called yolk pyramids (Dotterpyramiden, Fioroni 1987).

Whether these membranes disappear in advanced

1 stages is not clear. some sections it looks as if, but this might be just an artefact caused by the fixa-

tion. As a result, the cells have the appearance of roundly white isles surrounded by the purple yolk

M' 7, 8). The loses rror of (Figs. cytoplasm its protrusions pit ' images eggs at the 16-cell stage. The two ty- s o w t the are eggs 1 a | e j-t or even. 10 shows | | jente(j quadrant(arrow) (upper egg) edges Figure these events a tight orientedA quadrant (arrow) (lower egg). schematically. These morphological changes of the

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7. of the Formation ofthe in the ofthe of Fig. Stages gastrulation. A) gastrulation centre region retarded derivatives micromeres a, b

and tl These cells delaminate into the interior of the The derivatives towards This (arrow). start to egg. macromere migrate this area.

stage is characterised by a sickle shaped arrangement of the cells at the anterior margin of the gastrulation centre (dotted line). B)

ofthe formation in the and Beginning blastopore gastrulation centre. The derivatives ofmacromeres B D start to overgrow immigrating

and raesendoderm and cell The the the real invaginating germ precursors. arrows indicate migration of macromere derivatives. C) A

formed the disc. The is closed slit. blastopore (bp) has at the anteriorof forming germ D) blastopore (bp) almost forming an elongated

Germ disc after the closure ofthe The ofthe mesendoderm due the two cell E) blastopore. area appears lighter to layers (dotted line).

ofthe ectoderm formation the and The E) Beginning row (r) at boundary between the naupliar post-naupliar regions. dorsal organ (do)

anterior the lies to germ disc.

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Fig- 9. sections the of cell Blastomere at Histological showing change shape during development. A) an early cleavage stage (about

16-cell The stage). and the nucleus are located in the centre ofthe cell surrounded cytoplasm by yolk. B) Blastomere at a stage of around 30 cells. The cell contains still amount of but the and the nucleus have the a large yolk cytoplasm migrated to egg surface. C) Cells of an disc. some are found in some of the cells. The lies in the of early germ Only yolk droplets yolk centre the egg forming compartments surrounded by membranes.

macromere derivatives first in the towards the occur equatorial migration initial gastrulation centre.

area the Later the other With the surrounding egg. macromere formation, germ disc consists of about

derivatives follow. 120- Thereby all the macromere des- 130 cells. The extra-embryonic area includes cendants begin to migrate together toward the ini- about 30 blastomeres. These numbers correspond

tial thus gastrulation centre, forming a compact germ relatively good (even when the retardedblastomeres

disc and (Figs.7, 8, 10, 11). Correspondingly, the micro- A a, b, d are considered) with the number of mere becomes region extended. This it covers divisions, which took this time way cleavage place up to the biggest in the part of the egg surface (Figs. 8, 10, 11). macromeres (8/128) and in the micromeres hi contrast to the macromere derivatives, most of (6/32). the micromere derivatives do not extrude all of their yolk (Figs, 8E, 11). Figure 11 shows macromere

derivatives, which differentiated disc are as germ Gastrulation

cells, in comparison with micromere derivatives, which are part of the Orchestia extra-embryonic region (“ex- In gastrulation happens in three phases: tra-embryonic ectoderm” see Anderson 1973). 1. The of several immigration macromeres during hi summary, one can note three the major events, seventh cleavage probably forming vitello- which are undergone by the macromere derivatives, phages. the forming germ disc; (i)The of the nuclei 2. The in of the the migration sinking micromeres in a and ar*d the towards the surrounding cytoplasm egg adjacent regions forming the initial centre for Periphery. (ii)The extrusion of the yolk. (iii)The gastrulation (Figs. 7, 8, 12).

