Wilhelm Roux' Arehiv 169, 308--313 (1972) �9 by Springer-Verlag 1972

Experimental Changes of the Cleavage Pattern in the Eggs of a Gall Midge (Wachtliella persicariae L.)

after Local Ultrasonic Treatment

R a i n e r W o l f

Heiligenberg-Institut fiir experimentelle Biologie, Heiligenberg (Baden) und Zoologisehes Insti tut I der Universit~t Wiirzburg

Received November 25, 1971

Summary. To affirm the hypothesis that during the early cleavage in WachtlieUa persi- cariae L., central and marginal ooplasm do not only differ by their kinematic aspects (Wolf, 1969; Wolf and Krause, 1971) but also physiologically, the radial symmetry of the ooplasm has been destroyed experimentally. For this purpose a device has been developed permitting a local ultrasonic treatment of insect eggs limited to well defined regions of various sizes (Fig. 1). The changes in the egg architecture can be observed directly while the treatment is going on and their effects on embryogenesis are analyzed by time-lapse motion pictures.

Cleavage nuclei which have been transferred prematurely to the egg surface during low- intensity ultrasonic radiation of the whole egg length (Fig. 2a), move back into the central ooplasm after t reatment; the marginal plasm becomes populated only later at the same time as in untreated eggs. Local high-intensity ultrasonic treatment, too (Fig. 2 c), obviously does not cause any damage to the nuclei, while the ooplasm within the treated area becomes com- pletely mixed up. After exposure the untreated region may become partially coated by a sheath of mixed up ooplasm. The populating of the egg surface by the nuclei is restricted to those sites where the untreated marginal ooplasm remains at the surface. These are the only areas for the later formation of preblastoderm and blastoderm. For the normal distribution of the cleavage energides a certain ooplasmic component sensitive to ultrasonic t r e a t m e n t seems to be responsible, the ultrastrueture of which will be subjected to further investigations.

Zusammeu/assung. Um am Beispiel der Gallmiicke Wachtliella persieariae L. die tIypo- these zu erh/s dal3 sieh zentrales und marginales Ooplasma w~hrend der friihen Furehung nicht nur kinematisch (Wolf, 1969; Wolf und Krause, 1971), sondern auch physiologisch unterscheiden, ist experimentell die radi~re Symmetrie des Eies zerstSrt worden. Hierzu ist eine Versuchsanordnung entwiekelt worden, die eine gezielte Ultra-Besehallung eines mehr oder weniger groBen, scharf begrenzten Eibereichs ermSglicht (Abb. 1). Die Veri~nderungen der Eiarchitektur werden w~hrend der Behandlung direkt beobachtet und ihre Auswirkungen auf die Embryogenese mit Hilfe yon Zeitrafferfilmen analysiert.

Furchungskerne, die infolge schwacher Ultrabesehallung des ganzen Eies vorzeitig an die Eioberfl~che verlagert worden sind (Abb. 2a), wandern naeh der Behandlung wieder in da- zentrale Ooplasma zuriick; das !VLrginalplasma wird erst sp/iter, zur selben Zeit wie in uns behandelten Eiern, besiedelt. ~qach starker lokaler Ultrabeschallung (Abb. 2c) werden die Kerne offensiehtlich ebenfalls nicht geseh~digt, aber das Plasma wird im beschallten Eibereich v511ig durchmischt. Naeh der Behandlung kann ein Teil der unbeschallten Eiregion oberfl~ch- lich yon durchmischtem Ooplasma schlauchartig iiberzogen werden. Die Kerne kSnnen die Eioberfl~che nur dorb besiedeln, wo noch unbeschalltes 1Vfarginalplasma an der Oberfl~che liegen geblieben ist; nur in diesen Gebieten ist die Bildung eines Pr/iblastoderms und sp/~ter eines Blastoderms mSglich. Fiir die normale Verteilung der Furchungsenergiden im Eiraum muI~ eine besonders schallempfindliche Komponente des Ooplasmas verantwortlich sein, deren Feinstruktur noch elektronenmikroskopisch untersucht werden soll.

