Comparative analysis of the late embryogenesis of Sida crystallina (OF Müller, 1776) and Diaphanosoma brachyurum (Lievin, 1848)(Crustacea: Brachiopoda: Ctenopoda)

Hydrobiologia 380: 103–125, 1998. © 1998 Kluwer Academic Publishers. Printed in Belgium.

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Comparative analysis of the late embryogenesis of Sida crystallina (O.F. Müller, 1776) and Diaphanosoma brachyurum (Lievin, 1848) (Crustacea: Branchiopoda: Ctenopoda) Alexey A. Kotov & Olga S. Boikova A. N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninsky Prospect 33, 117071 Moscow, Russia Received 24 November 1997; accepted 7 May 1998

Key words: Ctenopoda, late embryogenesis, staging, embryonic instars, embryonic moults

Abstract The embryonic development of two ctenopods Sida crystallina and Diaphanosoma brachyurum has been investigated by observing living embryos removed from the female brood pouches. The sequence of morphological changes was analysed, as was the time at which the activity of certain organs began. The timing of these events at 21–22 ◦ C is documented for both species. During embryogenesis four membranes are cast off. The growth in length of embryos began only after the shedding of the external egg envelope. Growth rates throughout embryogenesis are documented. A new outline of ctenopod embryogenesis is proposed. It includes four stages (= instars) demarcated by the shedding of membranes (= moults), as is commonly accepted for juvenile and mature animals in ctenopods and other crustacean groups. These instars are well distinguished morphologically. Their characteristics are presented. Differences in the duration of embryogenesis in Sida and Diaphanosoma are explained by greater extension of the first, and especially the fourth, instars of Sida, while the duration of the second and third instars are approximately the same in both. A list of instar features is given which may be used to determine the instar (and approximate age) of embryos in the brood pouch of ctenopod females from natural populations. Abbreviations: aI – antenna I; aII – antenna II; ab – basal segment of aII; ae – endopodite of aII; ax – exopodite of aII; bs – basipodite seta; cm – heart; cp – carapace; do – dorsal organ; gn – gnathobase of thoracic limb; gt – gut; ec – eye capsule; en – endopodite of limb; ep – eye pigment; ex – exopodite of limb; fd – fat drop; is – incurved setae before the moult; lb – labrum; md – mandible; mI – membrane I (external egg membrane); mII – membrane II; mIII – membrane III; mIV – membrane IV; mxI – maxilla I; mxII – maxilla II; mz – maxillary zone; ns – ‘natatorial’ setae of postabdomen; oc – ocellus; pb – posterior border of the head; pc – postabdominal claws; pd – postabdomen; pe – presumptive ectoderm; pg – presumptive gut, mesoderm + primordial germ cells; pp – peripheral plasma; saI – segment of aI; saII – segment of aII; smd – segment of mandibles; smxI – segment of the first maxillae; smxII – segment of the second maxillae; st – setae of limbs and maxillae I and II; sw – swimming setae of antenna II; tl – thoracic limb; ts – thoracic segment; tz – thoraco-abdominal zone; yb – yolk blocks; yg – yolk granules Introduction Investigation of the embryonic development of socalled ‘Cladocera’ began in the last century, and the majority of both very old and more recent detailed publications on Anomopoda (Grobben, 1879; Lebe-

dinsky, 1891; Samassa, 1893a–c; Cannon, 1921; Wotzel, 1937; Baldass, 1941; Kaudewitz, 1950) and Ctenopoda (Samassa, 1893c; Sudler, 1899; Agar, 1908; Baldass, 1937) have not lost their value and significance. Unfortunately, most of them dealt mainly with early embryogenesis, which in the Crustacea re-

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104 sults in the formation of a germ band (Ivanova-Kazas, 1979). Embryonic development during this period in small branchiopods (‘Cladocera’) is very similar to that in other crustaceans (Anderson, 1973; IvanovaKazas, 1979). Differences in the cleavage, gastrulation and so on between groups of crustaceans are chiefly determined not by systematic position, but the amount of yolk in the egg. Late embryogenesis – morphogenesis – has been inadequately studied. Taking into consideration the possible polyphyly of the so-called ‘Cladocera’ (Fryer, 1987), it may be noted, that differences between the main orders of small branchiopods are quite obvious. Direct development (loss of naupliar stages) connected with a reduction in size, reduction of number of thoracic segments, and other changes could have occured independently in Ctenopoda, Anomopoda, Haplopoda, and Onychopoda. Thus Leptodora (unlike all other small branchiopods) has two types of development: with metamorphosis, when a nauplius hatches from the resting egg, and direct, when a juvenile with a complete number of segments and limbs hatches from the parthenogenetic egg. This is may indicate that direct development is a recent acquisition of the Haplopoda. According to Starobogatov’s (1986) extreme views, the Haplopoda and Onychopoda do not belong to the Branchiopoda at all. Most information on late embryogenesis in small branchiopods relates to the Anomopoda. These studies usually sought to determine stages of development (Obreshkove & Fraser, 1940; Fox, 1948; Hoshi, 1950, 1951; Green, 1956; Esslová, 1959; Murugan, 1975; Murugan & Sivaramakrishnan, 1973; Murugan & Venkataraman, 1977), sometimes the development of organs and their systems: ocellus, eye, nervous system (see review by Ivanova-Kazas, 1979), or the modification of embryonic external morphology. As yet no-one has succeeded in making a more detailed description of morphogenesis than did Grobben (1879) for Moina rectirostris more than a century ago. A analysis of the morphogenesis of Anomopoda elucidated some evolutionary problems. For example, Glagolev (1986) studied the late embryos of several species of Daphnia and found certain setae on the endopodites of their limbs, absent in adults. Thus the possibility of using embryological data for the reconstruction of the limb evolution was demonstrated, and the oligomerization of Daphnia limbs was proved. Kotov (1996) examined the morphogenesis of chydorids, bosminids and daphniids and found the second maxillae, which are absent in the adult.

