VOLUME 138 (1)

Numbers and distribution of Red-footed Falcons (Falco vespertinus) breeding in Voivodina (northern Serbia): a comparison between 1990-1991 and 2000-2001

Seasonal Variation in blood plasma sodium and potassium concentrations in the lizard Agama nupta (Agamidae)

Shell utilization by the land hermit crab Coenobita scaevola (Anomura, Coenobitidae) from Wadi

Wojciech NIEDBA/

LA

New Palaearctic species of Phthiracaroidea (Acari, Oribatida)

El-Gemal, Red Sea

^{Marc CALLEBAUT} AN INTERNATIONAL JOURNAL PUBLISHED BY

A review. Historical evolution of preformistic versus neoformistic (epigenetic) thinking in embry-

THE ROYAL BELGIAN SOCIETY FOR ZOOLOGY

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Jarosùaw WI ˛ACEK

Volume 138 (1) – January 2008

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RESULTS AND DISCUSSION

A total of 116 and 61 pairs of Red-Footed Falcon nested in Voivodina in 2000 and in 2001, respectively (Table 1). Ten years before there were 308 in 1990 and 124 pairs in 1991 (PURGER, 1996). The survey in 1991 indicated a 60% population decrease since 1990, whereas the number recorded in 2001 was 47% less than in 2000. This suggests that the population size along the southwestern margin of European area fluctuates greatly among years. Difference between the surveys in 1991 and 2000 was not big (124, 116 pairs), but whereas a total of 432 pairs of Red-Footed Falcon nested in the study area at the time of the first surveys (1990/91), there were only 177 pairs of them breeding during the second surveys (2000/01), which suggest population decline. Also, the number of nesting localities varied from year to year, being 19 in 1990 and 22 in 1991. Only 9 were used in both years (PURGER, 1996). Our survey revealed a further decline in the number of nesting localities used; 17 in 2000, 11 in 2001 and 6 were used in both years (Table 1). Only one nesting location used in the first survey was used again in 2000-2001 (2km south-east of Jazovo). In this colony 147 nesting pairs were counted in 1990 and 29 pairs in 1991 (PURGER, 1996), but only 12 pairs in 2000 and 6 pairs in 2001 (Table 1).

TABLE 1

Number of breeding pairs of Red-footed Falcon found in Voivodina during 2000 and 2001.

UTM	Locality	2000	2001
DR27 DR29 DR48 DR49 DR54 DR55 DR58 DR64 ER10	Bachka 1 km south of Sterijino Selo 2 km south of Sterijino Selo 2.5 km north-west of Zimonjić (Kapetanski rit) Banat 2 km south-east of Jazovo 1 km north-west of Podlokanj 1 km south-west of Vrbica 1 km west of Banatski Monoštor 1 km east of Banatski Monoštor 2 km east of Crna Bara 3 km east of Crna Bara 3.5 km east of Crna Bara 5 km south-east of Crna Bara Torda 2 km south-west of Torda 4 km north of Bašaid Mokrin (Vašarište) 1 km north-west of Mokrin 1 km south-west of Čestereg Banatsko Karađorđevo 1 km north-east of Banatski Dvor 1 km north of Banatski Dvor 7 km north-west of Vršac Total	1 2 6 12 1 1 1 -1 1 --25 4 1 42 9 2 -1 -6 116	--2 6 ---2 --1 1 14 2 -26 5 -1 -1 -61

the region (Table 1, Fig. 1). The shift of nesting locations towards the north-east coincided with a contraction of their range that was associated with declining numbers. However, the majority of Red-Footed Falcons in Voivodina (1990 – 99%, 1991 – 96%, 2000 – 92%, 2001 – 97%) bred in the northern, central and eastern parts of Banat (Table 1). After a period of ten years, not only the number of nesting locations, but also the number and size of larger colonies (with more than ten pairs of Red-footed Falcons) had declined. In 1990-1991, 88% and 72% of pairs nested in 5 and 4 large colonies respectively, and by 2000-2001 only 3 and 2 larger colonies (Jazovo, Torda, Mokrin) remained (Table 1). Thus, despite the population decline, 73% and 68% of Red-footed Falcons nesting in the areas beyond the Tisa River continued to nest in larger colonies (Table 1). The majority of nesting areas in Banat are about 10-20km from the Tisa, and are aligned parallel with the river (Fig. 1). No nesting Red-footed Falcons were found in the central parts of the region during the survey in 2000-2001. In 2000 there was still a smaller colony near Vršac (ER10), but it had disappeared by 2001 (Table 1). Overall, nesting areas in Voivodina have shifted to the north-east by 50-70 kilometres (Fig. 1).

The 1990-1991 results suggest that there were less birds nesting within Voivodina in 1991; only about a third of the 1990 total. Also, the number of solitary nesting pairs was significantly higher in 1991 (χ²=17.77, df =1, P< 0.001). In contrast, there was no significant difference (χ²= 0.16, df=1, ns) in nesting strategies between 2000 and 2001 (Table 2). Red-Footed Falcons nesting in Voivodina used Rook nests even if they were situated in human settlements. In 1991, 11.3% of nests were found in the villages Bašaid and Sakule (PURGER, 1996). This association has subsequently increased. In 2000, 59.5% of nests (Mokrin, Torda, Čestereg) and in 2001, 67.2% of nests (Mokrin, Torda, Banatsko Karađorđevo) were located within villages (Table 1).

TABLE 2

Numbers of Rook and Hooded Crow and Magpie nests occupied by Red-footed Falcons in 1990 and 1991 (according to Purger 1996), and in 2000 and 2001 (original data). No significant difference was found between the proportions of the two groups in the two study periods (χ² = 0.26, df = 1, ns).

1990 301 (98%) 7 (2%) 1991 108 (87%) 16 (13%) Both years 409 (95%) 23 (5%) 2000 107 (92%) 9 (8%) 2001 58 (95%) 3 (5%) Both years 165 (93%) 12 (7%)

In the Rook colony in Mokrin in 2001 (DR58, Table 1)

Breeding pairs were found only in Bachka and Banat the mean number of offspring per reproductive female (Table 1, Fig. 1). In the north-western and south-eastern was 2.46±0.76 (n=26) and was not significantly different parts of Bachka there were 4 and 5 pairs nesting in 4 and in either 1990 (t=0.95, df=48, ns) or 1991 (t=-0.398, 3 locations in 1990 and 1991, respectively (PURGER, df=56, ns). There was no significant difference (t=1.31, 1996). By 2000 there were 9 pairs in 3 locations and in df=54, ns) between breeding success in 1990 (2.66±0.76, 2001 only 2 pairs at 1 location in the north-eastern part of n=24) and 1991 (2.34±0.87, n=32) (PURGER, 1995).

From our results in 2000 and 2001 (116 and 61 pairs, respectively) it appears that HAM & RAŠAJSKI (2000) overestimated the size of the Red-footed Falcon population (250-336 pairs between 1994-1996) breeding in Voivodina. Several authors have presented various distribution maps of nesting areas (PURGER, 1996; PURGER & MUŽINIĆ, 1997; RAŠAJSKI, 1997; HAM & RAŠAJSKI, 2000), but those can not be used for assessing population fluctuations. The spatial distribution of recent data reveals that Red-footed Falcons have traditional breeding locations with larger colonies in Banat where there are more extensive steppe grasslands, but the boundaries of the distribution area do fluctuate. Such fluctuations in range can best be determined if the survey is repeated every year. Our data indicate that the Red-footed Falcon population is declining and its range contracting towards the north-east. Thus, both trends are similar to what has been observed in Hungary (HARASZTHY, 1998; TÓTH & MARIK, 1999; BAGYURA et al., 2001).

Nesting strategy appears not to have changed during the past ten years: the majority of the Red-footed Falcons in Voivodina nest in Rook colonies, even when those are located inside villages. In Hungary only one third of the birds breed in colonies and the rest nest solitarily or in semi-colonies (TÓTH & MARIK, 1999). If solitary nesters have lower breeding success than birds in colonies (TÓTH, 1994; TÓTH & MARIK, 1999; HARASZTHY & BAGYURA, 1993), then the decline of the Hungarian population may be assisted by the lower breeding success. In Voivodina, south from Hungary, Red-footed Falcons continue to nest in colonies, but the population is still declining. Moreover the proportion of birds nesting in Rook colonies was even higher in 2000-2001. Maybe if population size falls under a critical limit, colonial nesting results in growth again through the effect of higher breeding success? This question can be answered only if population size and changes in nesting strategy are monitored for several years. It is likely that the disappearance of nesting sites and feeding grounds greatly influences both nesting strategies and population trends (TÓTH, 1994; TÓTH & MARIK, 1999). The Red-footed Falcon population in Voivodina is threatened by the increase of monoculture plantations and by the use of biotoxins (HAM & RAŠAJSKI, 2000). Changes in agricultural practice are the main factor implied in the population decrease of The Lesser Kestrel (Falco naumanni) and other steppary birds of prey (TELLA et al, 1998). This is supported by SNOW & PERRINS (1998), the major causes of Red-footed falcon population decrease being the decline of insect prey because of pesticide use, and habitat changes.

D. Purger, J. Rašajski, B. Trócsányi and A. Zsulyevits for collaboration. I am grateful to S.J. Petty, P. Vickery and J. van der Winden and two anonymous referees who made invaluable comments on the manuscript. The research was supported by the Hungarian Academy of Sciences (Bolyai János Research Fellowship: BO/00473/00)

BAGYURA J, HORVÁTH Z & KALOCSA B (2001). A Magyar Madártani és Természetvédelmi Egyesület Ragadozómadárvédelmi Szakosztálya 2000. évben elért eredményeinek összefoglalója. Túzok, 6: 25-29 (in Hungarian).

BIJLSMA RG (1997). Red-footed falcon Falco vespertinus. In: HAGEMEIJER EJM & BLAIR MJ (eds), The EBCC Atlas of European Breeding Birds: Their Distribution and Abundance. T. & A.D. Poyser, London: 182-183.

