A N Khanaychenko - Species-specific differences in diatom-induced anomalies in calanoid copepod embryogenesis - страница 1
МОРСЬКИЙ ЕКОЛОГІЧНИЙ ЖУРНАЛ
A. N. Khanaychenko, Ph. d, s. Sc.
A. O. Kovalevski's Institute of Biology of the southern Seas of National Academy of Sciences of Ukraine, Sevastopol, Ukraine
SPECIES-SPECIFIC DIFFERENCES IN DIATOM-INDUCED ANOMALIES IN CALANOID COPEPOD EMBRYOGENESIS:
ARE THEY LINKED TO THE STOCK-PILED ANTIOXIDANTS?
Egg production and viability of co-occurring in nature copepods, Calanus helgolandicus and Calanoides carinatus, fed diatom, Thalassiosira rotula, and then switched to dinoflagellate Prorocentrum minimum, were compared in experimental conditions. Egg production of both species on diatom diet was similar (up to 17-22 eggs.female-1.d-1) but no viable nauplii were observed. Development was arrested at different stages depending on degree of anomalies. Abnormal embryos displayed various degrees of deteriorations in pigment distribution and organization of extracellular matrix. Reproductive responses of C.carinatus both to negative (diatom) and positive (dinoflagellate) diets were postponed in comparison with quick responses of C.helgolandicus. After 3 days of dinoflagellate diet C.carinatus still produced only abnormal embryos with strong residual effect of diatom diet, while C.helgolandicus produced 50 % of viable nauplii. Based on own and literature data, our hypothesis links degree of diatom-induced copepod embryonic anomalies to the disturbances in antioxidant properties of the membranes and the degree of lipid peroxida-tion in embryo membranes and cytoplasm attributed to imbalanced content and ratio of HUFAs and carotenoids from freshly assimilated diet. Species-specific differences in copepod reproductive responses to diatom feeding are supposedly related to different stock-piled material and pathways of these essential components to the late oocytes.
Key words: calanoid copepods, diatom, embryo, anomaly, extracellular matrix, pigment
Since the start of copepod investigations till now, diatoms, usually dominating in natural phytoplankton of temperate waters during winter, early spring and autumn, are considered as one of the basic food components for the herbivorous calanoids [11, 42] and thus, the basis for their recruitment [3, 18]. Still, the field and experimental data of the last decade came in contradiction to this classic view. It appears that food web "dia-toms-copepods" in many cases leads to reproductive failure of copepods. Several species of diatoms actively consumed by ovigerous females of copepods were proved to induce high egg production coinciding with low egg-hatching success resulted from abnormal embryo development [17, 31, 48, 52]. Low value of diatoms for copepod recruitment was observed during rearing of calanoid copepods through numerous generations [23, 25, 53]. Diatom-induced inhibition of reproduction in 16 copepod species was registered in 12 different environments by 15 laboratories . Reproductive failures of Acartia clausi [17, 38, 39], Calanus helgolandicus [17, 37, 39], C. fin-marchicus , C. pacificus , Centropages typicus , Temora stylifera  were observed in natural environment during diatom blooms. A similar effect was registered in laboratory conditions when different calanoid copepods were fed by a single or mixed diet including various diatom species: Phaeodactylum tricornutum [6, 16, 32], Chaetoceros curvisetum , C. difficilis , Cylindrotheca closterium (= Nitzchia closterium) , Ditylum brightwellii , Pseudonitzschia delicatissima [38, 50], Skeletonema costatum [17,
© A. N. Khanaychenko, 2004
38], Thalassiosira rotula [6, 17, 47], T. norden-scoldii , T. weisflogii .
A decrease in viability of eggs produced by copepods fed on diatoms was supposed to result from a deficiency in essential biochemical components . However, analogous anomalies in embryo development were related not only to consumption of diatoms in the field and laboratory conditions, but also to the action of water-soluble diatom extracts applied to the newly spawned eggs of copepods . Originating from diatoms, chemical toxins induce various disturbances in oogenesis and embryogenesis by the blockage of cell division [16, 32, 47, 48]. Antimitotic activity of compounds originating from free fatty acids released during oxidation of lipid components from diatom cells was first reported in 1972 . Recent research found out that antiproliferative action of diatoms is associated with low molecular weight aldehydes . Purified from diatom extracts, low molecular aldehydes produced from different diatoms were identified as 10-carbon aldehydes - decatrienal and decadienal for T. ro-tula  and 8-carbon aldehydes - octadienal and octatrienals for S. costatum . The degree of antiproliferative effect of purified aldehydes was found to be dose-dependent similar to the diatom feeding effect [6, 47].