Migration and of of descendants disc formation change the shape macromere during germ (compare Figs. 9, 10). Panels A to show the same egg at different The area of A is in the front. In C and D developmental stages. quadrant panels the egg is slightly rotated backwards. The cells Arvd and Arvv as landmarks because in this (arrows) serve particular embryo they showed an aberrant ve °pment (for some reasons did not divide and had with the extrusion oftheir The they they problems yolk). A) macromeres B to P the ,h undergo 7 is in the 6"’ The cell surface is still rounded cleavage (see Fig. 5). A cleavage. and the cytoplasm is surrounded by 0 B) Cell lh migration started. Macromeres B D least in the 8 A is in th • has The to are at cleavage, the 7 cleavage stage. The cells used fs an drnarks (arrows) have not divided. The cell surface is now flat and the is internalised. The yolk cells lie closer together. C) dtlon ° and yolk extrusion have and the disc is The to ■ proceeded early germ recognisable. arrows point the cells that serve as marks. Gastrulation stated Advanced of the has (see Figs. 7B, C). D) formation germ disc. The blastopore lies in front of the 1

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II). of the of disc Fig. Schematic representation germ disc formation.A) shows migration macromere derivatives during germ formation

the in and disc formation. row surface lower (arrows). B) depicts changes cell morphology during migration germ Upper view, row

lateral view of cells. See text for explanation.

II. in different of disc formation. disc The Fig. Sagittal sections through eggs stages germ A) Early germ formation. arrow points to

the anterior end ofthe disc. The arrowhead micromere derivative. of forming germ marks a typical dorsal B) Slightly advanced stage

disc. The the anterior end of disc. The derivatives to the show an early germ arrow points to the forming germ macromere right the

with their characteristic appearance typical for migrating cells. At the dorsal side the micromere derivatives shape are visible (arrowhead).

12. Fig. Histological sections through the gastrulation centre. A) Sagittal section through an embryo at the onset of blastopore

formation to the In the there several cell the ofcells (anterior left). anterior area of the germ disc, are layers indicating immigration

duringearly gastrulation, B) Transverse section through the blastopore in an advanced stage of gastrulation (compare Fig. 7).

3. The gastrulation sensu stricto by a combined tioned above, the retarded micromere descendants

and of anterior lie the anterior of the disc in invagination immigration germ at edge germ a deeper

disc cells which gives rise to the main part of position sunken into the yolk. From here a gastral

the mesendodermal mass is which (Figs. 7, 12). groove or blastopore forming propagates

the Consequently, two last phases are not strictly in posterior direction (Fig. 7). It has about 1/3 of

distinct. The last starts when the disc. phase just germ the length of the germ The gastrulation is a

disc consists of about 130 cells (Fig. 7). As men- combination of invagination and immigration of

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F ‘S- 13. Germ disc formation from macromere descendants Wolff& Scholtz of the cell (compare 2002). Tracing lineage using a vital ye. In every case ofthe 8-cell labelled. of B, one macromere stage was A,B) Labelling macromere A) shows the living egg with light

above. The anterior. shows the under fluorescent join dorsal organ (do) indicates B) same egg light. The labelledcells (descendants 0 B) form the side of the disc. C,D) of macromere D. the with morphological right germ labelling C) depicts living egg light from °ve. Slightly earlier in A. Anterior is the dorsal is not formed. The dotted line stage than panel up, organ yet indicates the mesen- ermal mass the The labelled cells - D) shows same egg under fluorescent light. (descendants of D) form the morphological left half o the germ disc. E,F) of macromere C. the with Labelling E) depicts living egg during gastrulation light from above. The blastopore P) marks the anterior end of the shows the under the germ disc. F) same egg fluorescent light. Cells at ofthe posterior margin germ ISC esc endants of the C) are labelled. of macromere A. G) depicts living with from G,H) Labelling egg light above (same stage as Panel C). The dotted line indicates the mesendodermal mass at the anterior ofthe the embryo. H) shows same egg under fluorescent Ofdy cells of the mesendodermal mass (descendants ofA) are labelled.

k erm d' s c cells rise mesendodermal In late this giving to the germ discs, can be recognized by the mass. The blastopore becomes closed of the anterior by growing brighter appearance region due to pettier °f the two the lateral edges in posteroanterior two layers of cells (Fig. 7E). The fate of the lrect >on. After closure the disc shows char- blastomeres germ a gastrulating A, a, ba, and da has been acteristie pear-like shape with a narrow anterior described in another part paper(Wolff & Scholtz 2002). ’8- VE). little The derivatives of A During gastrulation mitotic activ- give rise to the mesoderm of 1 Y can be seen within the The the germ disc. cells of naupliar region and to anterior parts of the mesendodermal mass and the cells endodermal primordial germ midgut glands, a forms the primordial re spiead under the ectoderm and the layer (Figs. 7E, F). germ cells, gastrulating descendants of ba