CADE TJ (1982). The falcons of the world. Collins, London.

CRAMP S & SIMMONS KEL (eds) (1980). Handbook of the birds of Europe, the Middle East and North Africa, Vol. 2. Oxford University Press, Oxford.

FOWLER J & COHEN L (1995). Statistics for Ornithologists. British Trust for Ornithology, Thetford.

HAM I (1977). Stanje i mere zaštite ptica grabljivica Vojvodine sa posebnim osvrtom na Deliblatsku peščaru. Priroda Vojvodine, 3: 49-52 (in Serbian).

HAM I & RAŠAJSKI J (2000). Siva vetruška Falco vespertinus (Linnaeus, 1758). In: PUZOVIĆ S (ed), Atlas ptica grabljivica Srbije, mape rasprostranjenosti i procene populacija 19771996. [Atlas of birds of prey of Serbia: their breeding distribution and abundance 1977-1996] Zavod za zaštitu prirode Srbije, Beograd: 153-155 (in Serbian with English summary).

HARASZTHY L (1998). Kék vércse Falco vespertinus. In: HARASZTHY L (ed), Magyarország madarai. Mezőgazda Kiadó, Budapest: 99-100 (in Hungarian).

HARASZTHY L & BAGYURA J (1993). A comparison of the nesting habits of the Red-footed Falcon (Falco vespertinus) in colonies and solitary pairs. In: NICHOLLS MK & CLARKE R (eds), Biology and conservation of small falcons. The Hawk and Owl Trust, London: 80-85.

HEATH M, BORGGREVE C, PEET N & HAGEMEIJER W (eds) (2000). European bird populations: estimates and trends. BirdLife Conservation Series No. 10. BirdLife International, Cambridge.

MAGYAR G, HADARICS T, WALICZKY Z, SCHMIDT A, NAGY T & BANKOVICS A (1998). Nomenclator avium Hungariae. Madártani Intézet – MME – Winter Fair, Budapest Szeged.

MARKOVIĆ JÐ (1990). Enciklopedijski geografski leksikon Jugoslavije. Svjetlost, Sarajevo. (In Serbian and Croatian). MATVEJEV SD & PUNCER IJ (1989). Map of Biomes – Landscapes of Yugoslavia. Natural History Museum, Belgrade.

PARABUĆSKI S & JANKOVIĆ M (1978). An attempted determination of the potential vegetation of Voivodina. Proceedings for Natural Sciences, Matica Srpska Novi Sad, 54: 5-20.

POSTUPALSKY S (1974): Raptor reproductive success: Some problems with methods, criteria, and terminology. Raptor Research Report, 2: 21-31.

PURGER JJ (1995). Breeding success of the Red-footed Falcon (Falco vespertinus) in Banat (Voivodina, Yugoslavia) based on ringing data. Ornis Hungarica, 5: 67-68.

PURGER JJ (1996). Numbers and distribution of red-footed falcon (Falco vespertinus) nests in Voivodina (northern Serbia). Journal of Raptor Research, 30: 165-168.

PURGER JJ & MUŽINIĆ J (1997). The breeding distribution and migratory movements of the Red-footed Falcon (Falco vespertinus) in Province Voivodina (southern part of Carpathian basin). Ring, 19: 65-73.

RAŠAJSKI J (1997). Ptice Srbije: sa kartama distribucija. [Birds of Serbia: with distribution maps]. Prometej, Novi Sad (in Serbian).

SNOW DW & PERRINS CM (1998). The Birds of the Western Palearctic, Non-Passerines. Vol. 1. Oxford University Press, Oxford.

STEVANOVIĆ V & STEVANOVIĆ B (1995). Osnovni klimatski, geološki i pedološki činioci biodiverziteta kopnenih ekosis

Population trends of Red-footed Falcons in northern Serbia

Majid Sampour

There have been a number of published studies with lizards showing that many species, but not all, experience increased blood sodium (hypernatraemia) and potassium (hyperkalaemia) concentrations in summer when supplies of water are at a premium (BRADSHAW, 1986; BRADSHAW, 1997). Hypernatraemia has been shown to result from excessive dietary intake of sodium ions (BRADSHAW & SHOEMAKER, 1967). This study is the first to examine water and electrolyte balance in the Iranian agamid lizard Agama nupta, and to examine the maintenance of electrolyte homeostasis during the hot dry season near Khoramabad. Another desert agamid lizard, Uromastix acanthinurus has been reported as being able to maintain electrolyte constancy in its blood throughout the summer (BENTLEY, 1976; GRENOT, 1976), but subsequent careful analysis of changes in plasma sodium levels from separate populations near Beni-Abbes in Algeria showed that this species also experiences hypernatraemia in summer (LEMIRE et al., 1979; LEMIRE et al., 1982; BRADSHAW, 1986).

The study of changes of the amount of electrolytes in the blood of different lizard species and during various seasons has attracted a considerable deal of attention. BENTLEY (1959), working with the skink, Tiliqua rugosa, reported the existence of high concentrations of Na⁺ in plasma during mid-summer. In addition, he found that sodium injections in the laboratory also resulted in hypernatraemia. BRADSHAW & SHOEMAKER (1967), carried out field studies on water and electrolytes balance in the agamid lizard Amphibolurus (now Ctenophorus) ornatus in Western Australia and found that high concentration of Na⁺ in the plasma of this lizard during summer, were associated with a significant isosmotic expansion of the extracellular fluid. Plasma sodium levels were found to increase to as high as 300mmol.L^-1 in extended droughts and were associated with significant mortality.

In lizards, an active nasal gland for discharging salt has attracted zoologists’ attention, and in some species the role of this active nasal gland in excreting ions has been investigated. In some lizards species however, no research has been performed on nasal glands and their function remains unknown.

Since the discovery of nasal salt glands in many species of reptiles some zoologists have shown that in natural and laboratory conditions, a group of terrestrial reptiles can exude the salt in a highly-concentrated solution from the nasal gland (SCHMIDT-NIELSEN et al., 1963; DUNSON, 1974; 1976).

This subject has been reviewed by CLOUDSLEY-THOMPSON (1971), and LEMIRE & VERNET (1982), reported that some species of terrestrial reptiles usually exude relatively smaller amounts of salt relative to marine and estuarine species. A nasal gland in the form of a special gland specialized for salt secretion is common in herbivorous desert lizards, in particular those where their only water sources are the plants that they eat (GABE & SAINT GIRONS, 1971; 1976). These animals ingest significant amounts of electrolytes particularly potassium and they must adapt to quantitative changes in ion concentrations in their food. The importance of nasal gland secretions in regulating plasma electrolyte concentrations in the family Iguanidae, residing in deserts of Northern America, has been demonstrated in both Sauromalus obesus (NORRIS & DAWSON, 1964; TEMPLETON, 1964; SHUTTLEWORTH et al., 1987); and Dipsosaurus dorsalis (TEMPLETON, 1966; SHOEMAKER et al., 1972; HAZARD, 2001). GRENOT (1976) showed that these lizards are similar in their ecology to the north-Africa agamid lizard, Uromastix. acanthinurus. However despite salt being gathered on the nasal cavities in normal conditions (GRENOT, 1968), and having a salt gland in the nose which has specialized morphologically (LEMIRE et al., 1970; 1972; LEMIRE, 1975), the manner of adaptation of the nasal gland in Agamidae against increasing salt ions in comparison with Iguanidae of Northern America has remained unknown. Nasal gland structure in U.acanthinurus has been studied by LEMIRE & GRENOT (1974), LEMIRE et al. (1970), and in Agama

Our investigation which carried out with A. nupta around Khoramabad has indicated that lizards fall into hibernation during the cold season, emerging to become active at the beginning of spring. The lizards are typical heliotherms, during the warm season. They use sunlight to regulate their body temperature. Typical thermoregulatory behaviour is indicated by A. nupta including the changes of dermal colour, which may assist in thermoregulation (RICE & BRADSHAW, 1980). A. nupta do not drink water and their required water is supplied solely through their insect diet.

A. nupta in Khoramabad indicate that plasma electrolyte concentrations increase significantly during the summer months with clear evidence of both hypernatraemia and hyperkalaemia. As in desert populations of lizards studied by LEMIRE & VERNET (1981) the plasma salt level changed according to season, increasing markedly during the warm season. In spring when environmental heat is lower than in summer, the concentration of sodium and potassium is lower in these lizards, and in summer through increasing environment temperature, the concentration of the ions particularly sodium goes up, and reaches a relatively high level in August and early September. Then, as September progresses, and temperatures fall, sodium levels decrease to reach essentially normal values. It should be considered that the percentage of humidity in spring is higher than in summer, and this factor may increase the amount of water present in the lizards’ food. In summer when the humidity is low, the rate of evaporative water loss from the lizards^’ body is high and there is a potential for plasma Na⁺ and K⁺ concentration to increase through dehydration. BENTLEY (1959) reported high levels of sodium in the Australian skink Tiliqua rugosa in the middle of summer. BRADSHAW & SHOEMAKER (1967) studied Amphibilurus ornatus in Western Australia and reported a similar case of hypernatraemia, but one induced by sodium loading from the diet, rather than from dehydration. Increasing sodium concentration in some of the species depends on environment conditions and type of feeding, and may reach to 300mmol/L^-1 and for potassium 12mmol.L^-1 (BRADSHAW, 1997).

The secretory capacity of nasal salt gland has been examined in some species of lizards (MINNICH, 1981; LEMIRE & VERNET, 1982) with experimental loads, but due to differences in morphology and in the doses used, it is not always easy to obtain a clear idea of the potentialities of the nasal gland.

The first investigation on salt secretion on reptiles indicated that many of terrestrial lizards have a special gland in their nose, which has the property of producing a hyperosmotic secretion (SCHMIDT-NIELSEN & FANGE, 1958; SCHMIDT-NIELSEN, 1964). In the desert lizard Uromastix acanthinurus, which feeds on plants, electrolytes excreted from the nasal gland assist in maintaining the water and electrolyte balance in the animal (LEMIRE, 1976). There is a nasal gland with high electrolyte excretion to adapt to environment in desert reptiles. The importance of the function of nasal gland in excreting salt and regulating electrolytes concentration in some species of North America Iguanidae has been highlighted by NORRIS & DAWSON (1964), TEMPELTON (1964; 1966), SHOEMAKER et al. (1972).