The mechanism of aldehyde production, considered to be a wound-activated chemical defense from diatoms, was defined as a cascade of sequential chemical reactions . Immediately after disruption of T. rotula, phopholipase A2 generates production of high local concentrations of unsaturated aldehydes from free eicosanoic acids released from the diatom cell phospholipids through the programmed mechanism of the typical lipid peroxidation process - aldehyde generating lipase/ lipoxygenase/hydroperoxide lyase cascade. Different diatom species and even strains  produce different reactive oxygen species (ROS) but the inhibitory effect was supposed to depend on a reactive structural element - a, P, y, 5 -unsaturated aldehyde  produced from free eicosapentaenoic acid (EPA, 20:5n-3) released from disrupted cells.
However, the mode of action of diatoms inhibiting cell division is only hypothesized. Even the data provided by the same authors could be controversial, being either pro negative impact of diatoms on copepod recruitment , or contra this postulate . Until now, it is not clear why various copepod species demonstrate different resistance to the same concentrations of deleterious diatoms. Moreover, inhibition of normal oogenesis is reversible in one species, while is likely irreversible in another [32, 39].
The objective of the present study was to assess and compare the effect of an experimental deleterious diatom diet, and its residual duration after a shift to a positive diet, on a species-specific reproductive response of different but related ca-lanoid copepod species co-occurring in the same natural environment. Our aim was to find out a possible explanation of modifications in diatom-induced embryonic anomalies on the basis of own experimental as well as literary data.
Two major Calanidae species from the Atlantic Ocean, Calanus helgolandicus, an indicator of the North Atlantic Central Water, and Ca-lanoides carinatus, an indicator of the South Atlantic Central Water , were considered to be the appropriate experimental models. These related species co-occur in the N.W. African upwell-ing area  and in the English Channel . The last is the central area of distribution of C. helgo-landicus and the approximate northern limit of distribution of C. carinatus. Both species are fine-particle filter-feeders, predominantly herbivorous, associated with phytoplankton blooms, have generalized life cycles and morphological similarity of larval stages, and do not need re-mating for production of fertilized eggs which develop to hatching in about 1 day [13, 31].
As during the mid-autumn period the food source in the coastal waters in the Western English Channel is scarce, it was considered that experimental diet effect on copepod reproductive response could not be obscured significantly by
feeding in situ. Both diatoms and dinoflagellates encountered as typical components of the natural environment in the English Channel and typical diet components for C. helgolandicus [11, 28, 31] and C. carinatus .
Differences in the diatom induced reproductive response of two related copepod species from the same habitat were never tested before simultaneously. Neither was the deleterious effect of diatom diet ever tested on C.carinatus. For the first time the same diatom maternal diet (T. rotula, actively consumed by copepods and known for its strong deleterious effect [17, 32, 48]) was proposed for simultaneous testing and comparison of the norm of negative reproductive response of C.helgolandicus and C.carinatus, related species from the same habitat. A dinoflagellate, P.minimum, which usually ensures the highest egg viability in copepods [32, 48], was selected to test the effect of positive maternal diet on recovery of reproduction in both species.
Material and Methods. Mature females of C. helgolandicus and C. carinatus were sorted within 2 h after delivery from the same zooplank-ton catch (gently towing a 500 цггі mesh plankton net obliquely from 20 to 0 m) offshore from Roscoff 48o 45' N and 3o 58' W, in the Western English Channel in October 2001. Females (6 replicates each species) were incubated individually in 150 ml of natural seawater (in 300 ml dishes) and daily transferred to the new dishes with fresh medium. In the first 24 h after delivery (day zero -D0), copepods were incubated in ambient water (filtered through 50-цгп) to assess initial fecundity in situ. During the following 48 h (D1-D2) cope-pods were transferred to a fresh suspension of diatoms (T. rotula, THA, 5x104 cells per ml determined by microscopic count) in FSW, simulating feeding under short-term diatom bloom conditions to achieve quick reproduction response of both copepod species. Thereafter, copepods were food-deprived for 48 h (D3 - D4) to remove diatom residuals from the maternal organism. From day 5 (D5) onwards, copepod females were switched to a suspension of dinoflagellate P. minimum, PRO, 5x104 cells per ml. Microalgae for experiments were used in the exponential phase of growth. Co-pepods were kept in dim-light conditions at 14±1oC, corresponding to the ambient field temperatures optimal for reproduction of both species [14, 32]. To reduce possible underestimation of (1) egg production (EP, eggs.female-1.d-1) resulting from any injury (cannibalism, or quick autoly-sis of abnormal eggs during early embryogenesis, etc.), EP was recorded every 4h, except at night, and eggs were transferred for incubation to culture dishes.