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& and da form the post-naupliar mesoderm and the ton 1973; Magniette Ginsburger-Vogel 1982).

posterior region of the endodermal midgut glands However, some students of amphipod development that the furrows After gastrulation has finished, some blastomeres came to the conclusion cleavage

undivided the are an central (see Wolff& Scholtz 2002) in front of germ only superficial, leaving

mass Valette 1860; van Beneden disc begin to differentiate into cells forming the yolk (La St.George

& Bessels Ulianin Della Valle dorsal For a detailed 1869; 1881; 1893; organ (Fig. 7F). description & Charniaux-Cotton 1973). Van Beneden of this structure see Meschenmoser(1996). The cell Mergault & Bessels concluded that the of all marking with Dil clearly reveals that the outer (ec- (1869) eggs and disc is marine gammarid species cleave totally, that, todermal) layer of the germ exclusively in contrast to this, the of freshwater species formed by derivatives of macromeres B, and D, eggs ( pulex and Gammaruspoecilurus) cleave and transiently of C (Fig. 13) (see Wolff & Scholtz This is not correct. In her which is superficially. hypothesis 2002). The only part of the germ disc partly investigation, Pereyaslawzewa (1888a) shows clear- formed by micromere derivatives is the most ante-

that Gammarus a total rior lower of the disc. Here lie the de- ly poecilurus undergoes layer germ of blastomeres and our own observa- scendants of micromeres ba and da shown separation a, (not tions of the eggs of the freshwater species Gammarus in this paper). Together with the derivatives of pulex reveal the same total cleavage type as in macromere A they form the mesendodermal mass Orchestia There is some evidence that cells (unpub.). and the primordial germ (see Fig. 13) (Wolff in the “superficial cleavage” amphipods is an arte- & Scholtz 2002). The complete germ disc is oval fact due to the problems with fixation of the yolky shaped and lies obliquely on the egg surface (Fig. and this also be the reason for the eggs, may opin- The mirror arc still discernible. 7). image germs ion that the total cleavage changes to superficial disc be- The ectodermal cells of the germ start to after the 16-cell stage (Heidecke 1904; Wey- the for- right come arranged in transverse rows during goldt 1958). We can clearly demonstrate by semi- sometimes mation of the dorsal organ or slightly thin sections and by the lineage tracing with Dil later (Fig. 7F). In the mesoderm the mesoteloblasts that the cleavage is entirely total until the yolk are differentiated. The further development of the compartments are separated from the blastomeres germ disc and germ band follows the same mode disc formation. during germ as described in detail for the amphipod Gammarus The later cleavage events, from the 16-ccll stage pulex (see Scholtz 1990; Scholtz et al. 1994; Wolff described in detail on, are only by Langenbeck & Scholtz 2002). (1898) and in parts by van Beneden & Bessels (1869)

who developed a schematic representation of later

cleavage divisions. The problem of the interpreta- Discussion tion of advanced cleavage stages is the variability

between the the and size eggs concerning position A secondary meroblastic radial despite cleavage ofindividual cells, the different division frequency

yolky eggs of the blastomeres, the high degree of cell migra-

tion found in the macromeres, and the tendency of

The reveals that the present investigation early the macromere derivatives to decrease in size so

embryonic ofthe Orchestia more development amphipod that they become more and difficult to dis-

cavimana follows the mode of a un- total, radial, tinguish from the micromeres and their progeny.

equal early cleavage. These results correspond, up A further complication is due to the mirror images

to the 16-cell the and of the which led stage, to descriptions figures germs (see below), investigators found in the papers of several authors who studied who did not notice this phenomenon to erroneous

& 1898: 2 amphipod development (e.g. van Beneden Bessels interpretations (e.g. Langenbeck Figs. and

1869; Rossiiskaya 1888, Pereyaslawzewa 1888a,b; 3). On the other hand, almost all cited authors re-

Rossiiskaya-Koschewnikowa 1890, 1896; Wagner port that from the 16-cell stage on the macromere

1891; Langenbeck 1898; Heidecke 1904; Rappaport divisions are in increasing advance compared to