Cleavage Pattern after Local Ultrasonic Treatment 309

The plasmic flow of doub le - foun ta in - type observed in the eggs of Pimpla dur ing intravitelline cleavage (Wolf and Krause , 1971) pe rmi t s one to dis t inguish be tween a " c e n t r a l " and a " m a r g i n a l " ooplasm b y the i r different kinetics. Super/icial cleavage only sets in a f te r the energides have popu la t ed the egg in- te r ior to i ts full length. Along wi th the popu la t ing of the marg ina l p lasm there seems to be a cer ta in ac t iva t ion of the nuclei, connected wi th the fo rma t ion of nucleoli (Meng, unpub].) . I n Waehtliella, too, the cleavage divisions are accom- pan ied b y flowing pulses of a centra l and a marg ina l ooplasm, if only local ly (Wolf, 1969). The manner of nuclear movemen t in cer ta in except ional cases speaks in favour of physiological dissimilarities between central and marginal plasm in add i t i on to the i r d i f ferent k inet ics : I f a t the 2 -4 nuclei s tage a divis ion spindle happens to bui ld up t r ansve r sa l ly to the longer egg axis, the fi l ial nuclei a t f i rs t do no t move as far a p a r t as usual , i.e. t h e y do no t reach the zone of marg ina l p l a sm as would be expected. A normal d i s t r ibu t ion of the nuclei (Fig. 3a) is a t t a i n e d only af ter the spindle has t u rned a round wi th in the egg; hereaf te r the marg ina l p l a sm a t the 16-nuclei s tage becomes popu la t ed as usual . Therefore a t leas t a t the t ime of ea r ly cleavage centra l and marg ina l p l a sm should differ in such a w a y as to cause l imi ta t ions of movemen t and t e m p o r a r y pos i t ion of the energides. This hypothes i s can be p roved expe r imen ta l ly b y defined changes of the no rma l egg a rch i tec ture which is more or less rad ia l , e i ther b y comple te ly mixing up centra l wi th marg ina l p l a sm or b y p r e m a t u r e l y dis locat ing the cleavage nuclei in to the marg ina l plasm. Centri/ugation of the eggs does no t lead to sa t i s fac tory resul ts : " s p i n n i n g " ( ro ta t ion axis wi th in the eggs) as well as " w h i r l i n g " (rota- t ion axis outs ide of the eggs; Bruhns, Krause , and Wolf , 1970) resul ts in t rans- locat ions wi th in t he eggs, b u t a t the same t ime the egg components become classified according to the i r densi ty . Ultrasonic treatment t u r n e d out to be a successful m e t h o d for the exper iments in question.

Methods During a total ultrasonic treatment of Drosophila eggs immersed in water, Counce and

Selman (1953, 1955; see also Goldman and Lepesehkin, 1952; Dyer and Nyborg, 1960) observed rotations of the plasm all over the egg. In the experiments to be described here, local treatment with ultrasound has been made possible by means of a special arrangement (Fig. 1). For this purpose the eggs have to be treated in air, whereby the transmission of the ultrasonic waves is restricted to the plane of contact. Thus, in eggs stuck upright at one of their pointed ends by means of their sticky chorion, the ultrasonic transmission becomes limited to their basal poles. Since a certain intensity Of ultrasonic treatment is required to initiate rotations of the ooplasm and since the intensity of ultrasonic radiation within the egg seems to decrease towards the apical pole, limited parts o/ the ooplasm can be caused to rotate.

According to the results obtained, no changes could be observed within the untreated parts of the egg during and after treatment, other than later mutual reactions between them and the treated parts. Thus, nonrotated regions may be called untreated by ultrasonic radia- tion. The technique applied corresponds to a local ultrasonic treatment, limited to distinct areas of various sizes within the egg. The power of ultrasonic radiation is regulated by different intensities of resonance, i.e. by varying the distance---and herewith the stationary waves within the water between the ultrasonic oscillator and the coverslip carrying the eggs (Fig. 1). The oscillator of the "Ultraschall-Nebler", made by Schoeller, has been used as ultrasonic generator at the frequency of 1 MHz and a maximum ultrasonic output of 20 VA. With high- intensity treatment (max. ultrasonic output) the maximum time of exposure amounts to 20 see, thus keeping the temperature of the transmission medium (H20) below 36 ~ C. The effects of ultrasonic radiation can be observed stereoscopically while the treatment is going on.

310 1~. Wolf:

micro - ..~ stereo- ~, manipulator -~ microscope ~i,

u . ~ ptankton V ~ . f / tube-

i

chamber

ultrasonic generator Fig. 1

- .~. - . . . , :

a

, ' ~ ' ~ � 9

. . . - - - ~ ' :