Energy resources and essential compounds stockpiled in the oocyte should self-sufficiently support all developmental transitions from the fertilized egg (i.e. cleavage, compaction, blastocyst formation and gastrulation) till hatching of the first larval stage (N1) and its molting into the "first-feeding" larva - nauplii N2. To determine (2) egg hatching success ( %H, percentage of hatched eggs from the total number of eggs) and (3) viability of hatched nauplii ( %Viab, percentage of N1, molted to N2, from the total number of eggs) batches of 10 freshly spawned eggs were incubated during (2) 24 h - (3) 48 h each in 2 ml of FSW at 14±1oC. Data were statistically analyzed and presented in figures as means and their standard deviations. Copepod late oocytes, embryos and nauplii were checked for morphological features and pigmentation patterns according to visible structures, distinguished under the light microscopy, on specific copepod development stages, as in Poulet et al. : (1) zygote (1 blas-tomere - 1B); (2) first cleavage (2 blastomeres: 2B); (3) 8-cell stage (8B); (4) morula stage (16B); (5) blastula stage (32B); (6) first naupliar stage N1; (7) second nauplii stage N2. Embryos obtained in experiments were compared with the normal ones described in literature. Normal intact newly spawned eggs of C. helgolandicus are characterized by transparent uniform cytoplasm and transparent double membrane ECM, as described by Poulet et al. . Normal eggs of C. carinatus present homogenous dark pigmentation of cytoplasm and transparent double membrane ECM,
"covered with branching wrinkles" described by Hirche . During the cleavage of normal embryo, blastomeres are similar, symmetrically organized and present well developed cell membrane between them.
Images were observed and digitized in bright field Nomarsky under the Olympus BH2 microscope equipped with a colour videocamera EuroCam Spot linked to a PC computer, using objectives: Splan 10 0.3 160/0.17 + Splan 20 PL
0. 46 160/0.17 and magnification 1.25X +(KOLCO) 0.76X.
Results. Adult females of both species (C.carinatus, 2,48±0,03 mm, C.helgolandicus -3,14±0,06 mm) from in situ failed to produce eggs without pre-feeding (D0, Fig.1A). Copepods started to spawn after 24 hours of diatom feeding (Fig.1). Egg production of C. helgolandicus (EP=10±1 eggs.fem-1.day-1), non-significantly differing from C. carinatus (EP=7±2,6 eggs.fem-
1. day-1), resulted from diatom (THA) feeding (D1, Fig.A), was typical for copepod late autumn populations. Still, all eggs spawned on D1 by both Ca-lanidae species were abnormal with species-specific differences in type and degree of embryo anomalies (Fig.2A-B). Eggs spawned by C.helgolandicus (D1), presented abnormal stratified blebbed membranes of extracellular matrix (ECM) and cytoplasm with irregular globular structures (Fig. 2A). They autolyzed within several hours at time-interval corresponding to morula-blastula stage in normal embryos, and resulted in 0 % hatching (Fig.1B).
Eggs produced by C. carinatus (D1) were characterized by normal ECM and relatively synchronous cleavage, presenting dark-brown pigmentation accented in proximities of cell membranes, particularly, within the cleavage furrows of dividing blastomeres, that is clearly distinguished by light microscopy on the morula stage (Fig. 2B). Egg hatching success was 100 % (Fig.1B) but all nauplii N1 revealed typical diatom-induced morphological deformities as is described in , i.e. asymmetrical development of the body, more reinforced on the right side, abnormally shortened and thickened appendages developed asymmetrically, resulted in crumpled movements of the nauplii, with asymmetrical distribution of brownish pigment granules in the body and appendages (Fig 3A). All nauplii N 1 died and did not molt into the N 2 stage. Thus, viability was 0 (Fig.1C).
