Chemical composition of spines and tests of sea urchins

The skeleton of spines and tests of the species of sea urchins Strongylocentrotus intermedius, Mesocentrotus nudus, Scaphechinus mirabilis, and Echinocardium cordatum from the Sea of Japan is composed of a spongy stereom, consisting of calcite with a high content of magnesium. It was found that the tests and spines of the skeletons of sea urchins are composed of calcium–organic composite materials inlaid with other metals: Mg, Fe, Zn, and Rb. In the four species of sea urchins studied, the strength and other mechanical properties of the tests and spines differ and depend on the chemical composition and structural organization of their components. It was shown that the content of volatile substances correlates with their fragility or elasticity. It is revealed that the chemical composition of the tests of two species of the spherical sea urchins S. intermedius and M. nudus indicates significant differences between these two species of sea urchins.     

Comparative ultrastructure of spermatozoa from two regular and two irregular New Zealand echinoids

Spermatozoa from four species of echinoids found in New Zealand had morphological characteristics typical of other echinoids, including a conical sperm head with an acrosome‐capped nucleus, a midpiece, and a single long flagellum. The spermatozoa of Fellaster zelandiae, Echinocardium cordatum, Evechinus chloroticus, and Centrostephanus rodgersii also showed statistically significant differences in species‐specific morphological characteristics. Evechinus chloroticus showed the most variable sperm morphology. The irregular urchins (F. zelandiae and E. cordatum) had short, wide sperm heads (head length:width ratios 2.93:1 & 2.97:1, respectively) with a long acrosome complex, while the regular urchins (E. chloroticus and C. rodgersii) had longer, narrower heads with a short acrosome complex (ratios 5.29:1 & 3.37:1). Spermatozoa of E. cordatum from the New Zealand population shared more characteristics with those of conspecifics from the Sea of Japan than those of conspecifics from the Baltic, reflecting the membership of the former two populations in a distinct Pacific clade. Volumetric calculations showed no evidence of phylogenetic grouping. Mitochondria of E. chloroticus spermatozoa were less than half the volume of those of C. rodgersii and E. cordatum, and those of F. zelandiae were intermediate in volume. These volume measurements will be useful in physiological studies of sperm performance and quality.     

Diversity of Polyhydroxynaphthoquinone Pigments in North Pacific Sea Urchins

Using high‐performance liquid chromatography with diode‐array detection and mass spectrometry (HPLC‐DAD/MS) we investigated the composition of polyhydroxynaphthoquinone (PHNQ) pigments from sea urchins Strongylocentrotus pallidus, St. polyacanthus, St. droebachiensis, Brisaster latifrons and Echinarachnius parma, collected in the Sea of Okhotsk and the Bering Sea. Identification of PHNQ pigments from sea urchins St. polyacanthus, B. latifrons, and E. parma was performed for the first time. Among the usual PHNQ pigments, mono‐ and dimethoxy derivatives of spinochrome E, not previously found in other sea urchins, were discovered in St. polyacanthus and St. droebachiensis. In St. droebachiensis, two monomethoxy derivatives of echinochrome A were detected, isolated previously from only tropical sea urchins. It was found that the composition and total content of pigments of St. droebachiensis depends on the collection area of the sea urchins and its depth and varies from 88 to 331 μg/g of dry shells. Sea urchins St. pallidus, B. latifrons and E. parma had average values for PHNQ pigment content, approximately 30 μg/g, and St. polyacanthus had a low PHNQ content, 13 μg/g.     

THE SURFACE EVENTS AT FERTILIZATION OF THE SEA URCHIN EGG I. EVENTS ON THE SURFACE OF THE VITELLINE COAT1

Sperm-egg interaction during normal fertilization in the sea urchins, Strongylocentrotus intermedius and Hemicentrotus pulcherrimus, was studied by scanning and transmission electron microscopy. Several seconds after insemination, acrosome-reacted spermatozoa were found attached to the surface of the vitelline coat on each egg. Soon, several bulges of the vitelline coat appeared surrounding the fertilizing spermatozoon. These bulges then spread over the surface increasing in number, while they became fewer and disappeared around the sperm head. Thin sections of the bulging areas revealed discharging cortical granules. As the bulging vitelline coat was elevated, the sperm head was incorporated into the perivitelline space, passing through a small hole in the coat that resulted from penetration of the sperm acrosomal process immediately before fusion of the gametes. When the spermatozoon disappeared beneath the fertilization membrane, a hole was left in the membrane and the cortical reaction had finished on the other hemispheric surface. Mechanical removal of the membrane at that time exposed a spermatozoon protruding perpendicularly from the egg plasma membrane surface. The anterior tip of the sperm head was smoothly connected with the egg surface, and neither microvillous projections nor cytoplasmic covering of the egg cytoplasm could be found around the spermatozoon.     