Few studies have been made on the late embryogenesis of ctenopods. Agar (1908) watched the formation of the second antennae in Holopedium gibberum and found, that the uniramous second antenna of the female appears as a typical biramous appendage in the embryo. Dejdar (1930) showed that all branchiopod larvae, like all ‘cladoceran’ embryos, have a dorsal organ, retained in the adults of some, disappearing in others. Löpmann (1937) gave a detailed description of eye development in Daphnia and Diaphanosoma, and the significant differences ascertained by him can be interpreted as evidence of the independent evolution of these two animals. An old article of Sudler (1899) on the development of Penilia still remains the most detailed description of ctenopod morphogenesis. Later, attempts were made to determine stages (Della Croce & Bettanin, 1965) and to compare the embryo’s development in the parthenogenetic and resting eggs (Onbe, 1974, 1978) of Penilia. However, Penilia is unique among ctenopods in having a closed brood pouch and a maternal placenta for the nutrition of the embryos. Of other Ctenopoda there are relatively good descriptions of early embryogenesis in Holopedium (Agar, 1908; Baldass, 1937) and a detailed study of Diaphanosoma development (Samassa, 1893c), also focused on early embryogenesis. Only two illustrations on late stages are known for Sida (Grobben, 1879). Some of the old papers are notable for the detailed description of particular stages, but the reconstruction of changes of morphology and anatomy was open to discussion. Thus subsequent authors often criticized their precursors (e.g. Grobben, 1879; Samassa, 1893c). Surely, it was impossible to determine the time of embryonic moults or the beginning of locomotory activity or heartbeats from a set of mounts. Our aim was to make a detailed study of late embryogenesis in two common freshwater ctenopods: Diaphanosoma brachyurum Lievin, 1848, and Sida crystallina O. F. Müller, 1776, which differ in habits, size and fecundity, by means of in vitro observations. This method was first proposed by Ramult (1926) and used repeatedly for Anomopoda (Obreshkove & Fraser, 1940; Fox, 1948; Murugan, 1975), but not once for Ctenopoda. We gave particular attention to embryonic moults and growth.

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105 Material and methods This investigation was conducted during summerautumn of 1994–1996 at the ‘Lake Glubokoe’ Hydrobiological station (Moscow Region, Russia). Adult females of both species collected from the lake were cultured individually in 5 cm diameter dishes, and watched every 15 min to detect the entering of eggs into the brood pouch. This event was regarded as the initial moment of embryonic development. The end of the embryogenesis was determined by the moult which took place within a few minutes of the release of the embryo from the brood pouch. This was accompanied by considerable morphological changes which also made it possible to detect this event, if the embryo developed in vitro. Some eggs were removed from brood pouches and their development followed in vitro, while others developed in the brood pouches under similar external conditions. At temperatures fluctuating between 21 and 22 ◦ C the development time of Sida ranged from 62–69 h (mean 66 h); of Diaphanosoma from 49–55 h (mean 52 h). The removing of eggs and their further cultivation was conducted according to Obreshkove & Fraser (1940) and differed only in use of filtered lake water in our experiments instead of a sterile physiological solution. The method of observation of Sida and Diaphanosoma was different in certain details. Eggs of the former have a firm envelope that is not damaged during their removal. Moreover, the substantial fecundity of Sida allowed us to observe the development of eggs from whole broods of certain females. A dish containing these embryos was placed directly under the microscope (care being taken not to overheat the water) and a water-immersion objective (magnification × 600) made it possible to distinguish the separate ectodermal cells, and even their nuclei. The observations were usually carried out either every one or two hours during most of the development of an embryo, or continuously during the periods of shedding of membranes, segmentation, and other significant modifications. In all, more than 300 embryos from 60 broods have been used for this study. An inverted microscope was used to observe 70 large adult females of Sida, which mostly attached themselves immovably to the dish bottom (dorsal side down) throughout the period of observation. Unlike Sida, females of Diaphanosoma in Glubokoe Lake during the periods of our investigations carried only one or two eggs. These had a very

thin, easily damaged outer membrane, which prevented us from obtaining a whole brood of embryos from one female. Survival of embryos was low, and no one embryo completed its development in vitro in our experiments. At a certain hour of development, an embryo was removed from the brood pouch and observed in vitro usually for not more than 6–10 h. Then the data for different embryos were summarized, and the sequence of events was reconstructed, using the logic of method of Polishchuk & Ghilarov (1981) for the estimation of growth of mature ‘cladocerans’. The differences in cultivation of eggs of Sida and Diaphanosoma are connected with a different approach to the study of embryonic growth of these species. In Sida, the length of embryos was measured at certain time intervals during their whole development, while for Diaphanosoma 20–50 randomly separated embryos at certain stages of development were taken for measurements. Seven stages, characterized by rather distinct morphological features, have been distinguished. All drawings were made by means of RA-4 drawing apparatus. In cases when an embryo of the previous stage of Diaphanosoma in our illustration was larger, than those of subsequent one, that means that two different specimens have been used for drawing.

Results Embryogenesis of Diaphanosoma brachyurum During the first 10 min after the release of the egg mass into the brood pouch, the eggs separate and assume their definitive form. A few oil droplets joint into a single large drop. Within 0.5 h after laying, the egg (Figure 1) is ellipsoidal in shape and almost completely filled by small greenish yolk granules (yg) except for a narrow zone of transparent peripheral plasma (pp) that is somewhat widened at the two poles. The large fat drop (fd) occupies the central region. An attempt to take the egg out of the brood pouch soon after its laying often results in exfoliation of the outer envelope (mI). This is the first (egg) membrane described in different Anomopoda and Ctenopoda and is shed during the further development of the embryo. An inner, very thin and fragile, membrane, no more than an ovum plasmolemma, is present beneath the outer envelope. Approximately an hour after laying, cleavage begins (Figure 2). This is poorly seen in the living

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Figures 1–15. Egg end embryo of Diaphanosoma brachyurum: Figure 1. Egg at 0.5 h of development. Figure 2. Beginning of cleavage at 1.5 h. Figures 3–4. Embryo at 9 h, dorsal and lateral. Figures 5–6. At 11 h, ventral and laterodorsal. Figures 7–9. At 11.5 h, ventral, lateral and dorsal. Figures 10–11. At 12 h, ventral and lateral. Figures 12–13. At 12.5 h. Figures 14–15. at 13 h.