Spawning continued during the second day of THA feeding (D2), with EP differed non-significantly between two species (17,7±3,1 and 22,7±8,6 eggs.fem-1. day-1, for C.helgolandicus and C. carinatus, respectively; Fig.1A), but egg hatching success diminished to 0 for both species (Fig.1B). The anomalies of Calanidae spp. eggs increased in variability and degree of embryo degeneration. General patterns for D2 embryos were severe degeneration of ECM membranes, asymmetry of cytoplasm structures and erroneous distribution of pigment granules in cytoplasm. Degree of anomalies varied between species and between different specimens within one species.
A small portion of C. carinatus eggs presented thick condensed (lacking transparency) blebbed membranes of ECM. They stopped development at the late embryo stage. Distinct asymmetry in the body and limbs and erroneous distribution of abnormal brown pigment granules in the embryo cytoplasm were observed (Fig. 2C).
The majority of the eggs spawned by females of both species on D2 displayed disintegration of thin outer and inner membranes of ECM lacking hyaline layer between. Half of these eggs underwent relatively synchronized cleavage until 16B (C. carinatus, Fig. 2E), or 32B (C. helgo-landicus, Fig. 2D) stage, and after 4-5 mitotic divisions development stopped. Blastomeres of these eggs did not flatten in the absence of normal cell adhesion at cell membranes, and, as a result, compaction did not occur. The embryos, instead of formation of normal morula, presented a "grape-like" group of round blastomeres disposed asymmetrically within the thin membrane. The other part of the eggs, with even more degenerated membranes, stopped development during
the first cell division and presented pigment granules either erroneously distributed within the blebbed cytoplasm in C. carinatus (Fig.2F) and C.helgolandicus (Fig.2G), or concentrated at the
poles of the polarized zygote (Fig.2H), or localized in the proximities of the first cleavage furrow (C. carinatus, Fig.2I).
- I ■ I
10 0 -
8 0 -
6 0 -
4 0 -
2 0 -
Via b % 10 0 -,
Fig.1. Egg production (EP, eggs.day-1.female-1) (А); hatching success (H %, percent of hatched eggs of the total number of spawned eggs) (B); viability % (Viab %, per cent of viable nauplii I of the total number of spawned eggs) (C) of Calanus helgolandicus (Ch) and Calanoides carinatus (Cc) during experimental feeding: in situ - D 0; Thalassiosira rotula - D 1-2; starvation - D 3-4; Prorocentrum minimum - D 5-9 . Values (means) and error bars (standard deviations) of 6 replicates.
Рис.1. A. Продукция яиц (EP, яиц.сут-1.самка-1) (А); процент выклева (H %, процент выклюнувшихся яиц к общему числу отложенных яиц) (B); жизнеспособность (Viab %, процент жизнеспособных науплиев к общему числу отложенных яиц) (C) Calanus helgo-landicus (Ch) и Calanoides carinatus (Cc) при питании: in situ D0; Thalassiosira rotula - D 1-2; голодание - D 3-4; Prorocentrum minimum - D 5-9. Приведены средние значения величин и их стандартные отклонения (6 повторностей)
Fig. 2. Types of embryo anomalies in C. helgolandicus (A, D, G) and C. carinatus (B, C, E, F, H, I) resulted from diatom Thalassiosira rotula "maternal diet" on D1 ( A, B, C) and on D2: (D, E, F, G, H, I). Only type B developed till hatching into abnormal nauplii I (Fig.3A). Arrows show abnormal dark-brown pigment distribution in eggs.
Рис. 2. Типы аномалий эмбрионов C. helgolandicus (A, D, G) и C. carinatus (B,C, E, F, H, I) на первые - D1 -(A, B, C) и вторые сутки - D2:- (D, E, F, G, H, I) при питании самок диатомовыми Thalassiosira rotula. Развитие до науплия I (аномального, рис.3A) происходит только из яйца типа B. Стрелки указывают на аномальное распределение темно-коричневого пигмента в яйцах.
Fig. 3. Residual effect of Thalassiosira rotula "maternal diet":
(A) Abnormal nauplii N I resulted from C. carinatus abnormal egg (Fig.2 C).