1960; Bregazzi 1973; Mergault & Charniaux-Cot- the micromeres and that the blastomeres of the small-

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est quadrant (micro- and show macromeres) some of early cleavages that the cleavage of Orchestia is retardation in their cleavage activity. Different views not of the all blastomeres spiral-cleavage type; are

are van Beneden presented by & Bessels a modified radial (1869) arranged according to cleavage. and Lalitha al. who et (1989) report synchronous This conclusion is supported by the tracing of the divisions and by Mergault & Charniaux-Cotton cells the lineage of individual at 8- and 16-cell stages (1973) and Bregazzi (1973) who write that the di- which shows remarkable differences between spi- visions of the micromeres are in advance - which ral cleavage and the cleavage mode of amphipods

is, at least for not In & Orchestia, correct. principle, (Wolff Scholtz 2002). Furthermore, it is clear the descriptions given cor- that the of by Langenbeck (1898) cleavage amphipods is highly derived

respond in several to those found malacostracans aspects very good among and crustaceans in general. by us in Orchestia. Nevertheless, the above sketched Again this is true for the cleavage pattern and the difficulties of lead to some fate of cells interpretation contra- single (this paper; Wolff & Scholtz dictions in her On 308 she writes, “The This raises the report. page 2002). question, what was the rea- subsequent cleavage the 8-cell son for the shift planes (after stage) evolutionary from superficial cleav- which divide the two macromeres alter- towards total larger are age cleavage in amphipods with such

meridional and nately equatorial, while the an elaborated planes cleavage pattern. There is a prob- dividing EF (our blastomere and the micromeres lem. All studied A) amphipods possess relatively large are always meridional”. The corresponding figures yolky eggs. Accordingly, one would expect that show different patterns more the con- the has be of resembling cleavage to the superficial type. More- ditions we have found in Orchestia, i.e., the mac- over, amphipods are phylogenetically nested within

romeres from the 16-cell stage on divide in prin- the malacostracan taxon Peracarida which is cha-

c,ple as shown in 5. The schematic Fig. represen- racterised by brood care with a ventral brood pouch, tation of advanced as direct cleavage presented by van development, yolky eggs, and superficial

Beneden & Bessels several (1869) for gammarid cleavage (Johnson et al. 2001; Richter & Scholtz species also shows differences to the results given 2001). here with the directionof the regard to spindle upper Evolutionary changes ofcleavage from holoblas- rnacromere derivatives, where the authors tic meroblastic or vice presume to versa are often thought to deviations from the rule of which be related size and perpendicularity, to the the yolk content of the We lound to occur in the lower Famous only large macro- eggs. examples for this are the transitions mere derivatives giving rise to their smaller sister from holoblastic spiral cleavage towards discoidal cells in one direction. only With regard to the men- cleavage in cephalopod molluscs or from holoblastic boned difficulties of of advanced radial towards discoidal interpretation cleavage cleavage in am-

cleavage stages, we that the mode niote vertebrates. In carefully presume both cases the change of cleav- °und in Orchestia might be typical for amphipods type is correlated with an increase of size age egg 111 general. the differentiation of the A and content. The Although yolk opposite evolution can be quadrant is recognisable in the of most am- seen within the figures isopod crustaceans which plesio- P lipod embryologists the specific role of these cells morphically show a superficial cleavage with yolky orming an formation initial centre for disc and only some forms germ eggs parasitic evolved early ‘lud gastrulation has been only partially described total cleavage correlated with small yolkless eggs °y Langenbeck and (1898) by Rappaport (1960). (Stromberg 1971). Flowever, Fioroni (1987: 173 le atter reports an inuression of cells in the a out that there f.) pointed is no simple one to one 8i0n after the 32-cell in correlation stage Marinogammarus. between size, content and cleav- Tu , egg yolk ns phenomenon

the - does not to situ- there are of correspond age type eggs prosobranch gastro- 1 'on in Orchestia, lie in where only a few cells a pods which are much than j larger those of some wer position. In other gammarid species Rappaport cephalopod representatives. the Nevertheless, proso- could not find such either. Per- branch follow the of , ingression eggs mode spiral cleavage and laPs, different species with this those of vary respect, cephalopods show a discoidal cleavage. t is evident from our data on the radial This pattern phenomenon can be interpreted as being due