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Figures 16–26. Embryo of Diaphanosoma brachyurum: Figure 16. at 12.5 h, dorsal. Figure 17–19. at 13 h, ventral, lateral, dorsal. Figures 20–21. At 13.5 h, and premature moult, leading to death of embryo. Figure 22. At 14 h, ventral. Figures 23–24. At 14.5 h, ventral and lateral. Figures 25–26. At 15 h, ventral and dorsal.

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Figures 27–40. Embryo of Diaphanosoma brachyurum: Figure 27. At 16 h, ventral; Figures 28–29. At 17 h, ventral, two phases of moult. Figures 30–32. At 18 h, lateral, ventral and dorsal. Figures 33–34. Two phases of carapace expanding during 18–19 h of development. Figures 35–36. Embryo at 19 h, ventral and dorsal. Figure 37. At 20 h. Figures 38–39. At 21 h. Figure 40. At 23 h.

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109 embryo due to its opacity. Early embryogenesis of Diaphanosoma was described in detail by Samassa (1893c). By the end of cleavage, the yolk is driven to the centre of the egg, and the peripheral zone widens considerably. Soon gastrulation takes place by means of immigration of blastodermal cells. About nine–ten hours after egg laying (Figures 3, 4) shortly before the beginning of morphogenesis, a large double rudiment of the apical plate appears at the anterior end of the embryo dorsally. The embryo is now covered by a layer of uniform ectodermal cells (pe) with well defined nuclei. The yolk granules have fused into large blocks (yb) of irregular shape. The yolk mass on the ventral side is pushed to the centre by cells of the gut, mesoderm and gonad rudiment (pg). This stage was drawn incorrectly by Samassa (1893c), because the yolk droplets on his drawing are not yet fused into one mass. The first signs of segmentation are small furrows on the lateral sides of the embryo at about 10.5 h – rudiments of future second antennae (aII). Later, these elongate and spread mostly to the ventral and less to the dorsal side. Somewhat later, at about 11 h (Figures 5, 6), the transversal lateral furrows appear at the sites of the future first antennae (aI). By this time the rudiments of the second antennae have expanded, and exfoliation of the egg membrane from the embryo surface becomes evident in these regions. By 11.30 h transverse swelling, the mandibular segment (smd), appears on the ventral face of the embryo (Figures 7–9) followed by mandibular rudiment (Figure 10, md). A pair of inconspicuous posterior furrows probably correspond to the edge of the mandibular segment (Figure 11). Already at this early stage of development, the rudiments of the second antennae are becoming biramous, their basal parts (ab) are separated from each branch (ae and ax) by a small incision (Figure 10, 11). By 12.30 h the lobe-like rudiments of the mandibles are clear between the second antennae, and the rudiment of the labrum (lb), a projection of the segment of the first annennae (saI), is recognisable (Figures 12, 13). By 12.45 h (Figures 14–16) the posterior border of head appears (pb) as a result of separation of the maxillary zone (mz) and thoracoabdominal zone (tz), but segmentation of the latter is not yet visible. By 13–13.30 h segmentation of the thorax begins, and the furrows, marking the posterior margins of the first (tsI) and second (tsII) thoracic segments, appear almost simultaneously (Figures 17–19). These encir-

cle the body of the embryo, but are more evident latero-ventrally. By 13.30 h (Figure 20) the furrow of the third thoracic segment (tsIII) appears, and a dorsoventral furrow appears at the posterior end of the embryo (pd). It is not clear whether this split marks an anus only, or a double rudiment of the postabdomen. At this time (or later) in the case of a damaged embryo the outer egg membrane may be shed prematurely (Figure 21), but this abnormal moult leads to death of the embryo. defFiguresFigures By 14 h (Figure 22) the mandible rudiments are displaced forward, and the labrum expands backward considerably reducing the ventral area of the segment of the second antennae (saII). The maxillary zone also expands forward, and invades the region between the mandibles. The furrow of the forth thoracic segments (tsIV) appears by 14.30 h (Figures 23, 24), and of the fifth segments (tsV) – by 15 h (Figures 25, 26). Now the furrows of the first (smxI) and second (smxII) maxillar segments are visible on the ventral face. Only an inconspicuous incision is present between the mandibular and maxillary segments on the dorsal side of the embryo. Soon (15–16 h) the embryo casts off the outer egg membrane (Figures 27–29). Details of this process are better seen in Sida and are described under that species. By 16 h progressive expanding of the posterior margins of the thoracic segments and as a result, the formation of limb rudiments (tII–VI) are observed. Commencement of the process coincides with the casting off of the membrane. By 17 h the gnathobases (gn), endo (en)- and exopodites (ex) can just be discerned on the limbs (Figure 30), the sixth thoracic segment with feebly developed limb rudiments can be seen. Differentiation of buds of the first and second maxillae takes place. Thus the gnathobasic line includes those of the maxillae I and II (mxI and mxII). Setal rudiments (sw) are differentiated on the second antennae. A small fold, the rudiment of the carapace (cp), appears on the posterior edge of the segment of maxillae II dorsally, confirming that, as in carapacebearing brachiopods in general, the carapace is of maxillary origin (Fryer, 1996b). By 17.30 h the posterior margin of the sixth thoracic segment is marked by a clear-cut furrow (Figures 31, 32). The elongated labrum covers the segment of the second antennae, reaching the mandibular segment. The rudiment of carapace expands dorsally and posteriorly. By 18–19 h (Figures 33–36) the cephalic region is already well separated, the first antennae shift to the ventral face, the labrum enlarges and begins to expand

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Figures 41–48. Embryo of Diaphanosoma brachyurum: Figures 41–43. At 24 h, 25 h and 25.5 h, ventral. Figures 44–45. At 28 h, ventral and dorsal. Figures 46–48. At 30 h, 32 h, 34 h.

posteriorly prior to covering the mandibles, and the segment of maxillae I encroaches on the surface of mandibular segment ventrally. Rudiments of maxillae I and II are now of more or less equal size and well developed. The carapace fold is enlarged, but it does not reach even the second pair of thoracic limbs. A patch of larger ectodermal cells – a rudiment of the

dorsal organ (do) – is observed on the posterior region of the head dorsally. By 20 h the gnathobases of all limbs are demarcated by constrictions, and form a row of hexagons (Figure 37). The second antennae reach the second pair of thoracic limbs. By 21–22 h an incision appears on the second maxillae (similar to that on the maxillae II of anomopods (Kotov, 1996)), the cara-

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Figures 49–56. Embryo of Diaphanosoma brachyurum: Figure 49. At 35 h, dorsal. Figures 50–51. At 36 h (during the moult) and 37 h (after the moult), ventral. Figures 52–55. At 39 h, 40 h and 41 h, dorsal, at 41 h, ventral. Figure 56. At 52 h.