Cultivation of the heart urchin Echinocardium cordatum and validation of its use in marine toxicity testing for environmental risk assessment

To study environmental risk assessment, echinoderms provide a useful model for ecotoxicological testing. However, limited knowledge of the life history of field collected heart urchins is a problem and the use of cultured urchins has been investigated here. The present study describes a culture method for the heart urchin Echinocardium cordatum under controlled laboratory conditions, providing organisms with a low biological variation. Based on our optimized growth protocol both larvae and juveniles have a growth rate comparable to E. cordatum in the wild. The toxicological response of cultured and field-collected E. cordatum was compared in standard saltwater toxicity bioassays. Using ammonium chloride as a water-soluble reference toxicant, mean 96 h LC₅₀ values for cultured heart urchins versus field collected animals were 37.4 ± 7.6 mg NH₄+/l (n = 5) versus 22.5 ± 4.9 mg NH₄+/l (n = 19), respectively. Additional toxicity experiments with tributyl tin (TBT) spiked sediments revealed 14d LC₅₀ values of 1,242 (95% confidence interval 986–1,564) and 964 (95% confidence interval 843–1,102) µg Sn/kg dw respectively in cultured and field collected E. cordatum. From this it was concluded that cultured heart urchins are less sensitive to TBT than field collected E. cordatum. Furthermore in whole sediment toxicity tests, survival of cultured sea urchins was higher or at least similar to that of field collected E. cordatum. The increased sensitivity of field urchins compared to cultured urchins in various toxicity tests may be due to multiple environmental stressors reducing their overall performance. Overall it was demonstrated that the use of cultured E. cordatum provides a significant advance for urchin-based bioassays for marine environmental toxicity testing, resulting in a more homogeneous, vital population with experimental data displaying reduced variability.     

MORPHOLOGICAL OBSERVATIONS ON THE SPERMATOZOA OF ECHINOTHURID SEA URCHINS*

The morphology of the spermatozoa of three species of echinothurid sea urchins, Asthenosoma ijimai, Araeosoma owstoni, Hapalosoma gemmiferum, was investigated by means of transmission and scanning electron microscopy. The spermatozoa of these three species of echinothurid sea urchins have similar fine structure, but they differ in several features from the more familiar regular sea urchins. 1) The external anatomy of the head region of the echinothurid spermatozoon is diagnostic in that it has a highly elongated head. 2) The spermatozoon of echinothurid sea urchins has a very long slender nucleus, protruding on its proximal end, so that the shape of the nucleus resembles a sperhead. 3) The acrosomal granule in the acrosomal vesicle of the echinothurid spermatozoon is not a mass of homogenous particulate material but an electron opaque rod condensed in the central part of the acrosomal vesicle. Scanning electron microscopic examination revealed that echinothurid spermatozoa form acrosomal processes similar to those of other regular sea urchins. 4) The basal body is situated just beneath the middle of the posterior protrusion of the nucleus. The distal centriole is located beside the basal body almost in contact with it. The axis of the distal centriole is almost but not quite parallel to that of the basal body. A satellite complex can be recognized around the posterior part of the proximal centriole.     

A novel procedure for obtaining denuded sea urchin eggs and observations on the role of the vitelline layer in sperm reception and egg activation

A procedure is described for the complete removal of the vitelline layer of the eggs of the sea urchin, Strongylocentrotus purpuratus. The method involves treatment of unfertilized eggs with an S. purpuratus cortical granule protease preparation followed by incubation in an alkaline dithiothreitol seawater solution. Eggs denuded of their vitelline layers react metabolically to parthenogenetic agents and sperm like unfertilized eggs, whereas the fertilizability of denuded eggs and receptivity to sperm is much less than controls. The present method is superior to previous methods using mercaptans in that all of the vitelline layer is removed and to procedures using other proteolytic enzymes in that no ¹²⁵I-labelled plasma membrane proteins are extensively modified. Thus the cortical granule protease dithiothreitol procedure is ideal for studies of the plasma membrane of the unfertilized egg and for studies on the role of the vitelline layer in normal fertilization and development.     