TtT1 Fig. 2

Fig. 1. Arrangement for polar (= "local") or longitudinal ultrasonic treatment of insect eggs. Ultrasonic transmission through water by stationary waves (above the oscillator) upon the base of a plankton-tube-chamber, produced by C. Zeiss. State of oscillation in case of

resonance; p sound pressm'e, v sound particle velocity, sound level I ,~ p.v Fig. 2a~-c. 4-nuclei-cleavage stage; rotation of the ooplasm during a) longitudinal low- intensity treatment, b) longitudinal high-intensity treatment and e) polar high-intensity

treatment; see also Figs. 3f, 4a, and 3h

Results and Conclusions

Depending upon the ultrasonic intensity applied, the treated parts of the ooplasm react by more or less rapid rotations, measuring between 3 rpm to more than 20 rps. The rotation occurs approximately in planes parallel to the direction of the ultrasonic radiation, i.e. vertically to the base of the egg's attachment. The direction of the rotation depends with high propability upon slight asymme- tries at the area of contact. No changes in the direction occur during the time of t reatment , though in different regions of the egg the ooplasm may rotate in opposite directions (Fig. 2b). The ultrasonic t rea tment was found to cause the following changes :

1. Prolonged low-intensity treatment (1-3 min) hitting the egg full length ( ~ " longi tudinal" ultrasonic t reatmenti i in the slowly rotating ooplasm (3 rpm; Fig. 2a) initiates a movement o/the cleavage nuclei towards the ultrasonic oscillator (Fig. 3f). The nuclei hereby leave the longer axis of the egg, reaching the marginal plasm prematurely, which normally is not at tained before the 16-nuclei stage. As can be seen from time-lapse motion pictures of t reated eggs, the energides at the/ollowing (3rd) cleavage division leave the marginal plasm by the shortest way, and only later do they return on schedule. Afterwards a normal blastoderm is built up.

2. Brie/high-intensity longitudinal treatment (1-20 see) causes quick rotations of the egg plasm (Fig. 2b). At 1-5 rps the ooplasm becomes completely mixed up (Fig. 4 a), while more than 20 rps lead to a separation of the ooplasmic components (Fig. 3g). After the t reatment , during the following cleavage division, the pig- mented halos of the nuclei become visible again (Fig. 4b). In typical cases the

Cleavage Pattern after Local Ultrasonic Treatment 311

4 8 16 ca. 128 ca. 2000 4 4 4 number of nuclei

a 90 b 115 c 130 d 190 e 330 f 95 g 95 h 95 i 900rain

Fig. 3 a--e. Cleavage stages o/ Waehtliella. a) 4 cleavage nuclei (CN), surrounded by pigmented halos of protein yolk, within the central plasm; b) 8 CN, PC pole cell; c) 16 CN; the energides enter the marginal plasm; d) preblastoderm; e) blastoderm, f-i E//eets o/ ultrasonic treat- ment at the l-nuclei stage: f) CN prematurely transferred into the marginal plasm; g) separa- tion of ooplasmie components; h) anterior egg region treated locally; i) same egg as in h, at the stage of blastoderm. Anterior egg pole above, timing from oviposition (breeding

temperature 23 ~ C). Alive, 145 •

initial region of cleavage is no t dislocated. Furthermore, the capaci ty for active nuclear movements (Wolf, 1969) is preserved. But unlike those in unt rea ted eggs all the cleavage energides remain in the central plasm (Fig. 4 e-g), instead of moving to the egg surface, and the pole cells fail to develop (for normal pole cell forma- t ion see Fig. 3b). Such eggs die at the end of the 9th or 10th cleavage division without having built up a blastoderm (Fig. 4h-i) . Only in rare eases, as a cause for which the increase of the temperature above 36~ during the ultrasonic t rea tment cannot be excluded, was the number of cleavage divisions reduced or lacking altogether. Nevertheless, some time after the t r ea tment and before the final degeneration of the egg, about 8 eentres of pigmented yolk material similar to the halos of the nuclei could be observed to build up near the longer egg axis.

3. High-intensity polar treatment leads to a rapid rotation and mixing up o/ the ooplasm within a distinct basal area which m a y va ry in its extent (Figs. 2 c, 3 h). The fur ther development of the t reated region corresponds to t h a t of eggs with their plasm completely mixed up, while the un t rea ted region sometimes follows the normal developmental pa t te rn by forming a part ial blastoderm (Fig. 3i). I n other cases the energides in the unt rea ted apical region, too, remain within the central plasm, a l though it is of normal appearance at the end of the t rea tment (Fig. 5). Since in these cases untreated, non-damaged, cleavage nuclei behave in the same way as t rea ted ones, the nuclei themselves do not seem to su//er any damage /rom the ultrasonic radiation. Apparent ly some plasmic influence,

312 R. Wolf:

4-8 8 8-16 16-32 32-64 128 ca. 500 - - number of nuclei

a 95 b 105 c 120 d 140 e 160 f 190 g 235 h 315 i 675min

Fig. 4a- - i . Development, of an egg whose plasm became mixed up at the 4-nuclei stage by medium-intensity ultrasonic treatment. Taken from time-lapse motion pictures; for further

explanation see text

2 2-4 4 8 16 ca. 64 ca. 200 - - number of nuclei

a 80 b 90 c 95 d 130 e 155 f 220 g 260 h 770 i 930min

Fig. 5a- - i . Development of an egg after high-intensityultra sonic t reatment of its posterior region at the 2-nuclei stage. Arrowheads indicate the cephalic limitation of the treated ooplasm, which is slowly moving forward along the egg surface (a-e) ; above the last arrowheads (f, g), a preblastoderm is formed, later on degenerating along with the rest of the oop]asm (h, i)