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Figures 57–59. Diaphanosoma brachyurum: Figure 57. Released embryo (neonata) before final moult. Figure 58. First juvenile instar after this moult. Figure 59. Adult female with one embryo shortly before release.

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113 pace extends to the second pair of thoracic limbs, the dorsal organ is inflated, and rudiments of claws (pc) and postabdomonal setae (ns) became visible on the postabdomen (Figures 38, 39). By 23 h rudimentary setae begin to grow on the inner and posterior margins of the thoracic limbs and maxillae I and II (Figure 40, st). The second maxillae decrease in size (as compared to the first pair) and are displaced laterally. The setae of the second antennae and postabdominal claws have enlarged. By 24 h the covering membrane is significantly exfoliated on the distal parts of first and second antennae and the postabdomen (Figure 41). By 25–26 h (Figure 42) slight movements of the second antennae occur. These soon become more synchronized with each other and stronger, but the membrane inhibits them. Probably these movements lead to the casting off of membrane II, which happens approximately at 27–28 h (Figure 43). The moulted embryo (Figures 44, 45) has free second antennae, which extend to the third pair of thoracic limbs, and whose movements become more various. At the same time the thoracic limbs as well as the carapace (which extends to the third pair of thoracic limbs) continue to be covered by a slip-cover (the next membrane, mIII). After this moult, the setae of the trunk limbs and second antennae elongate. The labrum partly covers the first maxillae and now extends to the second maxillae. The distal mandibular surface (future molar surface) faces its partner on the opposite side. By this time the double rudiments of the retina are united as a single block, eye capsule (ec). By 29–30 h eye pigment (ep) appears as two small red-brown dots (Figure 46, ep). By 32 h the embryo has small but distinct eyes. The second antennae (Figure 47) beat actively: they move as a reflex response when the embryo is touched by a needle). The postabdomen has expanded and flexed ventrally, recalling that of the embryo of Moina (Grobben, 1879). The second antennae almost reach the fourth pair of thoracic limbs. By about 33 h the first heartbeats, movements of the postabdomen and gut (gt) peristalsis begin. The third casting off of the old cuticle takes place at this time (about 35–36 h). The thoracic limbs and postabdomen are liberated from the membrane, and the swimming setae of the antennae II, and postabdominal setae are straight and elongate (Figures 49, 50). By 37 h the eyes are enlarged considerably but their pigment occupies less than half of the eye capsule. The labrum and first antennae have assumed the adult form (Figures 51, 52). The second antennae ex-

tend to the fourth-fifth pairs of thoracic limbs, and the carapace covers the third, and partly the fourth pairs. The dorsal organ is of semicircular shape. The heart (cm) beats rhythmically, and more frequently. Movements of the postabdomen, gut peristalsis and the first movements of mandibles and labrum are readily apparent. An embryo outside the brood pouch assumes the characteristic adult posture: its second antennae rise upward in intervals between strokes (Figure 53). By 40–42 h the eyes are larger, the eye capsule round and movable. The second antennae extends to the sixth, and the carapace, to the fifth, pair of thoracic limbs (Figures 54, 55). By 44 h the eyes draw together; the pigment occupies more than half the eye capsule. Slight twitchings of the thoracic limbs and maxillae I and II occur. The dorsal branch of the second antennae (exopod) bears 3 and 7 setae on its proximal and distal segments respectively, and the postabdominal claws have rudimentary basal spines. The volume of yolk is much reduced. By 48 h (Figure 56) the eyes are large and the second antennae reach the sixth pair of thoracic limbs. The carapace valves come together ventrally but still do not cover the two posterior pairs of limbs completely. The first antennae bear rudimentary aestetascs. The second antennae and postabdomen still have incomplete armature: the incurved setae (is) of the next stage – which presage the moult – are visible in both. At about 50–52 h the embryo leaves the brood pouch. The neonate (Figure 57) covered by the last embryonic membrane (mIV) makes strokes with its antennae II, then stops and moults during some minutes. This moult marks the end of the embryonic period. The moulted first juvenile instar (Figure 58) clearly differs from a neonate: it is larger and more graceful, the exopod of antenna II bears 3 and 8 long, setulated setae on its proximal and distal segments, the carapace covers all the limbs, and the postabdominal claws are elongated. Vannini (1933) and Herzig (1984) reported that the gut of Diaphanosoma neonates is filled with yolk, but our study does not confirm this. However, this instar still retains some ‘embryonic’ traits: unfused eyes, dorsal organ as a spot with a clearly visible margin, presence of yolk granules, and an incomplete number (3 v. 4) of setae on the proximal segment of the antennal exopod. The last point was noted by Vannini (1933).