Other investigations indicated that in intertidal lizard, Uta tumidarostra, as in other lizards, the nasal glands are able to eliminate of ions (HAZARD et al., 1998), and in desert Iguana, Dipsosaurus dorsalis, (HAZARD, 2001) nasal glands are capable to secret ions sodium, potassium, chloride and bicarbonate in different rates. The rate of secretions may be related to diet of lizards.

LEMIRE M (1976). Etude anatome-histologyque de 1’organe nasale du lézard saharien Uromastix acanthinurus Bell 1825 (Sauria, Agamidae). Thèse Doct. 3^e cycle, Univ. Paris, V1: 131 P.

LEMIRE M & GRENOT C (1973). La structure nasale du lézard saharien Agama mutabilis Merren (Agamidae). Comptes Rendus de l’Académie des Sciences, Paris 277: 2719-2722.

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Received: September 28, 2005 Accepted: May 25, 2007

Sallam WS1, Mantelatto FL2* & Hanafy MH1

Gastropod shells are clearly important in all aspects of hermit life cycle as the main source utilized by these crustaceans to protect their soft and vulnerable abdomen. The crabs prefer to occupy shells of certain gastropod species to others, a preference not necessarily based on previous experience with these shell species but on certain shell properties that vary among gastropod species (CONOVER, 1978). Shells may function as a limiting resource to these crabs when in low abundance or adequacy (KELOGG, 1976). They may restrict crab growth (VANCE, 1972; BERTNESS, 1981), enhance their predation risk (VANCE, 1972), reduce fecundity (BERTNESS, 1981; ELWOOD et al., 1995), and modulate reproductive activity (BERTNESS, 1981; LITULO, 2004) and success (HAZLETT, 1989; HAZLETT & BARON, 1989). The patterns of shell utilization vary between hermit crab populations and are influenced by the type and size of shells available in the survey, the locality and the hermit crabs’ shell preference (MANTELATTO & GARCIA, 2000; MEIRELES et al., 2003; MANTELATTO & MEIRELES, 2004).

On the other hand, research investigating aspects available worldwide on coenobitid hermits has focused on reproductive aspects (see TUDGE & LEMAITRE, 2006) and the mechanisms of migration (see BARNES, 2003 and NIEVES-RIVERA & WILLIAMS, 2003 for review). Nevertheless, the shell utilization approach for these land crustaceans has received little attention in recent years and is poorly known even though its significance. Since movement is particularly costly in the terrestrial environment, the crabs must carry their shells all the time and sometimes inadequate ones in function of their size and weight.

As an initial step to evaluate the ecological parameters affecting population of land hermit crabs in south Red Sea, we characterizes the shell utilization pattern of C. scaevola in a sandy beach in the protected area of Wadi El-Gemal at Marsa Alam, Red Sea.

Hermit crabs were obtained from the protected area of Wadi El-Gemal in Marsa Alam, Red sea on July 2003 and January 2004. The animals were collected by hand at low tide from a sandy shore in the early morning by one person during a 20min walk over an area of 300m long. They were fixed in 10% formalin in seawater and transported to the laboratory. Processing started by careful removal of crabs from their shells in an anticlockwise fashion. Crab specimens were sexed by observing the position of the genital opening (gonopores). They were weighed (WW) and measured for cephalothoracic shield length (CSL=measured from the tip of the rostrum to the V-shaped groove at the posterior edge) and width (CSW), left chelar propodus length (LPL) and height (LPH). Shells were weighed (SWW) and measured for shell aperture width (SAW) and length (SAL) and shell internal volume (SIV=determined by the amount of water required to fill the empty shell by means of a measuring pipette). Measurements were carried out using a 0.1mm venier caliper. Gastropod shell species were identified according to SHARABATI (1984). The normality of hermit crab size (shield length) data was checked by Kolmogorov-Smirnoff test (ZAR, 1996). The chi-square test (χ²) was used to compare the absolute frequency of occupation of shell species between sexes. To determine correlations between the dimensions of hermit crabs and occupied shells, regression analyses were performed (Spearman test) and by correlation coefficients using the power function equation (Y=a.X^b).

A total of 391 individuals of C. scaevola were collected (172 males and 219 females). No ovigerous females were observed in the samples. There was a unimodal size distribution for each sex and the size frequency distribution showed a prevalence of specimens measuring 3.5 to 8.0mm in shield length (Fig. 1). The size ranged from 2.5mm (males and females) to 9.0 and 9.5mm in shield length for females and males respectively. Overall sex ratio was 1: 1.2 in favour of females and was significantly different from the expected 1: 1 (χ²=5.64, d.f.=1, P<0.05).

The hermit crabs were found occupying ten species of gastropod shells in different percentages (Table 1). Nerita undata was clearly the most occupied (86%), with no difference in shell use between sexes (χ²=1.06, d.f.=1, P<ns), followed by Turbo radiatus (7.2%). On the other hand, there were significant differences in gastropod shell species occupation between sexes. Littorina scabra, Monodonta canilifera and Nassarius arcularius plicatus were occupied only by males (χ²=16.9, d.f.=9, P<0.05). Regarding to the shell versus hermit correlations, shell utilization in C. scaevola was strongly associated with shell internal volume.

Shell species occupation as a function of hermit crab size is illustrated in Fig. 2. The diversity of shells utilized increased with the increasing of individual size. Among the occupied shells, N. undata dominated and was occupied by a wide range of size classes (2.5–8.5mm CSL). Small sized crabs (2.5–3.5mm CSL) occupied Planaxis sulcatus and Nassarius arcularius plicatus while larger specimens (8.5–9.5mm CSL) occupied Turbo radiatus, Polinices milanostomus and Monodonta canilifera.

Since almost all individuals (86%) were found occupying N. undata, we presented the regression equations showing the relations between C. scaevola dimensions and N. undata (Table 2). The equations ranked the relationships between the crab dimensions and internal volume of the occupied shell as recognized by the high correlations (r>0.80).

The population of C. scaevola showed unimodality in the size-frequency distribution for the total individual analyzed. This life trait is the most common pattern among the hermit crabs (see MANTELATTO & SOUZA, 2000 for review) and reflects continuous recruitment and mortality of the species (DÍAZ & CONDE, 1989). Sexual size dimorphism was observed in C. scaevola, where males attained larger sizes than females. This dimorphism is a common one reported for marine hermit crabs (i.e, MANTELATTO & GARCIA, 2000; BIAGI et al., 2006), associated

Percentage

female %

male % 20 18 16 14 12 10 8 6 4

2.5 - 3 3 - 3.5 3.5 - 4 4 - 4.5 4.5 - 5 5 - 5.5 5.5 - 6 6 - 6.5 6.5 - 7 7 - 7.5 7.5 - 8 8 - 8.5 8.5 - 9 9 - 9.5

Size range (mm).

Fig. 1. – C. scaevola. Size frequency distribution (CSL) for the total number of individuals obtained from the protected area of Wadi El-Gemal in Marsa Alam, Red sea on July 2003 and January 2004.

Cerithium caeruleum								Littorina scabra
Monodonta canilifera								Nerita undata
Nassarius arcularius Plicatus								Natica gualteriana
Planaxis sulcatus								Turbo radiatus
Nassarius protrusidens								Polinices milanostomus
3	9	43	37	56	61	63	42	27	23	17	2	5	3

2.5 - 3 3 - 3.5 3.5 - 4 4 - 4.5 4.5 - 5 5 - 5.5 5.5 - 6 6 - 6.5 6.5 - 7 7 - 7.5 7.5 - 8 8 - 8.5 8.5 - 9 9 - 9.5

Cephalothoracic shield length (mm)

Fig. 2. – C. scaevola. Gastropod shell species occupation as a function of hermit crab size. The numbers above the bars indicate the total sample size for each size class.

TABLE 1

Total number and percentage of gastropod shells occupied by Coenobita scaevola in Wadi El-Gemal, Red Sea. (N=number of specimens).

Gastropod Shell Species	Males N (%)	Females N (%)	Total N (%)
Cerithium caeruleum (Sowerby, 1855) Littorina scabra (Linnaeus, 1758) Monodonta canilifera (Lamarck, 1816) Nerita undata (Linnaeus, 1758) Nassarius arcularius plicatus (Roding, 1798) Natica gualteriana (Recluz, 1844) Planaxis sulcatus (Born, 1778) Turbo radiatus (Gmelin, 1791) Polinices milanostomus (Gmelin, 1971) Nassarius protrusidens (Melvill, 1888)	3 (1.74) 2 (0.91) 5 (1.3) 2 (1.2) – 2 (0.5) 1 (0.6) – 1 (0.3) 138 (80.2) 197 (90.0) 335 (86.0) 5 (3.0) – 5 (1.3) 2 (1.2) 2 (0.9) 4 (1.0) 3 (1.7) 2 (0.9) 5 (1.3) 14 (8.1) 14 (6.4) 28 (7.2) 2 (1.2) 1 (0.5) 3 (0.8) 2 (1.2) 1 (0.5) 3 (0.8)
Total	172 (43.99) 219 (56.01) 391

TABLE 2

Regression equations for the relations between hermit crab Coenobita scaevola dimensions and the most occupied shell Nerita undata (R²=determination coefficient; CSL=cephalothoracic shield length; CSW=cephalothoracic shield width; LPL=left propodus length; LPH=left propodus height; WW=hermit crab wet weight; SAW=shell aperture width; SAL=shell aperture length; SWW=shell wet weight; SIV=shell internal volume; N=391).

