Proceedings of 6th International Fruit Symposium 6–10 May 2002, Stellenbosch, South pp. 55–58

Early developmental stages of capitata embryos

R.N. Stefani, D. Selivon* & A.L.P. Perondini Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, R. do Matão 277, 05508-900 São Paulo, Brazil

Early stages of embryonic development of the Medfly Ceratitis capitata are described following in vivo observations from egg deposition up to germ band extension.Additionally,nuclear behaviour was studied in embryos stained in toto with the fluorochrome DAPI. It was found that embryonic development follows the general pattern known for other high dipteran species, in relation to nuclear multiplication, displacement of nuclei up to blastoderm formation, gastrulation and germ band extension. The embryos of C. capitata do not present the phenomenon of extrusion of part of the egg-cell which occurs in species of fruit of genus Anastrepha. It was also observed that the duration of the examined period of embryonic development in C. capitata is about three times longer than in and slightly shorter than in Anastrepha, but the relative timing and duration of early stages are similar in all the three species.

INTRODUCTION embryonic development of Ceratitis capitata, in Comparative studies on ontogenesis have been comparison with similar stages of Anastrepha one of the main approaches to understanding fraterculus and . evolutionary processes in the last decades. In rela- tion to , new insights have been achieved MATERIALS AND METHODS mainly by studies on embryogenesis of Drosophila, A laboratory strain of Ceratitis capitata was used which have stimulated a large number of analysis in the present study. For in vivo observations of in other insects (Sander 1996; Tautz et al. 1994). embryonic development, eggs were collected at The tephritid flies are a group of insects poorly hourly intervals, dechorionated in a 3% chlorox studied in regard to embryonic development. solution and immersed in water. Embryogenesis Embryogenesis of Bactrocera (Dacus) tryoni was was followed from oviposition up to the initial describedbyAnderson(1962),andrecently,studies stages of germ band elongation. have been made on the development of the genus For analysis of nuclear divisions, dechorionated Anastrepha (Selivon et al. 1996, 1997; Perondini eggs were fixed by phase partition according to et al. 1998; Ribas 1999). In this genus, a peculiar Zalokar & Erk (1976), and after removal of the phenomenon was found during the early stages of vitelline membrane the embryos were stained with development.At initiation of the germ band exten- the fluorochrome DAPI according to Foe & Alberts sion, the embryos eliminate part of the posterior (1983).The stained embryos were placed in a drop end of the egg-cell.Later on,in some species,there of glycerol:PBS (1:1) containing the antifading occurs an additional elimination by the anterior n-propylgallate. For embryos at initial cleavages pole during head lobule development. Perondini the number of nuclei was counted directly.At later et al. (1998) suggested that the phenomenon of stages, owing to the impossibility of direct count- elimination might be restricted to the genus ing,the number of nuclei was estimated according Anastrepha since it was not observed in Bactrocera to Perondini et al. (1986). tryoni (Anderson 1962), B. carambolae (Selivon & Images were obtained using a Leica DC100 CCD Perondini, unpubl.) nor in C. capitata (Perondini device coupled to an Olympus fluorescence micro- et al. 1998). scope. These observations point to the necessity of further comparative studies on the initial stages of RESULTS AND DISCUSSION embryonic development in fruit flies in which the phenomenonofeliminationisorisnotpresent.The Nuclear multiplication comparisons would be useful to elucidate not As expected,embryos stained by the fluorochrome only the mechanism involved in this peculiar DAPI showed an increasing number of nuclei phenomenon, but also will contribute to a more during embryonic development.Based on analysis comprehensive understanding of embryol- of Drosophila embryos,a terminology to designate ogy. In this report we describe the early stages of the early embryonic stages according to the

*To whom correspondence should be addressed. number of mitotic cycles was introduced by E-mail: [email protected] Zalokar & Erk (1976), and refined by Foe & Alberts 56 Proceedings of the 6th International Fruit Fly Symposium