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114 Table 1. The sequence of some events in late embryogenesis of Sida crystallina and Diaphanosoma brachyurum. Event

Age of embryo in hours Sida Diaphanosoma

First instar Appearance of rudements of second antennae (beginning of head segmentation) Of first antennae Of mandibles Of labrum Separation of maxillary zone (marking of head border) Appearance of first thoracic segment (beginning of thorax segmentation Second thoracic segment Third thoracic segment Fourth thoracic segment Fifth thoracic segment Shedding off outer egg membrane (first moult) Second instar Appearance of fissures of both maxillae Sixth thoracic segment First movements of second antennae Shedding of second membrane (second moult) Third instar Appearance of ocellus pigment Fising of ocellus pigment Appearance of pigment in eyes First heartbeats First movements of postabdomen Gut peristalsis Casting of third membrane (third moult) Fourth instar First movements of mandibles Of labrum Of thoracic limbs Release of embryo from brood pouch Final (fourth) moult

Embryogenesis of Sida crystallina The embryonic development of Sida is similar to that of Diaphanosoma, so is described more briefly, except when structures absent or hardly distinguished in Diaphanosoma requir mention. The sequence of events in the development of both species is summarized in Table 1. By an hour after laying, the outer membrane is thickened and the egg has an elliptical shape. During

13–13.30 14 14.30 15

10 11 12 12.30

16

12.45

16.15 16.30 17 17.30 18

13 13.15 13.45 14.30 15

18

15

18.15 19 29–30 30–31

15.15 16 25–26 27–29

25 28 28 32–33 35 28 36

– – 29–30 33 33 33 33.30

(35) (35) 49 65–66 65–66

36 36 44 52 52

the first 1–3 h egg cleavage and blastoderm formation take place. By 3–4 h the yolk is concentrated in the centre of the egg. Gastrulation occurs by 8 h, and the differentiation of paired rudiments of the apical plate by 10 h. By 13–13.30 h the furrows of the second, and (somewhat later) those of the first, antennae appear. The mandibular segment becomes apparent by 14–14.30 h. Immigration of ectodermal material takes place at the posterior end, where the yolk is considerably reduced. By 15 h rudiments of the labrum and

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Figures 60–66. First moult and first membrane (egg envelope) of Sida embryo. Figure 60. Tubes of shed membranes in brood pouch of female. Figures 61–62. Phases of moult. Figures 63–64. Phases of rolling of membrane tubes. Figures 65–66. Tubes of shed egg membrane.

mandibles are distinguishable. The second antennae become biramous; incisions between their branches and basal parts appear. The posterior dorsoventral furrow appears at 15.30 h. The maxillary zone is separated by 16 h but still unsegmented and the posterior margin of the cephalic region can be distinguished. At about 16.30 h the lateral furrows of the first and second thoracic segments appear and, somewhat later, gradually expand ventrally. By 17 h furrows of the third, and by 17.30 h the fourth, thoracic segment can be seen. The second antennae grow and move the outer membrane aside from the embryo body. The border between the segments of the maxillae I and II also be-

comes distinguishable. At about 18 h the embryo casts the outer egg envelope (Figures 60–66), and furrows of the maxillae I and II and fifth thoracic segment appear. By 19 h the sixth thoracic segment is evident, and the posterior margins of the first five segments inflate. These are the rudiments of thoracic limbs with just discernible gnathobases. The second antennae have distal setae, and the embryo dorsum possesses a group of large cells, the rudiment of a dorsal organ, having a clearly visible border at 20 h. From the posterior part of the cephalic region arises the rudiment of the carapace.

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Figures 67–74. Second and third embryonic moults in Sida: Figure 67. Embryo at 30 h shortly before second moult. Figure 68. Second moult at 31 h. Figures 69–70. Distal part of the second antennae, and rudiment of postabdomen at 33 h. Figure 71. Embryo at 34 h. Figures 72–74. Phases of third moult.

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117 At 22 h the thoracic limbs have well marked gnathobases, endo- and exopodites. The first antennae have shifted to the ventral side, the rudiment of labrum extends over the mandibles, and the retinal rudiment has become double. By 23–24 h two rows of hexagons formed by the trunk limb ganothobases have appeared. The second maxillae have moved peripherally. The second antennae and carapace have elongated. At 25 h the ocellus pigment (oc) appears as two red dots. As is well known, the ocellus of Sida is reduced, being represented only by two lateral cups (Elofsson, 1966). We think that the two dots are rudiments of two cups. Endopodites and gnathobases of the thoracic limbs bear the small tubercules of future setae, and the postabdomen has very small rudiments of claws and postabdominal setae. The second antennae continue to enlarge, and the dorsal organ has inflated in the form of a hemisphere. At about 28 h two spots of eye pigmentation appear, and gut peristalsis begins. The second membrane has exfoliated at the head. By 29–30 h the first movements of the second antennae may be visible, and the second membrane is shed from the head, distal part of the second antennae, and postabdomen (Figure 67). The ocellus pigment fuses into a single spot. By 30– 31 h the second membrane is cast off (Figure 68), The second antennae become free while the thoracic limbs and postabdomen continue to be enclosed by the next (third) membrane. This next membrane already is being shed from the distal end of the second antennae, and the claws of the postabdomen (Figures 69, 70). Rudiments of the anchoring organs are represented as spots of small specific cells. The eye pigmentation of the Sida embryo resembles that of Diaphanosoma, but the second membrane of the latter is cast twothree hours later. Furthermore, Sida embryos lack the stage without eye pigment and with free movement of second antennae. By 32–33 h the first heartbeats are observed. The rudimentary carapace has enlarged, having the maxillary gland developed in contact with posterior anchoring organs. By about 35 h the first movements of postabdomen, mandibles and labrum take place, and the eyes are larger (Figure 71). About 36 h, after the next moult (Figures 72–74), the thoracic limbs, postabdomen, second antennae and their setae become free, the postabdominal claws and setae and the swimming setae of the second antennae elongate. Limb movements begin only at 49– 50 h. By 65–66 h the embryo is released from the brood pouch. It is more developed than the late embryo of