Induction of cross-fertilization between sea urchin eggs and starfish sperm by polyethylene glycol treatment

Cross-fertilization between sea urchin eggs (Strongylocentrotus nudus) and starfish sperm (Asterina pectinifera) was induced by treatment with polyethylene glycol (PEG). Without treatment with PEG, the denuded egg surface (jelly coat- and vitelline coat-free) engulfed the head of acrosome-reacted sperm; however, sperm penetration did not occur [Kyozuka and Osanai, 1988]. When these eggs were exposed briefly to PEG (molecular weight 3,000) in seawater, the sperm entered the egg by membrane fusion. Cortical granules were discharged, and embryogenesis began following sperm penetration. PEG did not induce parthenogenesis in Strongylocentrotus eggs. Egg activation is thus closely linked with gamete membrane fusion.     

Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

Growth characteristics of two HeLa S3 strains possessing low level glucocorticoid-inducible and high level suppressible alkaline phosphatase.

Sperm of the sea urchin, Anthocidaris crassispina loses its fertilizing capacity, without losing its motility, on prior exposure to both native and trypsin-digested, univalent Concanavalin A (Con A). Neither agglutination nor acrosome reaction is evoked by ConA treatment. Fluorescein-conjugated ConA binds to the apex of sperm head and to the midpiece. The observed effects of ConA are cancelled by methyl α-d-mannoside. ConA neither binds to sperm of Hemicentrotus pulcherrimus nor renders it infertile. Fertilizability of egg of both species is not reduced by ConA, though formation of the fertilization membrane and 1st cleavage are seriously affected. It is suggested that the species-specific polysaccharide component is situated on the apex of the sea urchin sperm head and constitutes the counterpart to the sperm-binding protein of the vitelline membrane of the egg which belongs to the same species.     

Does Hybridization Increase Evolutionary Rate? Data from the 28S-rDNA D8 Domain in Echinoderms

The divergent domain D8 of the large ribosomal RNA is very variable and extended in vertebrates compared to other eukaryotes. We provide data from 31 species of echinoderms and present the first comparative analysis of the D8 in nonvertebrate deuterostomes. In addition, we obtained 16S mitochondrial DNA sequences for the sea urchin taxa and analyzed single-strand conformation polymorphism (SSCP) of D8 in several populations within the species complex Echinocardium cordatum. A common secondary structure supported by compensatory substitutions and indels is inferred for echinoderms. Variation mostly arises at the tip of the longest stem (D8a), and the most variable taxa also display the longest and most stable D8. The most stable variants are the only ones displaying bulges in the terminal part of the stem, suggesting that selection, rather than maximizing stability of the D8 secondary structure, maintains it in a given range. Striking variation in D8 evolutionary rates was evidenced among sea urchins, by comparison with both 16S mitochondrial DNA and paleontological data. In Echinocardium cordatum and Strongylocentrotus pallidus and S. droebachiensis, belonging to very distant genera, the increase in D8 evolutionary rate is extreme. Their highly stable D8 secondary structures rule out the possibility of pseudogenes. These taxa are the only ones in which interspecific hybridization was reported. We discuss how evolutionary rates may be affected in nuclear relative to mitochondrial genes after hybridization, by selective or mutational processes such as gene silencing and concerted evolution.     

Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

The ultrastructural characters of the mature spermatozoon of Opechona bacillaris (Molin, 1859) (Digenea,Lepocreadiidae) a parasite of Scomber colias Gmelin, 1789 (Scombridae) off the coast of Dakar (Senegal)

This study presents the ultrastructure of the mature spermatozoon of Opechona bacillaris, a digenean belonging to the family Lepocreadiidae. The sperm cell of O. bacillaris exhibits the general pattern described in most of the Lepocreadioidea: two axonemes of the 9 + ‘1’ pattern of the Trepaxonemata, mitochondria, a cortical mitochondrion, a nucleus, electron‐dense material in the anterior extremity of the spermatozoon, external ornamentation of the plasma membrane with associated spinelike bodies, and granules of glycogen. However, particularities of O. bacillaris are the simultaneous presence in the anterior extremity of the spermatozoon of the electron‐dense material, a mitochondrion, and the absence of cortical microtubules. In the Lepocreadiidae, we describe for the first time in O. bacillaris spinelike bodies associated with the external ornamentation of the plasma membrane and two mitochondria. The first mitochondrion is moniliform and composed of a mitochondrial cord with joined mitochondrial bulges. The second mitochondrion shows a regular form. The posterior tip of the spermatozoon has only singlets to owing to the disorganization of the second axoneme and granules of glycogen as occurs in Hypocreadium caputvadum, the other studied species of the family Lepocreadiidae.     