Relations	Y= a.Xb	R2
SAW x CSL	SAW=0.6401CSL0.9586	0.49
SAW x CSW	SAW=0.4293CSW0.9681	0.49
SAW x LPL	SAW=0.7574LPL0.9469	0.53
SAW x LPH	SAW=0.4910LPH1.0645	0.50
SAW x WW	SAW=0.0018WW2.5603	0.48
SAL x CSL	SAL=1.3051CSL0.7578	0.67
SAL x CSW	SAL=0.8851CSW0.7631	0.67
SAL x LPL	SAL=1.7377LPL0.6804	0.62
SAL x LPH	SAL=1.1556LPH0.8064	0.66
SAL x WW	SAL=0.0124WW2.016	0.66
SWW x CSL	SWW=4.3139CSL0.3454	0.53
SWW x CSW	SWW=2.9382CSW0.3542	0.56
SWW x LPL	SWW=5.0633LPL0.3175	0.52
SWW x LPH	SWW=4.1154LPH0.3723	0.55
SWW x WW	SWW=0.2992WW0.9441	0.53
SIV x CSL	SIV=5.8882CSL0.4691	0.79
SIV x CSW	SIV=4.0399CSW0.4765	0.79
SIV x LPL	SIV=6.747LPL0.4384	0.77
SIV x LPH	SIV=5.7532LPH0.5065	0.79
SIV x WW	SIV=0.6855WW1.2696	0.79

principally to differences in energetic repartition between sexes (ABRAMS, 1988) and is important in selective pressure as intra- and interspecific fights for food, copulation, territory and shell (MANTELATTO et al., 2005). This dimorphism can be attributed to such factors as differential mortality and growth rates between sexes (ABRAMS, 1978) with males reaching larger sizes within a shorter time than females, but being influenced by shell limitation, a fact that may imply reduced survival (FRANSOZO & MANTELATTO, 1998; MANTELATTO & GARCIA, 2000).

Coenobita scaevola has the greatest number of zoeal stages (7 stages) and the longest zoeal life span (54-80 days) among the members of genus Coenobita (see ALAIDOROOS & WILLIAMSON, 1989 and WANG et al., 2007). Comparatively, the larval development of Coenobita clypeatus and C. rugosus require about 22-30 days to attain the glaucothöe respectively, and at least an additional month to the first crab, the presumed settling stage (PROVENZANO, 1962; SHOKITA & YAMASHIRO, 1986). Since in the studied period we have not found ovigerous females we may expect that juveniles are not being recruited to the population all over the year. This condition was corroborated by a low number of small sized crabs (2.5–3.5mm CSL) found in the Wadi El-Gemal. Thus we infer that settlement could take place in a different habitat other than the usual one for the adults, due to the fragility of these organisms and necessity of small shells supply. The presented pattern has been reported for other marine hermit crabs (MANTELATTO & SOUSA, 2000; GARCIA & MANTELATTO, 2001a; MACPHERSON & RAVENTOS, 2004). According to BALL (1972), it appears that small sized individuals of C. compressus are much more sensitive to desiccation than larger animals and great aggregations can be found under ledges, in small rocky caves where is slightly more moisture.

Differences in gastropod shells utilization can occur as a function of the area of occurrence of the hermit crabs (GARCIA & MANTELATTO, 2000). Coenobita species are frequently found in areas, such as sandy beaches, where shells are extremely scarce (BALL, 1972). Specimens of

C. scaevola of Wadi El-Gemal were found occupying ten species of shells with relative differences between sexes. Littorina scabra, Monodonta canilifera and Nassarius arcularius plicatus were occupied only by males. This fact probably indicates the resource competition/partition occurring mainly to guarantee a good adequacy of individual size to shells available in the survey. Differences in shell utilization between sexes were also observed by IMAZU & ASAKURA (1994) and BERTINI & FRANSOZO (1999). These differences may be due to differences in body size, competitive ability or reproductive behaviour, respectively (BERTNESS, 1981; BLACKSTONE, 1985; IMAZU & ASAKURA, 1994). Also, in semi-terrestrial hermit crabs a well-fitting shell is essential for maintaining low evaporation rates and carrying ample water. An appropriately sized shell in good condition allows invasion of inland environments offering more shade, food, and fresh water for C. clypeatus studied on Curaçao (WILDE, 1973). According to this author, the hermit crabs with broken, ill-fitting shells are restricted to the coast, must rely on drinking seawater, and appear to be in relatively poor conditions.

A population of C. scaevola studied almost 40 years late by VÖLKER (1967) in the close region of Hurgada were found occupying a greater diversity of gastropod shells species (n=29) than the population studied here on the sandy shore of Wadi El-Gemal (n=10). Several hypotheses are possible to explain this apparent discrepancy in gastropod shell occupancy rates found between the two populations: 1) Gastropod life cycle – availability of different shell types (species) in nature is determined by the relative abundance of different live gastropods and their mortality rates (MEIRELES et al., 2003); 2) Environmental conditions – differences in abiotic characteristics of these two areas in terms of water dynamics (wave activity, intensity of currents, food supply) are determinant of installation of some invertebrate species (FRANSOZO & MANTELATTO, 1998); 3) Predation pressure – several combined actions from natural (crabs) and artificial (human tourism) predators can act in different ways to reduce the diversity of gastropod shells in the region.

Similarly as other marine hermit crabs, C. scaevola shows highlight occupation of one species of gastropod shells, N. undata, over other ones. This shell was occupied by a wide range of individuals of both sexes with CSL varying from 2.5 to 8.5mm, while large sized crabs (8.5–9.5mm CSL) occupied a variety of shells. Although that shell availability was not evaluated, this intense occupancy would indicate active selection behaviour in C. scaevola in the field. Interestingly, in the past study VÖLKER (1967) found that shells generally used by C. scaevola were found in the same frequency in the gastropod fauna, revealing a close relationship between shell use and availability of the resources. According to the energy savings hypothesis, proposed by OSORNO et al. (1998), C. scaevola of Wadi El-Gemal preferred N. undata because it’s the lightest of the shells available thus reducing the cost of bearing and carrying a shell. Large sized crabs might have modified their shell preference and occupied other species when large shells of N. undata became no longer available. The energy saved by carrying a light shell may be used to increase growth rate and egg production of intertidal hermit crabs, which ultimately improve fitness (GUILLÉN & OSORNO, 1993; BERTNESS, 1981). Also, crabs occupying shells large enough that they can withdraw completely and block the shell aperture with the chelipeds are much harder to extract from their shells than crabs which are too large to withdraw completely, and for this reason they would presumably be less vulnerable to predators (BALL, 1972). In conclusion, this pattern should be associated with the availability of resources and/or to the better suitability of these shells species to the condition of the individuals in the natural habitat.

Competition for shells may not be as intense among land hermit crabs as among their aquatic relatives. ABRAMS (1978) reported no fights for shells among Coenobita compressus in the field in Panama, whereas marine hermit crabs frequently fight. In opposite way when compared with marine hermit crabs, larger individuals of land hermit crabs tend to inhabit shells nearer their preferred size, i.e., shells that are modified by previous hermit crab use. The new shells have too little interior volume and are enlarged by hermit use (WOLCOTT, 1988). In this connection it is interesting to note that the majority of the shells occupied by Coenobita seem to be missing the columella (BALL, 1972). A similar observation has been made by KINOSITA & OKAJIMA (1968) on shells of Nerita striata occupied by Coenobita rugosus from Japan. ABRAMS (1978) suggested that rather than competition, terrestrial hermits show “shell facilitation”; that is, larger populations of crab generate, through wear, larger numbers of shells suitable for adult crabs. He accounted for limited adult populations by limited “recruitment” of entry-level shells, or possibly by predation on or food competition between adult hermits.

Interestingly and on the contrary to the proposed by VÖLKER (1967), that affirm “land hermit crabs seem to have no relation to the shells of a given snail species”, we found that shell dimension constitutes mainly the determinant for C. scaevola shell utilization, adopting in the Red Sea similar strategy developed by some other tropical and subtropical marine hermit crabs (GARCIA & MANTELATTO, 2001b).

The absence of ovigerous females during the two sampled periods was also an interesting and important point in the present study indicating the need for further frequent and systematized studies on this population since this is a rare pattern for these crustaceans. The presence of ovigerous females in some populations studied around the world have been previously documented (BALL, 1972; WILDE, 1973; VANNINI, 1976; SHOKITA & YAMASHIRO, 1986). So, despite the fact that reproduction in coenobitids has covered a range of topics in recent years (TUDGE & LEMAITRE, 2006), the factors responsible for the absence of egg-bearing females are not clear and may involve methodology design (only two sampled periods), shell resource availability, interspecific competition for food and shelters, and others. The data obtained from two dates by daylight collection provided limited information about the reason for this absence. However, some hypotheses can be raised taking in account the limited range of the collection: 1) peaks of presence of ovigerous females probably occurs in different periods; 2) ovigerous females may be taking shelter at other points along this beach showing a cryptic habit during daylight and active at night as C. compressus (BALL, 1972), a common occurrence observed among ovigerous females of crustaceans; Particularly for C. scaevola, and other coenobitids, VANNINI (1975) showed either daily or seasonal migrations depending upon rainfall. Also, spawning females of C. clypeatus presented unusual pattern since they did not enter the water, but move toward the sea at low tide to drop or fling their eggs onto the wet rocks (WILDE, 1973).

As a result of the intensive unregulated tourist development in the Red Sea area, a shore-monitoring program was established for the protected area of Wadi-El-Gemal. Continuous surveying of shores and beaches by rangers has led to the observation that C. scaevola is the first organism to disappear from any shore when a new tourist establishment is constructed (W. SALLAM, pers. comm.). Accordingly, this study suggests the possibility to this species to be used as a key-species in the preserving program of this protected area. Its rarity or abundance on any shore could reflect the degree of healthiness of that shore and might assist in assessing the amount of deterioration of the shoreline which has a considerable impact on the different biota.