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be leads inverted shell in to some sort of historical “burden” which must spiral cleavage to an coiling

considered inaddition to mechanistic adaptive causes adult gastropods (van den Biggelaar 1991). About

(Riedl 1975). Can this explanation be applied to the causes for the mirror symmetrical eggs in crus-

the evolution of total If be One amphipod cleavage? so, taceans can only speculated. possible rea-

that we have to assume the amphipod stem species son could be oogenesis in the paired ovaries indi-

small with small amount of maternal axis determination possessed eggs only a cating through yolk

yolk. This led to the evolution of total cleavage distribution. Another possible cause could be the

of Hertzler Clark which was maintained (burden) even in the large entry sperm (see & 1992). At

find in do know how mirror is yolky eggs we most amphipod species. In- present we not symmetry

terestingly enough, the representatives of the Ingol- related to blastomere clonal relationships, i.e., whe-

fiel idea have about the smallest the ther the A and B are sisters or I eggs among quadrants always

considered whether is the sister cell D in which amphipods. Ingolfiellidea are often as A to eggs are

an early offshoot of the amphipod lineage. How- oriented to the right (see Figs. 2, 6). The high vari-

their of the first makes it difficult ever, cryptic lifestyle (meiofauna, stygobiont ability cleavage plane

etc.) caused several apomorphic adaptive specialisa- to trace this but it also offers the possibility that

clonal A tions such as the loss of eyes. Nevertheless, per- there is always a relationships between

haps we have to think of an amphipod stem spe- and B and C and D.

cies that was morphologically and with respect to

size and not far from the Recent lifestyle so away

is known disc ingolfiellid pattern. Unfortunately, nothing Origin of the germ by migrations of macromere about ingolfiellid development (see below). derivatives

In Orchestia, the early germ disc (before gastrula-

Mirror images tion) is mainly formed by macromere descendants.

cells in Only some immigrating the retarded mi-

become of the disc. From the 8-cell stage on one can distinguish two cromere area part germ About

the the micromere descendants remains types ofeggs which are mirror images to each other. halfof early

This is indicated derivatives by the position of the smallest extra-embryonically (the of c, bp, and

& quadrant, the A quadrant. The ratio between these dp) (see Wolff Scholtz 2002). Whether all mac-

mirror is about 1:1. Mirror derivatives are involved in disc image germs images romere early germ

are reported in the development of several crusta- formation remains unclear. There are some hints

the cean species e.g. in other amphipods Tryphoselis that at margin, and in particular, at the poste-

in rior of the de- (Bregazzi 1973), several species of euphausia- region germ disc some macromere

ceans (Taube 1909), in decapods (Hertzler & Clark rivatives are not involved in embryo formation

1992), in the cladocerans Daphnia and Holopedium (Wolff& Scholtz 2002). Only Langenbeck (1898),

(von Baldass 1937, 1941), and in the copepod Cy- Rappaport (1960) and Mcschenmoser (1987) de-

clops (Fuchs 1914). In all cases, the meaning is scribe the role of the macromeres for germ disc

unclear and there is in no obvious effect on adult formation a corresponding way. Bergh (1894)

morphology. Cell lineage tracing in Orchestia has and Weygoldt (1958) report a migration of blas-

shown that the A marks the anterior the disc but do quadrant re- tomeres forming germ they not say

of the gion embryo, C is at the posterior pole, and from which cells they arise. Rappaport (1960) con-

B D the and form the lateral firms the function of macromeres the quadrants parts (Wolff forming germ

& in Scholtz 2002, present study). This means that disc by destruction experiments the 8-cell stage

the of the asymmetry early embryonic stages in in Marinogammarus. When he destroyed the mi-

reflects the orientation free which amphipods oblique of the cromcres, yolk cells, are characteristic

A/C axis with to the axis of for the differentiated in advanced respect longitudinal germ disc, more the The itself is This stands When the macromeres such egg. embryo symmetric. eggs. were punctuated,

in contrast the to phenomenon that a mirror image cells were not seen. Nevertheless, most authors

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disc is suppose that the rise micromere is formed the macromere derivativeswhich germ given by by aggre- derivatives & Ulianin (van Beneden Bessels 1869; gate by cell migration towards this region. Further

1881; Heidecke Sheader & but in 1904; Chia 1970; Bre- cells immigrate addition invagination takes gazzi 1973; Mergault & Charniaux-Cotton in 1973; place the same region forming a true blastopore.