(B) Abnormal pigmentation in the late oocytes in the oviducts of C. carinatus fed T.rotula
(C) "Marine snow" formation from abnormal embryo
(D) Viable egg (20 %) of C. helgolandicus fed P.minimum (D7)
(E) Normal nauplii N I C. helgolandicus resulted from the egg Fig.3D
(F-G) Qeavage of abnormal embryo from C. carinatus female after feeding P.minimum (D8) Arrows show dark-brown pigment granules
Рис. 3. Остаточный эффект "материнской"диеты Thalassiosira rotula:
(A) Аномальный науплий N I C. carinatus, полученный из аномального яйца (рис. 2 C).
(B) Аномальная пигментация поздних ооцитов в яичниках C. carinatus после питания T.rotula
(C) Формирование "морского снега" при лизисе аномального эмбриона
(D) Нормальный эмбрион C. helgolandicus после питания динофлагеллятами P.minimum (D7)
(E) Нормальный науплий N I C. helgolandicus, полученный из яйца (рис. 3 D) (F-G) Дробление аномального эмбриона C. carinatus после питания P.minimum (D8) Стрелки указывают на темно-коричневые гранулы
Eggs with severe deterioration often accumulated the gases between the plasma and outer membranes and floated to the surface (while normal eggs sink), where they soon "burst" into the substance similar to "marine snow" (Fig.3C). Microscopic observations in vitro revealed that free-living "run-and-tumble"-swimming heterotrophic bacteria, typical for "marine snow" aggregates, attracted chemokinetically by organic solutes  leaking out from disrupted abnormal eggs, were observed colonizing the scattered layers of outer membranes within several minutes, destroying them actively, and penetrating through inner membrane into the embryo cytoplasm within 30 minutes.
Females of both species changed gradually initial species-specific "wild pigmentation" patterns during 2 days of THA feeding. C. helgo-landicus totally lost the rose-pink coloration, and intensive red pigmentation of C.carinatus was also significantly reduced. Erroneously distributed pigment granules in the late oocytes were easily detected in C.carinatus female oviducts through transparent integuments under the light microscope on D3 (Fig.3B).
During starvation in FSW (D3-D4) C.carinatus totally ceased spawning, meanwhile, C.helgolandicus reduced gradually the production from 7,2 to 1 eggs.fem-1. day-1 (Fig.1A) of abnormal eggs, lacking outer membrane and disrupting prior the start of cleavage.
After switching to the PRO diet, C. helgo-landicus resumed egg production (D7) (Fig.1A) to the level typical for autumn populations of this species , but only about 20 % of spawned eggs displayed normal membranes (Fig.3D). These underwent typical cleavage and hatched (Fig.1B) into normal viable nauplii N1 (Fig.3E). Viability increased up to 50 % on D9, coinciding with hatching success (Fig.1B, C).
C. carinatus fed PRO also resumed pulsatory egg production (Fig.1A), but hatching success was 0 % (Fig.1B). These eggs spawned D7-D9 still presented anomalies resulting from the D1-D2 diatom maternal diet (Fig.3F). The ECM of these eggs were similar to the ECM of normal eggs (inner and outer membranes with hyaline layer). The cytoplasm was uniformly pigmented, but blas-tomeres displayed reduced adhesion and compaction did not occur. The majority of the embryos stopped development after 3 synchronous cell divisions at 8B stage (Fig.3G). The rest never survived after the 16-cell stage (Fig.3H). None of eggs produced by C. carinatus females resulted in viable nauplii during the 10 days of the experimental period.
Thus, it was observed that experimental "diatom bloom" conditions resulted in total mortality of the progenies of both copepod species. C. helgolandicus showed quick reproductive responses: both negative on diatoms, and positive when switched to dinoflagellates (recovered quickly the egg production and viability). Both reproductive responses of C. carinatus were postponed, and the residual negative effect of diatoms on embryos was observed during the experimental period of dinoflagellate feeding.
Discussion. The state of cell membranes as one of the criteria for identification of anomalies in the eggs of C. helgolandicus was discussed in Poulet et al. . Abnormal eggs were usually characterized either by "dark brown, opaque"  or "darker and more granular"  outer membranes in comparison with that of normal eggs in early stages of development (zygote - 2 cells), or presented "malformations of the cellular membrane between daughter cells during mitosis", or even "the absence of a cell membrane between daughter cells" at 16- (morula) -32-cell (blastula) stage embryos.