The first author wishes to express her sincere gratitude to Ms Marwa Essam for her help. Fernando Mantelatto extends special thanks to Wafaa Sallam for graciously allowing combining data and observation in order to jointly prepare this report and is grateful to CNPq for on-going Research fellowship. The authors are grateful to Drs. Renata Biagi and Andrea Meireles, and anonymous reviewers, for valuable suggestions on the manuscript. All experiments conducted in this study comply with current applicable state and federal laws.

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Marc Callebaut

UA, Laboratory of Human Anatomy & Embryology, Groenenborgerlaan 171, B-2020 Antwerpen Belgium To the memory of Professor Doctor Lucien Vakaet sr

Corresponding author : Prof. Dr Marc Callebaut, Laboratory of Human Anatomy and Embryology UA, 171 Groenenborgerlaan, B2020 Antwerpen. Phone: ++3232653390; fax: ++3232653398; e-mail. marc.callebaut@ua.ac.be.

ABSTRACT. In the classical embryology there exist two main concepts to explain the rising of a living being. At one hand there exists the theory of preformation i.e. all the parts of the future embryo would already exist preformed during the preembryonic or early embryonic period. On the other hand there is the epigenetic view in which it is propounded that all parts arise by neoformation from interacting (induction) previously existing, apparently simpler structures. We found evidence that initially a kind of preformation (or pre-existence), disposed in concentric circular layers, exists in the full grown oocyte and early germ. After normal development these circular layers will settle successively, from the centrum to the periphery, into the central nervous system, the notochord, somites, lateral plates (coelom) definitive endoderm. Under influence of mechanical and gravitational forces an early epigenetic development starts by unequal oblique uptake (unidirectional chaos) and segregation of ooplasmic determinants in the germ. So an epigenetic cascade of early general body plan formation occurs in the vertebrate embryo. Recently, we demonstrated by hemi-sectioning of avian unincubated blastoderms that both a kind of preformational (mosaicism) as epigenetic (regulation) behaviour depends on the spatial, ooplasmic distribution of Rauber’s sickle material, homologous to the also sickle-shaped Wnt expressing gene region in ascidians. This clearly brings preformation and epigenesis much closer together since there is a common influencing ooplasmic factor. An ooplasmic continuity bridging the premature oocyte ooplasm to the embryonic primordial germ cells of the following generation is demonstrated. Both nuclear and ooplasmic continuity in the oocytes, present in the ovary of birds and mammals, is shown by radioactive premeiotic DNA labelling.

KEY WORDS : preformation, mosaicism, epigenesis, predispositions, ooplasmic determinants.

Among all biological topics, embryology (developmental biology) presents most uniformity in the animal and even in the vegetal kingdom. So, not with standing the pronounced structural differences between species, it forms a common link among all living organisms. All embryological processes are based on cell biological, genetical and biochemical principles since every organism is formed by isolated or associated cells. In embryology one studies the very complex processes by which these cells differentiate and associate from one single initial more or less voluminous cell: the egg. The egg is the link between two generations and already by its volume it gives the impression to contain more then an ordinary cell. Our mind needs, already from its earliest manifestation, to know how a young animal, a child or a plant makes its appearance. It is still one of the first questions that children ask for when they begin to think and realize their own existence. Moreover their own origin, as is the case for most mammals is most difficult to observe since the mammalian embryo is completely hidden from direct observation. By contrast, avian embryos are more accessible to direct observation. For instance chickens lay eggs, incubate them by their own body heat and after approximately 20-21 days, as touched by a magician, hatching of a young bird takes place. It is easy to open the eggs at the successive stages of development, sometimes even without killing the embryo. This has been and is still one of the bases of modern embryology.

Ancient Arabs and old Germans thought that only mothers have the possibilities to give origin to the development of a child, whilst people in Greece considered that the father was only responsible (SHORT, 1977). Greek natural philosophers generally interpreted female seminal fluids as menstrual blood. The female usually was considered inferior to the male: due to a lack of internal heat, this menstrual blood did not form true seed. This did happen in males, leading to male seed or sperm, which also was thought to form out of blood (generally conceived as a life giving principle). Hippocrates (460-380 B.C.) “the father of medicine” was the first to control theories with real experiments. He made from embryology a separate science and made comparative embryological studies between the chicken and the human embryo. He accepted that both the female as the male contributed equally to the formation of the germ. ARISTOTELES in the 4^th century before J.C., also called “father of the natural history”, was the first to describe the “punctum saliens”, the early beating heart of the avian embryo and followed its evolution, day by day during further incubation. While he observed the evolution of the chicken embryo and it’s appearing organs, he concluded that the organs appear successively and not at the same moment. Therefore he proposed the so-called epigenetic development in contrast to the preformation hypothesis. Aristoteles did not consider uterine blood but the male seed as the essential factor for germ formation. Aristoteles saw it as an unmaterial principle of movement “triggering” the menstrual blood to further selforganize. The female “seed” is to be considered as the material cause of any embryological process. Ancient people must have observed the existence of egg sacs with large eggs in the ovaries from birds. The yellow aspect of the large follicles in the ovary and a similar aspect in the laid egg were the first indication that eggs are formed in the ovary. So the link between ovarian oocytes and the avian germ in the laid egg (oviparity) was established. But the principle of viviparity (development of an embryo in the female organism) could originally not been explained. ARISTOTELES in the 4^th century before J.C. has made embryological observations on much different kinds of animals. He followed not only methodically the development of the chicken embryo but has also observed the embryos of dolphins and larval stages of insects (CAULLERY, 1957). The role of the male and female respectively in the generation process could be explained according to Aristoteles by the example of the chicken. Indeed, the latter taken apart from the rooster still continues to lay eggs in which however no embryos develop. Therefore he concluded that generally speaking the female furnished the constituting material of the embryo whilst this material was vitalized by the male. The place where this occurred in the chicken egg would be the cicatricular region where the germ disc, forming the base of the future embryo develops. Departing from Aristoteles’s natural philosophy, VAN SPEYBROECK et al. (2002) have historically shown that epigenesis gained alternating attention from the 17^th century onwards. It was considered to be the opposite of the preformationist tradition. Where preformationism stated that the germ cells of each organism contain preformed miniature adults that unfold during development, epigenesis held that the embryo forms by successive gradual exchanges in an amorphous zygote. CLAUDIUS GALENUS (131-201) one of the founders of anatomy and physiology was born in Pergamon. He made dissections of animals, mainly apes, because it was not allowed to study human corpses. His influence has been very important until in the 16^th century. Galenus described the female ovaries as testes and propounded that the male sperm was secreted by the testes. So the notion of gonads (genital glands) male or female was established. Galenus holds a very naturalistic view on the embryological processes in terms of growth, nourishment and genesis (change and shaping). After Galenus during 13 centuries, science and particularly embryology were completely influenced by religious ideas and no new discoveries were made.

PERIOD
OF THE EARLY ANATOMISTS

Fabricius Ab Aquapendente (teacher of William Harvey) had a chair of anatomy at Padua and made an enormous contribution to embryology (ADELMANN, 1942). He

Fig. 1. – Preformation concept during the 17^th century: drawings of the early developmental stages of the chicken embryo in which, according to Fabricius Ab Aquapendente, already the adult form could be seen.

was probably the first investigator to give an exact account of the role of the ovary in the formation of the hen’s egg, for he observed that besides that the yolk was formed in the ovary that the egg white, the shell membrane and the shell were all formed during the transit of the egg down the oviduct. This key observation was the first indication that the egg might be produced directly by the female rather than as the result of the union of male seed “and female soil” as supposed by Aristoteles. He also thinks that the avian egg is fecundated by the so-called “aura seminalis” (an emanation from the semen of the rooster). FABRICIUS was also a preformationist since he pretended that a “little birdie” was visible in the egg during the earliest developmental stage (1637) (Fig. 1). However the initial developmental stages of most of the embryos have a size which makes them invisible to the naked eye. The first microscopes appear in the 17^th century, but only in the 19^th century a higher degree of perfection permitted their real use for embryological observations. Embryos in advanced developmental stages are more or less visible to the naked eye and already have more or less the form and structure of the adult animal. This suggested the attractiveness of the theory of preformation i.e. that development was only the result of the progressive enlargement of the germ, having from the beginning its final constitution and complexity. The preformation theory seemed to be first propounded by the arab scientist, AVERROES (also called Ibn-Roschd) who was a teacher at Cordoba (1115-1198). Contrary to this theory, the theory of epigenesis was formulated: the complex structure of the definitive adult forms and even the embryo is only realized progressively by transformation of more simple structures. In the course of the history of embryology, as a research discipline, it appeared that a kind of progressively build up preformation (pre-existence) exists in the egg (already before fertilization), which is indispensable for early embryonic development. Initially, epigenesists defended the view that the embryo progressively forms out from a homogeneous matter.

Aristoteles, but also HARVEY (1653) claimed that it is most certain that in the egg there is no prepared material at all. It takes until the studies of Albrecht Von Haller (1708-1777) first adhering to epigenesis, later becoming a strong ovist-preformationist, to conclude that the egg does contain differences in viscosity.

WILLIAM HARVEY (1578-1657), medical doctor from the king (Charles I) of England, discovered the blood circulation (1628) and made dissections from female deer’s after coitus and states that the young germs of the mammals are present in the form of eggs. In 1653 appears his book: “Exercitationes de generatione animalium”.