Diaphanosoma: the eyes are partly fused, and carapace covers all thoracic limbs (Figure 75). The next moult, that follows quickly, marks the end of embryogenesis. After that (Figure 76), the size enlarges significantly. There are 2 and 7 long setae with developed setules on the second and third segments of the upper branch of antenna II, the basal setae of antenna II are directed not laterally but posteriorly, postabdominal setae and its armature enlarge, and the yolk mass is divided in two parts. The anterior and pair of posterior anchoring organs are well developed looking like triangular outgrowths in lateral view. As in Diaphanosoma, the first juvenile instar of Sida retains some embryonic traits (Figure 77): an incomplete number of setae of second antennae (2 and 7 instead of 3 and 7 setae on the segments of the upper branch of antennae II), many yolk droplets, unfused eyes, dorsal organ behind the large anterior anchoring organ (this dorsal organ is present in the second juvenile instar as well). The embryonic membranes and their shedding During embryonic development, Sida crystallina and Diaphanosoma brachyurum successively shed four membranes (Table 1). The moults of Sida are more conspicuous due to its larger size and thicker membranes, and the following refers to this species. The laying of parthenogenetic eggs in Sida was described in detail by Weismann (1876–1879). Their formation was studied by Rossi (1980). The egg mass enters the brood pouch as ‘porridge’ (Müller, 1867) or like a ‘toothpaste from a tube’ (Green, 1956). Egg contours are not visible: their formation follows some minutes later. Probably the ‘cuticular hardening of the outer protoplasmic layer’, stimulated by separate oocytes, causes disintegration of the egg mass into separate eggs (Weismann, 1876–1879). Undoubtedly the formation of the outer membrane begins in the brood pouch. An egg is initially sausage-shaped, then it looks like a biscuit (Weismann, 1876–1879), and only after about half an hour does it reach its final elliptical shape. At first the outer membrane is very thin and easily broken but in an hour it becomes hard and elastic. Sometimes the membrane exfoliates at the poles revealing a new, extremely delicate, second membrane (plasmalemma). The outer membrane forms a hard ‘shell’, making growth or change in form practically impossible. The embryo surface is covered by a thin second membrane fitted closely within the outer membrane, but during

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Figures 75–77. Sida crystallina: Figure 75. Released embryo (neonata) before final moult. Figure 76. The first juvenile instar after this moult. Figure 77. Adult female with embryos shortly before release.

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119 segmentation it somewhat exfoliates in the vicinity of furrows. At approximately 16 h the enlarged second antennae exfoliate the first egg membrane significantly. The outer membrane is shed after the appearance of the fourth thoracic segment and before or during that of the fifth. It splits along the dorsal and ventral midlines (Figure 61), rarely along the lateral midline. The duration of the moult varies greatly. Some embryos quickly escape from the membrane, each piece of which rolls up instantly (Figures 63–66). In some embryos, however moulting takes up to two hours, sometimes with serious consequences. One (Figure 62) or sometimes both, halves of the membrane may jam and deform the embryo’s body. The embryo may remain in this state for a long time, and the final shedding of the membrane may takes place only after further increase in size (probably due to osmotic water absorbtion), which restores body shape. Rarely embryos of Sida failed to shed the membrane and died. Cast membranes form characteristic rolls. Whose presence or absence in the dish makes it possible to know whether a moult in vitro was missed. They were found easily in the brood pouches of females (Figure 60). Earlier Agar (1908) mentioned ‘conspicuous objects’ in brood pouch of Holopedium. The second, very thin, membrane presents already in the first hour of egg development but its hardening only takes place shortly before shedding of the outer membrane. At first it fits close to the embryo. After shedding of the egg membrane it exfoliates from the antennules, antennae and postabdominal rudiment. By 25 h the growing postabdominal claws and setae move it aside (Figure 67). Until that time, in non-sterile water bacteria populate the membrane surface, usually appearing shortly before its shedding. Shedding of the second membrane is promoted by movements of the second antennae which begin two hours before the moult and gradually intensify (Figure 68). At first the second membrane splits in the cephalic region, then the crack extends backward laterally. Although the membrane is very thin, its splitting can easily be followed because of the bacteria that are present. It is sloughed gradually from the cephalic region, aided by the second antennae, but on the thorax it may be retained for a long time (Sometimes until the next moult, but do not confuse with the third membrane!). Yet before the shedding of the second membrane, the next (third) membrane, already exfoliated on the apices of the second antennae, postabdominal

Table 2. The relative length increase (%) of Sida and Diaphanosoma embryos at different stages of development (in parenthesis the duration of stages in hours). Developmental stages

Sida crystallina

Diaphanosoma brachyurum

I + II (first instar) III (second instar) IV + V (third instar) VI + VII (fourth instar)

0 (18) 26.9 (12–13) 35.6 (6–7) 37.4 (28)

0 (15) 24.0 (12–13) 33.0 (6–7) 43.0 (18)

claws, and sometimes on whole postabdomen, may be seen beneath it. After the shedding of second membrane, the second antennae are movable and free (the third membrane mimics the form of this appendage), while the thoracic limbs and postabdomen are still enclosed within a cover formed by the third membrane. The gradual elongating and ventral flexing of the postabdomen causes considerable exfoliation (Figure 73). Finally postabdominal and antennal movements promote the shedding of the third membrane (Figure 72). During this moult antennal setae, postabdominal setae and claws evert and elongate. The last embryonic moult involves the shedding of the fourth membrane and takes place soon after the embryo is released from the brood pouch. It abruptly stretches its second antennae along the body and brings them together on the ventral side, widely opens its carapace valves, twitches with the postabdomen, and slightly moves its limbs. The old cuticle initially exfoliates on the first antennae, later on second antennae and splits along the cephalic region, then posteriorly. Abrupt movements of the second antennae promote the shedding of membrane from the cephalic region, turning it inside out, while the postabdomen promotes the same process in the thorax where the old cuticle is sloughed as an indivisible piece without being turned inside out. Apparently this moult is very similar to those in the postembryonic period.

Growth of the embryo In Sida, the length of embryos from one brood was measured at regular time intervals (Figure 78). Egg size varies considerably during the first hour after laying and then becomes stable. Undoubtedly the thick, firm, but elastic outer egg membrane prevents its growth. After its shedding the embryo begins to grow

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Figures 78–79. Figure 78: Change in average length of embryos of a single brood of Sida crystallina throughout their development in vitro. Glubokoe Lake, 18–20.08.96. Figure 79: Size-frequency distribution of seven stages of development of Diaphanosoma embryos from Glubokoe Lake 10–12.08.96. See explanation in the text. The number of measured animals and average length (in µm) are indicated.