(1983). The first stage corresponds to the fertilized egg after nuclear syngamy, thus showing a single nucleus. Each new interphase determines the next stage, hence, each one has a characteristic num- ber of nuclei corresponding to 2n–1,where n is the number of the stages. Figure 1A shows an embryo during the maturation division of the female pro- nucleus.In the next stages,embryos depict- ing 1 to 64 nuclei always showed a number of nuclei as a power of 2, according to a geometric progression. Beyond 64 nuclei, a variation in the number of nuclei per embryo was found,but when the data were arranged in classes, the modal number of the classes followed a geometric progres- sion up to embryos with 256 nuclei. Hence, up to this stage, eight nuclear divisions must have occurred. During these pre- blastodermic stages, the nuclei divide syn- chronously immersed into the endoplasm, and show characteristic displacements. At stage 5, the nuclei are distributed in the anterior two-thirds of the embryo, and lie Fig. 1. Embryos of Ceratitis capitata stained by the fluoro- parallel to the surface, as a hollow mono- chrome DAPI. A, stage of maturation division of the female layer ellipse (Fig. 1B). During the next four pronucleus; B, stage of nuclei located at the anterior 2/3 of the divisions, the nuclei undergo two move- embryo; C, stage where the nuclei have dispersed toward the posterior pole and had already start migration to the surface; ments:migration toward the posterior pole D, multiplication of the nuclei at the surface; E, embryo at (the ellipse elongates) as shown in Fig. 1C, stage 13, cellular blastoderm. In these figures, dorsal is top, and a movement toward the egg’s surface anterior is at left. Scale, 1 cm = 90 µm. (Fig. 1D).When the embryos are in stage 9,a large proportion of the 256 nuclei reaches pole of the embryos. the periplasm of the egg,marking the beginning of Nuclear divisions and migration observed in the syncytial blastoderm. The rest of the nuclei do C. capitata are similar to those described in not migrate, remaining inside the endoplasm, Drosophila melanogaster (Zalokar & Erk 1976; Foe forming the so-called yolk nuclei. They give rise to & Alberts 1983) and Anastrepha fraterculus (Ribas the vitellophages,and their ensuing multiplication 1999), where 13 nuclear divisions (14 stages) oc- occurs out of cycle with the nuclei in the periplasm. curred before cellularization of the blastoderm. In the periplasm, the nuclei undergo five new rounds of divisions, forming about 5000 nuclei Early embryonic stages when cellularization of the blastoderm occurs At oviposition the egg-cell of C. capitata fills the (Fig. 1E). space delimitated by the external coverings, the During the syncytial blastoderm stage, the divi- vitelline membrane and the chorion.Typically, two sions of the nuclei at the periplasm are no longer basic regions may be observed in the egg-cell synchronous within each embryo. An incipient structure by an in vivo analysis: a thin translucent parasynchrony may already be seen at stage 10, periplasm and the internal region filled by vitellum, and it is quite evident in most of the embryos after the endoplasm.Soon after oviposition the egg-cell nuclear cycle 11. In C.capitata,most embryos show starts a retraction at both of its extremities,leaving two or three distinct phases of mitosis, as in reduced spaces between the cell and the egg other Diptera (Zalokar & Erk 1976; Perondini et al. coverings (Fig. 2A). Most of eggs between 0 to 1 h 1986). The ‘wave of mitosis’ generally starts at the after oviposition (a.o.) are at stage 2 (two nuclei), anterior pole since the most advanced phases of while some eggs are still eliminating the second the mitotic cycle are usually found at the anterior polar body (Fig. 1A), and others at stage 1 (the Stefani et al.: Early developmental stages of Ceratitis capitata embryos 57 single nucleus of the zygote).Up to 2.5 h a.o. few alterations are observable by an in vivo analysis. However, the embryos have pro- gressed, actively multiplying their nuclei, the most advanced embryos possessing 64 nuclei (stage 7). The nuclei at this stage have already dispersed themselves along the embryo,but are still far from the surface. At about 3.5 h a.o., the surface of the em- bryos became irregular with a series of small protuberances, and clear spots ap- underneath these protuberances, caused by the arrival of the nuclei sur- rounded by yolk-free cytoplasm at the sur- face. At this moment, which marks the beginning of the syncytial blastoderm, the embryos are in stage 10,and about 156 nu- clei arrive at the surface, while the other nuclei (about 100) remain inside the endoplasm. At the posterior pole, the pole cells appear at about 4 h a.o. (embryos at stage 11), as shown in Fig. 2B. The number of nuclei continues to grow and by 8.5 h a.o. Fig. 2. Images taken in vivo of dechorionated embryos of cellularization of the blastoderm occurs Ceratitis capitata. A, freshly laid egg showing retraction of the (Fig. 2C). egg-cell at both ends; B, enlarged posterior pole of an embryo By 9.5 h a.o.the cephalic furrow begins to at 4 h a.o. showing the pole cells (arrow); C, cellular blastoderm; D, embryo at gastrulation showing the beginning of the cephalic form, indicating that gastrulation was initi- furrow (arrow). In this picture, dorsal is up, anterior pole at left. ated (Fig. 2D). Embryos at this period Scale in A, C and D, 1 cm = 120 µm; in B = 70 µm. showed full development of the cellular blastoderm and were well advanced into tion (Ceratitis/Drosophila, r2 = 0.9847; Ceratitis/ stage 14. In the next hour, 10.5 h a.o., most of the Anastrepha, r2 = 0.9802). The main differences embryos have started the process of germ band among the three species is relative to the slopes of elongation. the curves, 3.0215 and 0.8978, respectively, Comparison of initial development. Since the meaning that the duration of initial embryonic embryonic development of Drosophila is better development of Ceratitis is about three times understood and has been used as paradigm of longer than in Drosophila and slightly shorter development, at least for dipterans, in the next than in Anastrepha. Despite these differences, the analysis the data on C. capitata described here will relative timing and duration of each stage is similar be compared with the information on Drosophila for all three genera, as shown by the significant (Foe & Alberts 1983). It will also include a compari- correlations of the data. son with the data on Anastrepha fraterculus, a The fact that the timing of the early embryonic species of fruit fly for which data are also available stages of C.capitata described here is similar to that (Selivon et al. 1996; Ribas 1999). of other fruit flies,namely (Ander- In this analysis, six unequivocal events of early son 1962) and Anastrepha fraterculus (Selivon et al. embryonic development were used: migration of 1996;Ribas 1999),and to Drosophila,which belong nuclei toward the posterior pole, initiation of to a different dipteran family (Drosophilidae),is not syncytial blastoderm, appearance of pole cells, unusual since similarities in these stages are known cellularization of the blastoderm, appearance of to be conserved even in more distantly related the cephalic furrow and beginning of germ band species of flies (Tautz et al. 1994; Sander 1996). elongation. The data on the species were corre- The comparison also indicates that the phenome- lated in graphic plots of Ceratitis versus Drosophila non of elimination of part of the egg-cell during and Ceratitis versus Anastrepha (Fig. 3). As can be germ band elongation which occurs in Anastrepha seen, in both cases, the points fall on almost (Selivon et al. 1996, 1997; Perondini et al. 1998), straight lines, showing highly significant correla- does not result in gross modifications in the 58 Proceedings of the 6th International Fruit Fly Symposium