He did point out a place where fecundation would occur, but at the same time, his research on deer did not allow him to conclude that the male semen made any material contact with the egg (or left any visible trace) on the female egg. According to him the little white cicatricula on the top of avian eggs is the place where fecundation occurs. After incubation this region extends and forms a semi-liquid mass (colliquamentum) in which soon the first traces of the embryo and particularly the heart appear. From him comes the aphorism “ex ovo omnia”: every living being develops from an egg (1651). REGNIER DE GRAAF (1672) who was an excellent experimentator (LINDEBOOM, 1973) observed that after killing, female rabbits at different moments after coitus present a progressive evolution in the vesicles bulging at the surface of the ovary, followed by scars after ovulation. These “eggs” (sic) were found afterwards in the uterus. He thinks erroneously that the vesicles bulging at the surface of the ovary before the coitus were really the eggs themselves (SAWN, 1997). He observed that once they were found in the rabbit uterus they are first small but later larger, adhere to and become fixed into the wall of the uterus (implantation). Now we know that the ovarian vesicles described by Regnier De Graaf are not the eggs but are follicles surrounding the real, much smaller eggs. These follicles are now still called Graafian follicles in modern literature. At nearly the same moment, thanks to the use of the first microscopes, spermatozoa were seen in semen (perhaps by HAMM, or HARTSOEKER, 1694; fig. 2) but they were surely seen, drawn and described by the Dutch researcher ANTONIE VAN LEEUWENHOEK (1678; 1683). The main discovery of Van Leeuwenhoek was his finding in 1677 of “little animals” (animalcules) in semen (LA BERGE, 1999). He found these animalcules in semen from men but also from roosters and in the semen of different mammals. So Van Leeuwenhoek became an “animalculist” or “spermist” who thought that later the embryo was formed from one of these little animals (Fig. 2) or spermatozoa (Von Baer used this word). According to Van Leeuwenhoek, De Graaf’s egg, if it did anything, provided no more than nourishment to the embryo. During the next century little further research was done on the topic, although there were much theoretical discussions between the animalculists and the ovists (who considered the egg as the location in which the preformed embryo resides). De Graaf’s ideas on the egg as being the precursor of the embryo fell in some disfavour because the evidence for the animalculists was easy to obtain by simple observation of semen under the microscope, which seemed to demonstrate the animalculist preformation theory (LINDEBOOM, 1973). The ovistic hypothesis of De Graaf was on the contrary more difficult to control. It was in fact only much later, 100 years after De Graaf’s death, that CRUIKSHANK repeated and confirmed his studies on embryonic development in rabbits. Marcello Malpighi (1628-1694) described the capillary circulation in the lungs of frogs and also studied the development of the chicken embryo. His study “De formatione pulli in ovo” appeared in 1672. This biologist used systematically a microscope (fabricated by JANSSEN in 1590). He described the cicatricula as the most important part of the avian egg for the development of the future embryo after incubation. With Malpighi the microscope became an indispensable tool for biological research (LAMS, 1935; LÜTHY, 1996) and he adhered to ovism. In 1740, at the age of 20, CHARLES BONNET (Switzerland) describes for the first time the phenomena of natural parthenogenetic development (from a virgin egg) in a plant-louse. These observations of BONNET on virginal reproduction were confirmed by RÉAUMUR and BEGUELIN, TRIMBLEY and ALBERT VON HALLER. This discovery of parthenogenesis was strongly in favour of the ovist theory of preformation. However, the eventual “emboîtement” of the successive generations could hardly been explained. We must also mention the theory of organic molecules developed in 1745 by MAUPERTIUS (DOLLANDER & FENART, 1973). Seminal liquids both from the mother as from the father, coming from all parts of the body, should mix after mating in the uterus. So, Maupertius tries to explain that some characteristics of the father or mother are recognized in the children. By contrast both ovists and spermatists (or animalculist) believed in the theory of preformation i.e. that the egg or the spermatozoon contains a miniature individual with all the parts of the adult. FABRICIUS AB AQUAPENDENTE (1637) and MALPIGHI (1669) were convinced preformists and according to them the avian germ disc contains a miniature organism already in its definitive form (Fig. 1) (ADELMANN, 1942; 1966). By growing, its different parts unfold progressively into an adult form. Albertus Von Haller (1753) had a more evoluated idea (with epigenetic background) about preform-ism. He wrote: “nulla est epigenesis, nulla in corpore animali pars ante aliam facta est et omnes simul creatae existunt”. He found no recognizable organs in the chicken germ but considered that only their Anlagen were present and that these will progressively develop into their adult form. Von Haller was an authority in embryology and studied the chicken embryo at early stages after incubation. He wrote a book in French and described the development of the heart, the eye and respiratory phenomena (see the title page of his book: Fig. 3). He uses the chicken embryo to explain the formation of some anatomical structures in the human.

Fig. 2. – Drawing of the microscopic aspect of a human spermatozoon of animalculist Hartsoeker, in which he seems to see a miniature baby as was also accepted by Van Leeuwenhoek.

Fig. 4. – Early composition and disposition of the avian blastoderm and cicatricular region on the avian egg yolk ball, represented by PANDER in 1817: the so-called nucleus of Pander is seen as a triangular mass below the unincubated blastoderm (upper figure) and after incubation below the three germ layers (lower figure), clearly indicating epigenetic phenomena (neoformation).

In 1821, for the first time, VON BAER detected mature unfertilized eggs (oocytes) in the ovary of the female dog. In his paper “De ovi mammaliae et hominis genesis” (1827) he notes that they were identical to the ova found in the oviducts. Later, Von Baer started to analyze ovaries in rabbits, pigs and humans and described the human egg and the structure of the Graafian follicle. Von Baer developed and enlarged Pander’s ideas about germ layers and studied their fates during early embryonic development. He was able to demonstrate that the so-called “cutaneous” layer (external layer) transformed into superficial epidermis and central nervous system, that the so-called “muscular” layer (now merely somites) formed muscles, skeleton and connective tissues; that the internal “covering” layer of the digestive tract developed from the so-called “mucous” layer. From these three germ layers all body structures are formed according to an orderly process. By the study of chick embryos he detected a new embryonic organ for the first time i.e. the backbone “cord” (chorda dorsalis or notocord). Based on detailed comparative studies of embryonic development in different vertebrate embryos he formulated some conclusions: during embryonic development more specified characteristics appear later than the more general features and the general features of a large family of animals appear earlier than the features of a species. Von Baer recognizes a certain similarity only between the early embryos of different animal groups.

Fig. 5. – Schematic drawing representing the onion peel-like localization of the four ooplasms (α, β, γ, δ) at the end of oogenesis (modified after CALLEBAUT, 1975): the ooplasms will play a fundamental role, during the early development of the blastoderm; from peripherally and superficially to more centrally and deeply we see: the α ooplasm which after a centripetal movement functions during the cleavage stage (see Fig. 6); the β ooplasm is mainly incorporated in the primitive streak and in the embryo proper; a part of the γ ooplasm, surrounding the nucleus of Pander (NP), is spatially obliquely taken up caudolaterally in the blastoderm and forms Rauber’s sickle (see also Fig. 8); part of the superficial part of the δ ooplasm (from the nucleus of Pander) is also taken up in a spatially oblique manner by the more central part of the blastoderm and forms the sickle shaped endophyll and primordial germ cells (see also Fig. 8); CHR: spherical postlampbrush chromosomes seen in the centre of the flattened germinal vesicle; SCO: perinuclear subcortical cytoplasmic organelles or ticos; WG: wedge granules layer; PG: polar granules; NL: latebra neck.

The germinal vesicle (the large nucleus of the oocyte) in the large ovarian oocytes of birds is transparent (Fig.5) in contrast to the white opaque structure of the surrounding cicatricula. PURKINJE (1830) describes for the first time the avian germinal vesicle and gives it the name of “vesicula germinativa”: Habet itaque cicatricula ovi ovarii partem specialem et sibi propriam, vesiculam sphaericam subcompressam, membranula tenerrima constantem, lympha propria, fors generatrice repletam (inde vesiculam germinativam appellaverim), in fossam cumulo albo mammaeformi e globules composito. (Eggs in the avian ovary present in their cicatricular region a special characteristic structure with a spherical, partly compressed aspect. It contains its own liquid which is always surrounded by a tinny membrane and it is localized in a mammiform mass of globules, it has the power to generate a future germ (sic), therefore I give it the name of germinal vesicle). Thus for Purkinje the germinal vesicle was the germ-generating-structure. By contrast for Von Baer (1828) it was not yet clear if the avian embryo was only formed from the disintegrated germinal vesicle together with the spermatozoa, or that also material from the surrounding cicatricula (ooplasm) plays a role in germ formation. He speaks from “Umbildung” i.e. a series of transformations from simple towards more complex. It was COSTE (1850) who described for the first time the cleavage furrows in the avian blastoderm, formed in the cicatricula shortly after fertilization. So he demonstrated that the oocytal ooplasm of the cicatricula also gives rise to the avian germ. The germinal vesicle in birds, reptiles and cephalopods is according to COSTE (1850) always included in a fine granular layer, which forms the substrate for the later blastoderm from which the embryo will develop. SCHWANN (1839), who developed the general cell theory, was the first to understand that the oocyte (unfertilized egg) of a mammal must be considered as one cell. Owing to their enormous volume he still considered the large oocytes in the ovary of birds as multicellular. HOYER in 1858 clearly observed that the yolk of large avian oocytes contained no cells. Thus large intraovarian avian oocytes are single giant cells.

In 1865 JOHAN MENDEL discovered the fundamental laws of genetics. However his study has been negated and ignored for more then half a century (KRUMBIEGEL, 1957). The reason seems to be that in that period; Darwin described and propounded the variability of species whilst the work of Mendel just demonstrated a constant previsible evolution (DE VEER, 1969). Chronologically we can distinguish three steps in the pairing mechanisms of animals, resulting in the formation of a germ: pairing of the chromosomes during meiosis in the germ cells of the gonads of the parents, copulation of the parents, pairing of the gametes (unfertilized eggs with spermatozoa) at the moment of fertilization. By progression of the cytological techniques, at the end of the 19^th century, chromosomes in meiocytes (during oogenesis or spermatogenesis) were seen as most prominent structures. So VAN BENEDEN (1883) in the eggs of Ascaris megalocephala and VAN BAMBEKE (1885) found, that at the end of the meiotic division, only half of the number of chromosomes was found in the gametes (mature oocytes and spermatozoa). At the moment of pairing of the gametes (fertilization) the specific number of chromosomes is restored in the zygote. This demonstrated for the first time that both the father and the mother afford the same quantity of chromosomal material during the formation of the zygote, which gives rise to all the cells of the embryo. According to Van Beneden (University of Liège) and Van Bambeke (University of Ghent) this suggested that embryonic development is bound to chromosomal material charged with hereditary characteristics. However it is only in 1902 that SUTTON makes the link between the meiotic dissociation of chromosomes and the segregation of hereditary characteristics described by Mendel. The localization of genes on the chromosomes of Drosophila by MORGAN (1910) permitted further development of genetical knowledge. In recent decades the study of expression of genes during early developmental processes has become a powerful tool. Evolutionary and developmental biologists have joined forces to create a new field, unravelling the mysteries of evolution by studying the genes that control how an embryo develops (DEPEW & WEBER, 1995).