Cleavage Pattern after Local Ultrasonic Treatment 313

s ta r t ing f rom the t r e a t ed egg region, causes the u n t r e a t e d nuclei to follow the same a b n o r m a l deve lopmenta l p a t t e r n as the t r e a t e d ones. The reason for th is phenomenon becomes ev iden t in t ime- lapse mot ion p ic tures : S ta r t ing f rom the in ter face be tween the t r e a t e d and the u n t r e a t e d area, the t r e a t e d p lasm is seen to move along the surface, coat ing the ad j acen t u n t r e a t e d region p a r t i a l l y b y a ve ry th in p lasmic shea th of mixed up ooplasm (arrowheads Fig. 5a - f ) . The sites o/untreated marginal plasm remaining at the sur]ace are the only ones where the nuclei move out o/the central plasm, and only a t these sites do centra l p lasm and marg ina l p l a sm develop the t yp i ca l f lowing pulses in the r h y t h m of cleavage. E x c e p t for being coa ted b y the th in l aye r of mixed up ooplasm, the a d j a c e n t pa r t of the egg keeps i t s original r ad ia l a rchi tec ture . Bu t af ter hav ing lost con tac t wi th the egg surface, the u n t r e a t e d marg ina l p l a sm seems to lose i ts ab i l i t y to form a p reb las toderm, for no "inner b l a s t o d e r m " was seen to develop as descr ibed b y Ki i the (1966), who t r e a t e d the surface of eggs f rom Dermestes with UV radia- t ion. Regard ing the damage caused b y ul t rasonic rad ia t ion , ano ther conclusion refers to di f ferent th resho ld values for nuclei and ooplasm. Obvious ly the ooplasm contains some component(s) o/special sensitivity to ultrasonic treatment and essential /or the normal nuclear distribution, bu t unessent ia l for the cleavage divisions themselves. Before being discussed extensively , the above resul ts call for fur ther exper iments , especial ly to de te rmine whether or no t the u l t rasonic t r e a t m e n t des t roys speci]ic ultrastruetures of the ooplasm which in t u rn ac t upon the energides.

Acknowledgments. This investigation was supported by the Deutsche Forschungsgemein- schaft. I would like to thank Professor Dr. G. Krause for critically reading the manuscript and Professor Dr. L. Schneider for kindly providing me with the ultrasonic generator. The help in the preparation of the manuscript of Dr. E. Wolf and Mrs. Dorothea Wolf is grate- fully acknowledged.

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Biol. Zbl. 89, 473-479 (1970). Counce, S. J., Selman, G. G. : The effect of ultrasonic treatment on embryonic development

of Drosophila. J. Embryol. exp. Morph. 3, 121-141 (1955). Dyer, H. g., Nyborg, W. L.: Characteristics of intracellular motion induced by ultrasound.

Prec. 3 rd Int. Conf. on 1Vfed. Electronics, London, 445-449 (1960). Goldman, D. E., Lepeschkin, W. W. : Injury to living cells in standing sound waves. J. Cell.

and Comp. Physiol. 40, 255-268 (1952). Kfithe, H. W. : Das Differenzierungszentrum als selbstregulierendes Faktorensystem ffir den

Aufbau der Keimanlage im Ei yon De~'mestes/rischi. Wilh. Roux' Arch. Entwickl.-Mech. Org. 157, 212-302 (1966).

Selman, G. G., Counce, S. J. : Abnormal embryonic development in Drosophila induced by ultrasonic treatment. Nature 172, 503-504 (1953).

Wolf, R. : Kinematik und Feinstruktur plasmatischer Faktorenbereiche des Eies yon Wacht- lietla persicariae L. (Diptera). I. Teil: Das Verhalten ooplasmatischer Teilsysteme im normalen El. Wilhelm Roux' Archiv 162, 121-160 (1969).

Wolf, R., Krause, G. : Die Ooplasmabewegungen w~ihrend der Furchung yon Pimpla turionellae L. (Hymenoptera), eine Zeitrafferfilmanalyse. Wilhelm ]~oux ~ Archly 167, 266-287 (1971).

Dr. Rainer Wolf Zoologisches Institut I der Universit/~t D-8700 Wiirzburg R5ntgenring 10 Deutschland