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121 slowly but two or three hours before the shedding of the second membrane its growth increases considerably. The second and third membranes are cast successively at this time. They are very thin and elastic, and do not prevent growth by elongation of the cephalic region and postabdomen. After shedding of the third membrane growth slows again but continues for 28 h up to release from the brood pouch. A sharp increase in size takes place only after the shedding of the fourth embryonic membrane, after release. During the period of the development in the brood pouch, the length of the embryo increases approximately 2.1 times. Growth of Diaphanosoma embryos is shown in Figure 79). The following stages can be recognised: (I) Unsegmented egg. (II) Embryo in egg envelope, with no more than four thoracic segments. (III) Embryo with five or six thoracic segments and surrounded by a shell-like membrane. (IV) Embryo with free movable second antennae; thoracic limbs covered by membrane; eyes without pigmentation. (V) Similar embryo having small red-brown eyes. (VI) Embryo with free thoracic limbs, comparatively small eyes (pigment occuping less than half of eye capsule); second antennae not extending beyond fifth pair of thoracic limbs. (VII) Embryo with large movable eyes and long second antennae. At stage I and II the embryos are covered by egg membranes, at stage III these have already been shed and the embryo is covered by the second membrane, at stages IV and V by the third and at stages VI and VII by the fourth membrane. During the period of the development in the brood pouch, the length of the embryo increases approximately 1.7 times. Table 2 compares the growth of embryos of Sida and Diaphanosoma. In both animals more than 30% of size increase falls on the short period (about six hours) in the middle of development. Discussion Embryonic membranes On the status of the membranes The shedding of embryonic envelopes in Anomopoda and Ctenopoda was observed long ago (Grobben, 1879; Lebedinsky, 1891; Agar, 1908) but hitherto we

did not know their number or timing. Agar (1908) suggested that there are no differences in origin of outer and inner egg envelopes (he observed only two membranes) in either Ctenopoda or in arthropods in general. He called both membranes ‘cuticles’ and the shedding of both membranes (and the external envelope of egg too!) ‘moults’. For anomopods this question has been frequently mentioned. As more than two embryonic moults have never been recorded before, only their nature was discussed. Two opinions were pointed out: Lebedinsky (1891) and later Obreshkove & Fraser (1940) and Shuba & Costa (1972) considered the outer membrane as a chorion and inner one as a vitelline envelope of the egg, while others (Seidman & Larsen, 1979; Zaffagnini, 1987), based on histological studies, considered only the former as a vitelline envelope. To us, the second position appears best substantiated. For example, our observations show that the outer egg membrane of Sida and Diaphanosoma differ sharply from all other embryonic membranes: it is relatively thick and elastic, does not follow the change of the embryo’s shape and shedding is due to the protoplasmic pressure from within and its own elasticity. In addition it is stained well by basic stains. The second membrane, by contrast, is more similar to the third and fourth, and there is no reason to attribute special status to it. During the first hour after egg laying, the first membrane is thickened and becomes strong. This is not connected with secretions of the brood pouch, but with vital activity of the egg itself. Histological studies of the formation of parthenogenetic eggs (Rossi, 1980; Zaffagnini, 1987) show almost complete degeneration of feeder cells at the time of egg release into the brood pouch. If they take part in the synthesis of the outer membrane, their participation is minimal. No signs of extraembryonic ectoderm have been found. Just a plasmalemma, not a special membrane is, present beneath the outer egg membrane in the first hours after its laying. According to Seidman and Larsen (1979) the term ‘chorion’ cannot be applied to the outer membrane of resting eggs either. The second membrane of Ctenopoda and Anomopoda is sometimes called the naupliar membrane (Hoshi, 1950, 1951) or larval cuticule (Esslová, 1959) which is also incorrect. No crustaceans hatch at an earlier stage than a nauplius. In all studied species of large Branchipoda the shedding of two membranes secreted by the embryo has been observed (Fryer, 1996a). Apart from the egg envelope, secreted by the

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122 mother, the mature egg has a vitelline membrane and a hatching membrane from which the nauplius (covered by its own cuticle) is hatched. For this reason the identity of the second membrane of parthenogenetic egg of anomopods and ctenopods with the naupliar membrane of large branchiopods seems problematic. Possibly just the third membrane of the ‘cladoceran’ embryo is a homologue of the naupliar membrane of large branchiopods while the second membrane, forming the spherical cocoon around body of the embryo, is a homologue of the hatching membrane. However this question requires further investigation: at present it seems best to eschew specific terminology. Signs of the embryonization of development may be seen in the appearance membranes and in embryonic moults. The second membrane covers the whole embryo as a ‘cocoon’ and exfoliates at the time of segmentation, following which the embryo tries to move with the antennae II, which stimulates shedding of the membrane. The third membrane covers as a ‘shell’ only the thoracic limbs and postabdomen, while on the head it mimics the form of appendages. As a result the antennae II are free and movable. Such an embryo resembles a larva with movable cephalic appendages, but no thoracic limbs – a nauplius. The fourth membrane covers an embryo much resembling a free-living juvenile, and differs from the cuticle of the latter only by stronger elasticity and less firm nature through it is shed normally. Differences in outer membrane shed between parthenogenetic and gamogenetic eggs All authors describing the hatching from resting eggs of Anomopoda (Murakami, 1961; Shan, 1969; Fryer, 1972, 1996a; Onbe, 1974; Makrushin, 1989), Ctenopoda (Smyly, 1977; Onbe, 1978; Makrusin, 1989; Korovchinsky & Boikova, 1996) and Onychopoda (Onbe, 1974; Mordukhai-Boltovskoi & River, 1987) agree that the outer egg membrane splits in the transverse plane either in the middle; or somewhat closer to one of its poles. The uniformity of envelope shedding testifies to a similar initiation of splitting. It is considered that the entry of water increases pressure in the egg and splits its envelope. Such a mechanism is typical not only for ‘Cladocera’ and Branchiopoda in general, but for many other groups of invertebrates (Davis, 1981). Shedding of the outer membrane of the parthenogenetic egg has not been described in detail either for Anomopoda (Berill & Henderson, 1972) or for Ctenopoda. We have found that in Sida and Di-