The theory of epigenesis, based on neoformation, as has been proposed by C.F. WOLFF since 1759, considers the egg not as a mosaic of territories with an achieved fate but as a progressive realization of stages which are conditioned by an earlier more simple stage (VAN SPEYBROECK et al; 2002). According to this epigenetic hypothesis the half of a germ can still give rise to a complete normal embryo of a smaller size. The egg is thus capable to regulate development of its parts. However in some species there is evidence for preformistic behaviour as the result of factors or territories present in the ooplasm. The link between ooplasmic structures (before fertilization) and structures in the germ (after fertilization) is not always obvious and differs from species to species. So the first experimental investigations in embryology by CHABRY (1887) and ROUX (1888) and CONKLIN (1905) suggested prelocalization – preformation mechanisms by mosaicism. Indeed in some species (ascidian or amphibian) an isolated hemisected egg (containing one of the two first blastomeres) will develop only in the corresponding half (left or right) of the embryo. The term mosaic development is used as originally defined by CONKLIN (1905) in ascidian species: each region of the whole fertilized egg would be able to form more or less independently on its own. The development of the entire embryo was regarded as being the sum of the development and interaction of the individual parts. Here the concept of “preformation” more particulary mosaicism no longer refers to the strong preformationistic theory described earlier in this review (pre-existence of parts), but as a soft preformation or preexistence, inclining to a more sophisticated version of preformation, due to ooplasmic determinants. OSCAR HERTWIG introduced in 1916 the concept of “preformed epigenesis”: the development of multicellular organisms from a fertilized egg is an epigenetical process whose species-specific course is firmly determined by the preformed hereditary substance which serves as its basis. In other species (echinoderms) an isolated half of an egg will produce a complete miniature embryo (DRIESCH, 1891) as the result of so-called regulation phenomena. This can however been explained by the all or not existence in the isolated halves or parts of all the different kinds of ooplasm in the blastomeres. Indeed during a study of the early embryonic avian development (CALLEBAUT, 1987), I found preformation evidence for the role of the four ooplasms (α, β, γ, δ) which present an onion-peel distribution in the oocytal germ disc region (Fig. 5). I demonstrated that α ooplasm plays a fundamental role during the cleavage stage by penetrating along with the cleavage furrows into the underlying ooplasms (Fig. 6). The β ooplasm originally mainly localized in the peripheral region of the area centralis becomes concentrated in the primitive streak by converging phenomena. The γooplasm finally gives rise to Rauber’s sickle. So for instance in the large blastomeres of the early avian germ disc the four fundamental ooplasms can still be observed as is the case in the whole younger germ (CALLEBAUT, 1987) (Figs 5; 6). That the caudocephalic axis of a vertebrate embryo is not preformed in the egg but can develop under influence of gravity was probably first observed by WINTREBERT (1922) in Selachians. Indeed, Wintrebert described the relationship between the spatial orientation of the early Selachian blastodisc on its egg yolk with respect to the vertical and the ensuing development of its caudo-cephalic axis. Also in the Selachian egg any part of the periphery of the blastodisc can give rise to the embryonic caudal edge: the only condition is that it should correspond temporally to its highest point. Still in the 20th century preformistic views concerning the craniocaudal axis of the future vertebrate embryo, persisted. So BARTELMEZ (1912; 1918) claimed that the craniocaudal axis of the future pigeon embryo was already established in the ovarian primordial follicle, visible as the long axis of the oocyte. According to VAKAET (1953; 1955) and FAUTREZ & VAKAET (1954) the plane of bilateral symmetry of the ovoviviparous teleostean fish, Lebistes reticulatus, is already predetermined in the oocyte during previtellogenesis. This view, is contested, however by CLAVERT & FILOGAMO (1957 a; b). Indeed, after fertilization the Lebistes reticulatus egg becomes movable within its follicle and undergoes an orienting rotation by gravity whereby the future germinal disc is turned to the upper pole of the egg. By contrast to vertebrates the cranio-caudal axis of the Drosophila embryo seems to be preformed by the localisation of ooplasmic determinants within the oocyte (ST. JOHNSTON & NÜSSLEIN-VOLHARD, 1992). Bicoid mRNA localised in the cranial pole is translated after fertilisation to give rise to a morphogen gradient of Bicoid protein that patterns the head and thorax (BERLETH et al., 1988). Similarly, oskar mRNA is localised into the caudal pole of the oocyte where it directs the assembly of the pole plasm, which contains the caudal and germ line determinants (EPHRUSSI & LEHMANN, 1992). J. BRACHET (1933; 1941; 1961) and CASPERSON (1941) discovered the role of RNAs in the synthesis of proteins. In 1953, Watson & Crick found the basis of our knowledge about the replication mechanism of DNA synthesis, giving rise to the development of molecular biology. Particularly in oocytes, intense synthesis of RNA’s and proteins takes place (BRACHET, 1941). These molecules (messenger RNAs) will in part pass into the ooplasm of the zygote after fertilization and will play a fundamental role during early embryonic development. According to the hypothesis of DALCQ & PASTEELS (1937) the original localization of the ooplasmic constituents in the egg, even not genetically determined, can influence early stages of embryonic development. Thus a kind of molecular preformism, present in the ooplasm, must be accepted. The term epigenetics was introduced by C.H. WADDINGTON (1940; 1952) over 60 years ago as the study of those processes involved in the unfolding of development. The discovery of the role of DNA in inheritance has cast a shadow over this discipline for decades (CAVALI, 2006). It was found that molecular machines act on the chromatin to regulate gene expression. These epigenetic regulators play a crucial role in the global shaping and maintenance of developmental patterning. Histone modifications seem to play a major regulatory role. Epigenetics has then been redefined as the study of heritable traits that are not dependent on the primary sequence of DNA (CAVALI, 2006). Ooplasm remains indispensable for embryonic development, since until now in the absence of ooplasm no cloning of individuals can be obtained. The transplantation of somatic nuclei into maturing amphibian oocytes by GURDON (1968; 1969) demonstrated the fundamental effect exerted by the ooplasm and eventually the nucleoplasm of the germinal vesicle on an implanted nucleus for further embryonic development.

Fig. 6. – Schematic representation of the localization of the ooplasms in the first large “closing” blastomeres; the forming subgerminal space is lined by a narrow α layer; F: cleavage furrow; NP: nucleus of Pander containing δ ooplasm (after CALLEBAUT, 1987).