Magniette & Ginsburger-Vogel 1982). However, An anterior gastrulation has also been described in in of these most investigations the cleavages after Gammarus pulex (Bergh 1894; Weygoldt 1958; the 16-cell either stage are traced incompletely or Scholtz 1990), inParhyale hawaiensis (Gerberding not at all. The differentiation of the early retarded et al. 2002), and from the figures in several publi- micromeres and the size and of the cations it be deduced that appearance early can this is the case for micromere area may have led to the opinion that other amphipod species as well (e.g. Heidecke 1904; the form the disc. micromeres germ All this makes Pereyaslawzewa 1888b). Older reports about a meso- the conclusion evident that in formation amphipods in gen- derm by immigrating cells spread all over eral the disc is built germ mainly up by macromere the germ band (e.g. Ulianin 1881; Langenbeck 1898; descendants. The formation of the Heidecke have been germ disc is 1904) shown to be incorrect accompanied dramatic of the Scholtz Sometimes by migrations mac- (Weygoldt 1958; 1990). a pos- romere derivatives towards the area of gastrulation. terior gastrulation is reported (e.g. Langenbeck 1898;

Charniaux-Cotton Furthermore, during migration the macromeres get Margault & 1973; Lalitha et al. nd of their and yolk content by a mechanism which is 1989). The figures the description of Langen- still not completely understood. Rappaport (1960) beck (1898) are somewhat ambiguous and a rein- describes the phenomenon as cell division without vestigation of the gastrulation in Microdeutopus nuclear division. Our data are in agreement with seems justified. In the case ofOrchestia gammarel- this view. lus the posterior blastopore is an erroneous obser-

vation because the authors oriented the germ disc

upside down (Margault & Charnier-Cotton 1973: Gastrulation in amphipods - the only “protostome” Blanche 4) Similarly, Lalitha et al. (1989: Fig. 8)

the caudal malacostracan crustaceans mistook forming furrow as blastopore.

all other Interestingly, malacostracans show a blas-

Gastrulation in Orchestia or in the is a highly complex pro- topore gastrulation centre posterior re- cess of the disc with showing some more general features such as gion forming germ a close spatial •he subdivision the into several phases (Weygoldt 1979; relationship between gastrulation area and the Fioroni 1987) that is shared with other arthropods proctodaeum (e.g. Weldon 1892; McMurrich 1895; d an some features Taube 1909, typical for amphipods. Gastru- 1915; Manton 1928,-1934; Hickman Fition in Scholl Dohle Orchestia involves the immigration or 1937; 1963; 1970, 1972; Zilch 1974, delamination of Scholtz single cells and an invagination. 1978, 1979; 1984, 1992; Hcrtzler & Clark The first In cells that participate in gastrulation are 1992; Hertzlcr 2002). other words, amphipods v which are the ‘tellophages are presumably formed by bias- only true “protostomes” among malacost- tomeres which racan whereas the other immigrate during early cleavage. crustaceans, groups show e “deuterostome” gastrulation sensu stricto is started by the immi- a type of development. gration of the derivatives ofmicromeres a after the

’-cell stage followed by neighbouring cells. As is s’own by Wolff & Scholtz (2002) the descendants The embryonic development of amphipods shows a are the deriva- characters primordial germ cells whereas many apomorphic tlVes ot micromeres b and d and of macromere A aie 'esponsible for the formation of the mesoderm The of the taxon monophyly has never and endoderm. The of the is been area blastopore spa- seriously questioned. However, there are dra- 13 re lated to the of the formation of the matic differences between the region morphological rep- stornodaeum and marks of the resentatives of the the anterior pole large amphipod taxa Gammaridea,

turning germ disc. The and major part of the germ disc Ingolfiellidea, Caprcllidea, Hyperiidea (Gruner

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1993), and there is just one single apomorphic cha- character (Bergh 1894; Langenbeck 1898; Dohle

of all racter adults which occurs consistently in & Scholtz 1988; Scholtz 1990, 2000). However,