aphanosoma it splits longitudinally (not transverse as in resting eggs) and goes through both poles of the egg, suggesting another shedding mechanism. Probably simply the pressure of the gradually enlarged second antennae suffices. Final moult as the termination of embryogenesis There is no agreement between different investigators of small branchiopods as to whether a moult takes place after the animal’s release from the brood pouch. Some authors did not observed it, including Parejko (1992) who used video in his investigation. Kotov (1997) found this moult in all studied representatives of different anomopod families. Now it has been recorded in two ctenopods belonging to different tribes. This moult also raises the question about the end point of embryonic development. Steuer (1933) observed a moult in Penilia soon after release of the embryo from the brood pouch and distinguished the short period before the moult as a special instar of postembryonic development, thus recognising four juvenile stages. Pavlova (1959) did not observe this moult in Penilia and distinguished only three juvenile instars. We think that this moult terminates embryogenesis. A very short period before, it cannot be distinguished as a special instar of postembryonic development because animals released from the brood pouch do not differ morphologically from the late embryo, cannot feed themselves, and (in the case of Sida) cannot attach to a substratum. At the same time, the term ‘neonata’ cannot be applied to the first juvenile instar (Kotov, 1997). Periodization of ctenopod embryogenesis The evolutionary approach is traditional for the majority of the previous detailed descriptions of ‘cladoceran’ embryogenesis. Different stages, have been considered as embryonized larval phases of ancestors. This approach was established in the last century by Müller (1867) and Claus (1876), who found the ‘naupliar’ stage in ‘cladoceran’ embryogenesis. Hoshi (1951) successfully elaborated the embryogenesis of Simocephalus and described the embryonic moults. He subdivided development into four stages: egg, gastrula, nauplius, and hatched embryo. But such an approach is undoubtedly subjective, because the degree of embryonization of different structures varies significantly. For example, in Sida the formation of the ocellus (naupliar organ) takes place after that of

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123 the thoracic limbs, and the dorsal organ appears after the completion of segmentation while in acient primitive branchiopods it is well developed in the nauplius (Walossek, 1993). Innumerable schemes of periodization of Arthropodan embryogenesis, have been suggested. Most of those proposed for Anomopoda (Green, 1956; Shuba & Costa, 1972; Murugan, 1975; Murugan & Venkataraman, 1977) represent modifications of that of Fox (1948). The size and degree of eye merger were especially useful to him, and anticipated the widely used ‘eye index’ in Malacostraca (Perkins, 1972; Helluy & Beltz, 1991). However, it is not possible to use this feature to distinguish stages in ctenopods because their eyes become fully fused only during the postembryonic period. Attempts to elaborate the periodization of ctenopod embryogensis relate to Penilia (Della Croce & Bettanin, 1965), but the distinguishing of embryos with one, two, etc. segments by these authors as succesive stages is neither convenient, nor correct, because the duration of the segmentation period (six stages by Della Crosse & Bettanin) is very short compared with that taken by full development. We propose a completely new scheme of embryogenesis (and probably of the entire life cycle) of Sida and Diaphanosoma based on the recognition of successive instars, separated by moults (in our opinion shedding of egg membranes are moults too) as it is accepted for juveniles and adults specimens for ctenopods and many other groups of crustaneans. The moults do not vary in time in different individuals of a given species and occur at precisely definable events in the course of development (see Table 1). Thus the entire process of embryological development can be objectively subdivided into well separated instars. The first instar. This is prolonged from the entry of the egg mass into the brood pouch to shedding of outer egg membrane, with two phases, early embryogenesis (egg organisation, blastula and gastrula formation), and the beginning of later embryogenesis. The instar continues for 18 h in Sida and 15 h in Diaphanosoma. The time differences are correlated with the longer period of cleavage and gastrulation in Sida. The second instar. Its peculiarities are: short rudiments of second antennae extending to the first, sometimes the second thoracic segment; the entire body closely surrounded by an envelope. This instar may be separated into two phases: a short period of segmentation (till the formation of the sixth thoracic segment)

and a long period of formation of the thoracic limbs. It lasts 12–13 h in both species. Third instar. Peculiarities of the embryo are: the second antennae are free and movable, while the thoracic limbs and postabdomen are enclosed in an envelope. There are two phases in Diaphanosoma development: embryo without eye pigmentation and embryo with small red-brown eyes. Sida lacks the former phase. Instar duration is 6–7 h in both species. The fourth instar. The traits of the embryo are: long second antennae, free thoracic limbs and pigmented eyes. In less developed embryos the second antennae do not extend beyond the fifth pair of thoracic limbs and the eyes are comparatively small (pigment occupies less than half the eye capsule), while more developed embryos have longer second antennae and more large and movable eyes). The last embryonic moult takes place outside the brood pouch soon after the embryo is released. Instar duration is 28–29 h in Sida and 18– 19 h in Diaphanosoma. The durations of the second and third instars are similar in both animals. The longer duration of embryonic development in Sida is due to its longer phase of cleavage and gastrulation but mainly to a much longer developing fourth instar. We suggest that the longer duration of the last embryonic instar in Sida is correlated with the longer duration of the first and other juvenile instars (Herzig, 1984; Bottrell, 1975a, b). During this instar the embryo is becoming similar to the juvenile. May be, it is evidence of the embryonization of this instar in the past? Objective subdividing of the period of embryogenesis into separate instars with sharply different features, as in instars of juveniles or adults, means, that ecological questions concerning ctenopods and anomopods may now embrace embryos. Examples include the dependence of duration of different stages on temperature in different species; the construction of cohort life tables dealing with embryos and, as a result, with the whole life cycle including eggs, embryos, juveniles and adults in experiments; the measurement of the number of embryos in different instars in natural populations, the construction of life tables for natural populations and so on. Our list of instar characteristics may be used for the determination of the instar number (and approximate age after simple experiments of embryos in the brood pouch of ctenopod females from natural populations.

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124 Acknowledgements We are indebted to Prof G. Fryer and Prof N. N. Smirnov for discussion of principal points and editing of text, to Dr N. M. Korovchinsky for his criticism and I. N. Korovchinsky for help with the English translation of an early version of manuscript. This study was partly supported by the Russian Foundation of Fundamental Investigations (grant N 96-04-48063) and Biodiversity Program (grant 02.0001.97).

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