During the last decades it became clear that induction phenomena not only occur as the result of interaction between embryonic structures (in the sense of SPEMANN & MANGOLD, 1924) but also extraembryonic ooplasmic structures, all or not nucleized, play a fundamental role in early embryonic induction phenomena. Indeed in 1969 NIEUWKOOP discovered that during early blastulation in amphibians, signals are required from a region which is localized vegetal to the prospective dorsal blastopore lip, to initiate development of the mesoderm (Spemann’s organizer). This vegetal region is now designated as Nieuwkoop’s centre. In amphibians, Wnts seem to be the primary axis (formation of head and trunk-tail regions) inducing substances. Indeed in Xenopus, microinjection of several Wnts into the ventral cells of the early embryos leads to complete duplication of the body axis (CADIGAN & NUSSE, 1997; DEARDORFF et al., 1998). This duplication is believed to arise from the formation of a second Nieuwkoop’s centre (dorsal vegetal cells) of the early blastula which then induces overlying tissue in order to it might become the Spemann’s organizer (homologous to the avian primitive streak and nodus, WADDINGTON, 1932). Indeed in birds and mammals the properties of the Spemann’s organizer are performed by the node (Hensen’s node in birds) which is the rostral end of the full grown primitive streak. We found that in birds, RAUBER’s sickle (1876) (Figs 7; 8) has a homologous function as Nieuwkoop’s centre in amphibia. It functions indeed as the primary major organizer, initiating gastrulation phenomena and primitive streak formation (CALLEBAUT & VAN NUETEN, 1994; CALLEBAUT et al., 2003a). The localization and function of Rauber’s sickle present a strong similarity with the localization and function of the ascidian Wnt gene, Hr Wnt-5 from Halocynthia roretzi (SASAKURA et al., 1998). Indeed HrWnt-5mRNA is present in the vegetal cortex of unfertilized eggs. After fertilization, HrWnt-5 moves to the equatorial region to form a sickle-shaped structure after which this mRNA is concentrated in the most caudal region of the embryo. That Vg1 (the axis inducer) present in the avian Rauber’s sickle material (inclusive in the sickle horns) and not in the caudal marginal zone has recently been demonstrated (BERTOCCHINI & STERN, 2007). Hensen’s node must be considered as a secondary major organizer linked to Rauber’s sickle via a rostral outgrowth of the latter i.e. sickle endoblast. At the moment of the sickle-shaped bilateral symmetrization (characterized by the appearance of Rauber’s sickle), there oocurs a spatially oblique, sickle-shaped uptake of γ and δ ooplasms by oblique position in utero which become incorporated into the deeper part of the avian blastoderm (Fig. 8). This provokes an unidirectional chaos (radial symmetry breaking) in the ooplasm according to the principle of PRIGOGINE (PRIGOGINE & LEFEVER, 1968) which is indispensable for further development. These ooplasms seem to contain ooplasmic determinants (CALLEBAUT, 2005) which initiate (perhaps by Wnt signalling) either early gastrulation or neurulation phenomena by positional information (CALLEBAUT et al., 2003a). First we found (cytological) evidence for a radial predisposition (preformation) in the premature avian oocyte i.e. radially symmetric and concentric distribution of groups of mitochondria (CALLEBAUT, 1972) (Fig. 9). After the eccentric sickle shaped tilting of the yolk and ooplasmic layers in the fertilized egg by oblique positioning (CALLEBAUT et al., 2000), we observed the presence of predisposed sickle-shaped anlage fields in the upper layer (CALLEBAUT et al., 1996). There is a strong similarity with the gastrulation and neurulation phenomena described by VANDEBROEK (1969) in selachian germs (also developing on very large eggs). Indeed, in the latter vertebrate group there exist also analogous sickle-shaped anlage fields, localized in the upper layer in the same succession order as in birds. These fields are localized in the concavity of Rauber’s sickle in unincubated diblastic avian blastoderms which thus present a sickle-shaped bilateral symmetry (Fig. 10). Finally from this sickle-shaped bilateral symmetric disposition a primitive streak and neural plate will develop by convergent extension movements under influence of signalling molecules secreted by Rauber’s sickle (CALLEBAUT et al., 2003a) (Fig. 11) so a triblastic one-axis-containing embryo is formed. The early neural plate inducing structure which forms a deep part of the blastoderm is the δ ooplasm-containing endophyll (primary hypoblast) (Fig. 8). Together with the primordial germ cells it is derived from the superficial centro- caudal part of the nucleus of Pander which also contains δ ooplasm. It is indeed known that the prelaid nucleus of Pander (as is also the case for the endophyll) can induce the upper layer to form an early neural plate (CALLEBAUT et al., 2004b). The other structure (γ ooplasm) which is incorporated into the caudolateral deep part of the blastoderm forms Rauber’s sickle (Figs. 7; 8). It induces first gastrulation (intramuros) and later blood island and coelom formation (extramuros). Rauber’s sickle develops by ingrowth of blastodermal cells into the γ ooplasm (CALLEBAUT, 1994), which surrounds the nucleus of Pander (Fig. 8). The Rauber’s sickle constitutes the primary major organizer of the avian blastoderm. Rauber’s sickle generates only junctional and sickle endoblast and by positional information, organizes and dominates the whole blastoderm (first gastrulation, neurulation, and later coelom and cardiovascular system formation) (CALLEBAUT et al., 2003b). Fragments of the horns of Rauber’s sickle extend far cranially into the lateral quadrants of the unincubated blastoderm, so that often Rauber’s sickle material forms three quarters of a circle (Fig. 7). This explains the so called regulative capacities of isolated blastoderm parts with the exception of the anti-sickle region and/or the central blastoderm region, where no γ ooplasm and no Rauber’s sickle material is present (which again demonstrates the influence of the γ ooplasm) (CALLEBAUT, 2005). Recently we demonstrated that in the one and the same species (Gallus domesticus) and the same unincubated blastoderm both mosaic development or regulation phenomena can be obtained (CALLEBAUT et al., 2007). Indeed after hemi-sectioning of unincubated chicken blastoderms and culturing both halves formatted in vitro, two kinds of development can be discerned: 1. When the unincubated blastoderms were hemi-sectioned according to the plane of bilateral symmetry, going through the middle region of Rauber’s sickle (Fig. 12), we obtained two hemi-embryos (a left and a right one) containing each a half primitive streak (starting from the most median parts of Rauber’s sickle) giving rise to a half mesoblast mantle and a half area vasculosa, which differentiate incompletely thus indicating mosaic development. This (mediosagittal) hemi-sectioning of the avian blastoderm is comparable with the unilateral destruction experiments of the first two ascidian blastomeres by CHABRY (1887) and CONKLIN (1905). Indeed, the ascidian two-cell embryo already presents a left-right asymmetry visualized by the natural mediosagittal cleavage plane through the caudal sickle-shaped Wnt gene expressing zone (SASAKURA et al., 1998), homologous with Rauber’s sickle. 2. When the unincubated blastoderm is hemi-sectioned more obliquely going through a more lateral part of Rauber’s sickle (sickle horn), two complete bilaterally symmetrical miniature embryos will form indicating so-called regulation phenomena (Fig. 13). We demonstrated that those two types of development are in reality due to the different spreading and concentration of Rauber’s sickle tissue around the area centralis (CALLEBAUT et al, 2007). Embryonic regulation must thus not be considered as a kind of totipotent regeneration capacity of isolated parts of the unincubated avian blastoderm but depends on the spatial distribution of a kind of extraembryonic tissue (Rauber’s sickle) build up by the late oblique uptake of γ ooplasm at the moment of bilateral symmetrization (CALLEBAUT, 1994; CALLEBAUT et al., 2000) forming an ooplasmic mosaic. Thus not only embryonic gene expression phenomena take place during early development but also uptake of extraembryonic preformed ooplasmic determinants play a fundamental role for the initiation of gastrulation, neurulation and cardiovascular development. So finally three ooplasms are respectively found in the three elementary tissues (not germ layers !) of the early, unincubated avian blastoderm (Figs 7; 8): the upper layer from which the embryo proper develops containing mainly β ooplasm (forming the embryonic stem cells), the Rauber’s sickle containing γ ooplasm and the endophyll (primary hypoblast) containing δ ooplasm both forming the extraembryonic tissues. In mammals also the embryonic ectoderm (upper layer) constitute the real stem cells which under influence of the surrounding extraembryonic tissue transform in mesendodermal and neural cell lines (HADJANTONAKIS & PAPAIOANNAOU, 2001). So the general body plan (bilaterally symmetric) is established in the diblastic germ. This confirms, in the case of birds, the existence of a similar master plan for the early development as was proposed for all chordates by EYAL-GILADI (1997). According to the hypothesis of Eyal-Giladi, the speed at which axialization of the embryo proper takes place, depends on the translocation speed of oocytal determinants from the vegetal pole towards the future dorsocaudal side of the embryo. After arrival at their destination, the activated determinants form in all chordates, an induction center homologous to the amphibian “Nieuwkoop center” and the ascidian Wnt expressing sickle shaped region, which later organize the formation of the intraembryonic “Spemann’s organizer”. Thus in birds by using radioactive or trypanblue induced fluorescence oocyte labelling we could demonstrate that a kind of evolutive preformism exists which follows more or less the main evolution of the animal kingdom: from a radial symmetric disposition in the premature oocyte (as exists in coelenterates) via a sickle-shaped diblastic bilateral symmetric germ to a triblastic one-axis-containing embryo (gastrulation induced in aves by Rauber’s sickle). Finally, we observed the formation of the coelomic cavity with associated cardiovascular system (typical for coelomates; DOLLANDER and FENART, 1973) also induced by Rauber’s sickle material (CALLEBAUT et al., 2002; 2004a). An ooplasmic influence on spatial patterning of the mouse blastocyst has been demonstrated by GARDNER (1997), by using the localization of the polar body as a marker for the animal pole. Also PIOTROWSKA and ZERNICKA-GOETZ (2001) showed that the sperm entry position products the plane of initial cleavage of the mouse egg and can define embryonic and abembryonic halves of the future blastocyst. In addition, the cell inheriting the sperm entry position acquires a division advantage and tends to cleave ahead of its sister.

Fig. 10. – Schematic representation of the mean localization of the predisposed (not definitively committed) anlage fields (in good order but with possible partial overlapping of neighbouring parts) in the upper layer of the area centralis of a chicken (Gallus domesticus) unincubated blastoderm (slightly simplified after CALLEBAUT et al., 1996). Note the general eccentric sickle-shaped aspect of the anlage fields in the area centralis after the radially symmetry breaking eccentricity of the subgerminal ooplasms. There is an obvious parallelism between the sickle shape of the anlage fields in the UL and the ovoid central sub-germinal ooplasmic layers (CALLEBAUT et al., 2000). The curved arrows on the anlage fields indicate the logically previsible converging movements of the upper layer during the ensuing gastrulation (WETZEL, 1929) and neurulation (BORTIER and VAKAET, 1992).

Fig. 11. – Combined schematic drawing representing: 1. the hypothetical diffusion of morphogens or signalling molecules indicated by small dots) emanating from Rauber’s sickle (RS) and its sickle horns (SH) into the neighboring tissues of the avian blastoderm, i.e., into the area centralis (AREA CENTR), into the caudal marginal zone (CMZ) and into the caudal germ wall (CGW), where they can influence (induce or inhibit) ectopically placed structures (endophyll, Rauber’s sickle fragments, sickle or junctional endoblast) to form or not to form a second streak; 2. the broad movements (indicated by curved arrows) of cell groups in the upper layer of the area centralis, in the direction of the median primitive streak (partially after WETZEL, 1929). The curved legs of the U-shaped lines indicate moving fronts of cell groups (corresponding to local temporal primitive streak anlagen) that will ingress after fusion into the final median primitive streak.

Fig. 12. – Schematic representation of the hemi-sectioning through the middle of the sickle of Rauber (double lined) of an unincubated blastoderm; both halves of the sectioned blastoderm are represented at some distance (ready for culture); SH: sickle horns of Rauber’s sickle extending far cranially; R: right half blastoderm and L: left half blastoderm will each transform in a right and left half embryo, indicating mosaicism; the thick half arrows represent the formation of hemi-primitive streaks at the cut edge of the blastoderm after incubation.

Fig. 13. – Schematic representation of the oblique hemi-sectioning through the lateral part of the Rauber’s sickle of an unincubated avian blastoderm; the arrows 1 and 2 indicate the place where after culture a bilateral symmetric primitive streak will appear respectively under the influence of the middle part of the Rauber’s sickle (double lined) or under influence of the remaining fragmentary sickle horn (SH), indicating regulation.

E. Goeman for artwork, Mrs E. Van Nueten for excellent technical assistance and Mrs V. De Maere and Mr H. Callebaut for careful typing the manuscript. The author is also very grateful to Prof. Dr. H. Bortier and to Prof. Dr. F. Harrisson for the possibility to work in their laboratory.

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