- the that amphipod subgroups the basal fusion of first Scholtz (1990) mentions the same character

thoracic appendage, the maxilliped (Richter & occurs in Hyperia galba (Hyperiidea) and figures

Scholtz 2001). In contrast to this there are several 28 and 41 in the article ofPereyaslawzewa (1888b)

embryonic characters that exhibit a number of clear indicate that ectoteloblasts are also absent in Ca-

apomorphies for the Amphipoda, although at present prellidea.

the available data are still somewhat patchy. In 4) The 90° spindle orientation to the midline of the

particular, there are no embryological data at all cell c2 during the first differential cleavage of de-

for ectodermal in the the Ingolfiellidea but this group is of great in- rivatives of each row post-naupliar

since the size of the in the in band of Other malacostracans terest eggs ovary germ amphipods.

relation to the size of the oostegites (Siewing 1963) show an angle of the spindle direction of the cor-

indicates that they do not carry the eggs in the brood responding cells ofabout 45° (Dohle 1976; Scholtz

pouch but rather deposit them in the environment 1984; Dohle & Scholtz 1988). Our knowledge about

(Gruner 1993). this character in amphipods is currently restricted

The list of(potential) developmental apomorphies to several species ofthe Gammaridea (Scholtz 1990;

for Amphipoda is as follows: Scholtz et al. 1994).

1) The early cleavage pattern with the characteris- 5) A delay of the expression of the segment polar-

tic size with relations, the arrangement of the blastomeres ity gene engrailed respect to cell division in

in the 4- 16-cell and the the ectodermal of the band. to stages increasing asyn- rows post-naupliar germ

A the is chrony after the 16-cell stage. comparable pat- In amphipods engrailed expression switched

tern is not found in any other malacostracan or non- on one cell cycle later than in decapods, mysids,

malacostracan Patel crustacean group. This cleavage pat- and isopods (Scholtz etal, 1993; 1994; Hejnol

has been This has been shown for tern shown for many representatives of 2002). phenomenon sev-

the Gammaridea (van Beneden & Bessels 1869; eral gammaridean species including Orchestia cavi-

I960 Gammarus Langenbeck 1898; Rappaport Bregazzi 1973; mana and pulex (Scholtz et al. 1993;

Gerberding et al. 2002; Wolff and Scholtz (2002); Scholtz & Dohle 1996).

present study). Pereyaslawzewa (1888b) describes Although not all characters are investigated for

the same pattern for Caprella ferox (Caprellidea). all amphipod subgroups, and although the mono-

For Hyperiidea there is only one short description phyly of the amphipod taxa Gammaridea, Ingol-

without figures for Parathemisto gaudichaudi by fiellidea, Caprellidea, and Hyperiidea is not in all

Sheader but this and the discus- cases not about sister rela- (1977) description proven, to speak group

sion between these suggest that the cleavage follows the same mode tionships groups, this list is promis-

as in other amphipods. ing in establishing a set of powerful amphipod

Anterior 2) gastrulation with the blastopore spa- apomorphies. However, this list also demonstrates

tially related to the position of the stomodaeum. the incompleteness of our knowledge. For instance,

As mentioned above other malacostracans about the of the aber- posses investigations embryology

a gastrulation centre at the posterior, more closely rant group Ingolfiellidea are urgently needed. related to the forming proctodaeum. This character has been demonstrated so far only for gammaridean

species (Weygoldt 1958; Scholtz 1990; Gerberding Acknowledgements

et al. 2002; & Wolff Scholtz 2002; present study), but the in figures Pereyaslawzewa (1888b) indi- We thank Fred Schram for the invitation to contribute to this

issue and for of cate that the situation in caprellids is comparable. special comments on the text. The great efforts

Frank Schubert who did the video-tape are 3) A band with recordings gratefully germ a growth zone lacking ecto- acknowledged. Sebastian Holzapfel and Peter Lederer helped dermal teloblasts. All other malacostracans seem with ofthe The studies sectioning eggs. were supported by grants to possess ectoteloblasts (Scholtz 2000). the Again, ofthe Deutsche Forschungsgemeinschaft (DFG Scho 442/5-2,3). Gammaridea best studied are with respect to this

Downloaded from Brill.com10/09/2021 12:09:51AM via free access Contributions to Zoology, 71 (1/3) - 2002 27

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