Earlier, Hirche  described abnormal eggs of C. carinatus from Cape Bojador "without space between inner and outer membranes" with the "substance ... divided into many smaller spheres" (similar to Fig. 2E, present paper) attributed by the author to "decrease of supply of viable sperm". Still, these anomalies could be more reasonably supposed to be also related also to "maternal diet effect", as, according to the same author (Table 2, 13]), 100 % egg hatching success re
sulted from C.carinatus fed dinoflagellate (Gym-nodinium sp.) diet in comparison with 75 % hatching success of eggs from females fed diatom (P. tricornutum).
We ranked diatom-induced anomalies observed during Calanidae embryogenesis (our own and literature data) by a bottom-up principle, arranging them according to increase of degree of embryo degradation (DED) identified by microscopic observations and related to the stage of embryo death:
DED 1. Eggs display a normal transparent multilayer extracellular matrix (ECM), and undergo normal cleavage, compaction and gastrula-tion. Still, slight deterioration is observed in pigmentation, emphasized in the proximities of membranes (in cleavage furrows) of dividing cells (Example: C. carinatus, Fig.3B, present paper). Development of such eggs result in hatching of abnormal nauplii N1 with shortened massive bodies flexed dorso-ventrally; asymmetrically, shortened, abnormally in shape and segmentation appendages, antennule, antenna and mandible with atypical reduced number and length of bristles and darker opaque body colour with erroneous distribution of brownish pigment granules (Examples: C. helgolandicus: Fig.4F, Fig.5C-D ; C.finmarchicus, Fig.4C ; C.euxinus, own data, unpubl.; C. carinatus: Fig.3A, present paper). Development is arrested at stage nauplii I.
DED 2. Eggs display multilayer but non-transparent (opaque) thick ECM with blebbed membranes, undergo synchronized cleavage and gastrulation, but the late embryo displays severe asymmetrical cytoskeleton with abnormal distribution of pigment granules. Development stopped prior to hatching (Examples: C.helgolandicus, Fig.4B ; C.finmarchicus, Fig.4B ; C.euxinus, our own data, unpubl.; C.carinatus, Fig.2C, present paper). Development is arrested at the late embryo stage.
DED 3. Eggs show deterioration (stratification) of the multilayer ECM. The cytoplasm of the embryo is characterised by scattered, irregular, asymmetrical globules corresponding to nuclei in
Морський екологічний журнал, № 3, Т. III. 2004
the absence of normally organized cell membranes between dividing cells (probably resulting from deficiency of hyaline to form normal cell membranes). On the borders, the abnormal brownish pigment is concentrated. Karyo- and cytokinesis are desynchronized from the start of cell division (after ). (Examples: C.helgolandicus, Fig.3E, G ; Fig.2A, 2G, present paper; C.finmarchicus, Fig.4A ; C.carinatus, Fig. 2F, present paper). Development is arrested at a time corresponding to the morula stage in normal embryos.
DED 4. Eggs lack a multilayer ECM (transparent outer and inner membrane and hyaline layer between), i.e. "the space between inner and outer membranes" , and often display a severely deteriorated thin one-layer outer membrane. Cleavage takes place until the 16-cell or 32-cell stage. Cell adhesion is absent (presumably, the cell membranes lack tight junctions as a result of the absence of a hyaline layer or deformation of microtubules or both) and blastomeres do not flatten ("the substance is divided into many smaller spheres" . (Examples: C.carinatus, Fig.3B, ; Fig.2E, present paper; C.helgolandicus, Fig.2D, present paper; C. euxinus, our own data, unpubl.). Compaction does not take place, the morula does not develop. Development is arrested during cleavage, after 4 - 5 cell divisions.
DED 5. (diatom residual, observed during recovery diet, intermediate). Eggs present a multilayer ECM, undergo synchronized doubling of cell numbers until the 8-cell (rarely 16-cell) stage, cell adhesion is reduced, and blastomeres do not flatten. (Example: C.carinatus, present paper, Fig. 3F-H; C). Development is arrested after 3 - 4 divisions (prior to cell polarization).
DED 6. Eggs display a high degree of cytoplasm and ECM deterioration with stratification of membrane from the cytoplasm. Abnormally scattered pigment granules are disposed on different poles of the 1-cell embryo (zygote), or concentrated near the developing first cleavage furrow. (Examples: C.carinatus, Fig.2H-I, present paper). Development is arrested prior to the 2B stage.