AB

Fig. 3. Scatterplots of the time of initiation of certain developmental events in Ceratitis capitata compared to Drosophila (A) and Anastrepha (B). Numbers within the graphs indicate the stages used: 1, nuclei dispersing along the embryo; 2, syncytial blastoderm; 3, appearance of pole cells; 4, cellularization of blastoderm; 5, appearance of cephalic furrow; 6, initial germ band elongation. pattern of initial development. Whether or not hatching at the posterior egg pole in tephritid fruit more subtle differences in specific mechanisms, flies (Diptera). International Journal of Insect Morphol- ogy and Embryology 27: 249–254. like pole cell formation in Ceratitis capitata, are RIBAS, C.C. 1999. Estagios iniciais da embriogenese e present in contrast to Drosophila (Riparbelli et al. padrão da expressão de genes de segmentação em 1996), remains to be demonstrated. uma especie de Anastrepha do grupo fraterculus (Diptera, ). M.S. dissertation, Universidade ACKNOWLEDGEMENTS São Paulo, São Paulo, Brazil. RIPARBELLI, M.G., CALLAINI, G. & DALLAI, R. 1996. This study was supported by FAPESP (98/10701- Primordial germ cell migration in the Ceratitiscapitata 4, 01/07049-8). R.N.S. has a doctoral scholarship embryo. Tissue & Cell 28: 99–105. from CAPES, and D.S. and A.L.P.P. are research SANDER, K. 1996. Variants of embryonic patterning fellows of CNPq. mechanisms in insects: Hymenoptera and Diptera. Cellular and Developmental Biology 7: 573–582. SELIVON, D., MORGANTE, J.S. & PERONDINI, A.L.P. 1997. REFERENCES Egg size, yolk masses extrusion and hatching behav- ANDERSON, D.T. 1962. The comparative embryology of ior in two cryptic species of Anastrepha fraterculus Dacus tryoni (Frogg.), the Queensland fruit fly. Jour- (Wiedemann) (Diptera:Tephritidae). Brazilian Journal nal of Embryology and Experimental Morphology of Genetics 20: 587–594. 10: 248–262. SELIVON, D., MORGANTE, J.S., RIBEIRO, A.F. & PERONDINI, FOE, V.E. & ALBERTS, B.M. 1983. Studies of nuclear and A.L.P. 1996. Extrusion of masses of yolk during cytoplasmic behavior during the five mitotic cycles embryonary development of the fruit fly Anastrepha that preceed gastrulation in Drosophila embryo- fraterculus. Invertebrate Reproduction and Develop- genesis. Journal of Cell Science 61: 31–70. ment 29: 1–7. PERONDINI, A.L.P.,GUTZEIT, H.O. & MORI, L. 1986. Nuclear TAUTZ, D., FRIEDRICH, M. & SCHRÖDER, R. 1994. Insect division and migration during early embryogenesis of embryogenesis – what is ancestral and what is Bradysia tritici Coquillet (syn. Sciara ocellaris) (Diptera: derived? Development (suppl.): 193–199. Sciaridae). International Journal of ZALOKAR, M. & ERK, I. 1976. Division and migration of and Embryology 15: 155–163. nuclei during early embryogenesis of Drosophila PERONDINI, A.L.P., SELIVON, D. & MORGANTE, J.S. 1998. melanogaster. Journal de Microscopie et de Biologie Facultative polar extrusion of yolk masses and of Cellulaire 25: 97–106.