CHANGES IN THE SPERMATOZOON DURING FERTILIZATION IN HYDROIDES HEXAGONUS (ANNELIDA) : I. Passage of the Acrosomal Region through the Vitelline Membrane

In the previous paper the structure of the acrosomal region of the spermatozoon was described. The present paper describes the changes which this region undergoes during passage through the vitelline membrane. The material used consisted of moderately polyspermic eggs of Hydroides hexagonus, osmium-fixed usually 9 seconds after insemination. There are essentially four major changes in the acrosome during passage of the sperm head through the vitelline membrane. First, the acrosome breaks open apically by a kind of dehiscence which results in the formation of a well defined orifice. Around the lips of the orifice the edges of the plasma and acrosomal membranes are then found to be fused to form a continuous membranous sheet. Second, the walls of the acrosomal vesicle are completely everted, and this appears to be the means by which the apex of the sperm head is moved through the vitelline membrane. The lip of the orifice comes to lie deeper and deeper within the vitelline membrane. At the same time the lip itself is made up of constantly changing material as first the material of the outer zone and then that of the intermediate zone everts. One is reminded of the lip of an amphibian blastopore, which during gastrulation maintains its morphological identity as a lip but is nevertheless made up of constantly changing cells, with constantly changing outline and even constantly changing position. Third, the large acrosomal granule rapidly disappears. This disappearance is closely correlated with a corresponding disappearance of a part of the principal material of the vitelline membrane from before it, and the suggestion is made that the acrosomal granule is the source of the lysin which dissolves this part of the vitelline membrane. Fourth, in the inner zone the fifteen or so short tubular invaginations of the acrosomal membrane, present in the normal unreacted spermatozoon, lengthen considerably to become a tuft of acrosomal tubules. These tubules are the first structures of the advancing sperm head to touch the plasma membrane of the egg. It is notable that the surface of the acrosomal tubules which once faced into the closed acrosomal cavity becomes the first part of the sperm plasma membrane to meet the plasma membrane of the egg. The acrosomal tubules of Hydroides, which arise simply by lengthening of already existing shorter tubules, are considered to represent the acrosome filaments of other species.     

Glycosidases,proteases and ascidian fertilization

Spermatozoa should bind to and then penetrate the vitelline coat for fertilization in ascidians and many other animals. There is substantial evidence that the binding of ascidian sperm is mediated by a sperm glycosidase and complementary saccharide chains of glycoproteins in the vitelline coat. Involvement of a sperm proteasome in the binding is also suggested. For the penetration, sperm proteases such as chymotrypsin-like enzyme, acrosin, spermosin and proteasome are suggested to play essential roles. Sperm glycosidase, that is translocated from the tip of sperm head to the surface overlying the mitochondrion, anchors the mitochondrion at the outer surface of vitelline coat. Therefore it assists sperm to penetrate the vitelline coat and traverse the perivitelline space. For fusion with egg plasma membrane, sperm metalloendoprotease seems to be involved. Egg glycosidases and proteases serve for some steps after fertilization, such as the prevention of polyspermy, expansion of perivitelline space and regulation of cell cycle.     

Ultrastructure and potential taxonomic importance of euspermatozoa and paraspermatozoa in the volutid gastropods Zidona dufresnei and Provocator mirabilis (Caenogastropoda, Mollusca)

The ultrastructure of mature spermatozoa is investigated for the first time in the Volutidae, based on the commercially significant South American species Zidona dufresnei (Donovan, 1823) (fresh material) and supplemented with observations on testicular (museum) material of the deep sea New Zealand species Provocator mirabilis (Finlay, 1926). Euspermatozoa of Z. dufresnei (ex sperm duct) consist of: (1) a tall-conical acrosomal vesicle (with short basal invagination, constricted anteriorly) which is flattened anteriorly and associated with an axial rod, centrally perforate basal plate and short accessory membrane; (2) a rod-shaped, solid and highly electron-dense nucleus (with short basal fossa containing centriolar complex and initial portion of a 9 + 2 axoneme); (3) an elongate midpiece consisting of the axoneme sheathed by 5–6 helical mitochondrial elements, each exhibiting a dense U-shaped outer layer; (4) an elongate glycogen piece (axoneme sheathed by nine tracts of putative glycogen granules); (5) a dense annulus at the junction of the midpiece and glycogen piece and (6) a short free tail region (axoneme surrounded only by plasma membrane). Paraspermatozoa of Z. dufresnei are vermiform and dimorphic: the first type contains approximately 14–20 axonemes (arranged peripherally and interspersed with microtubules) and numerous oblong dense vesicles, numerous less dense (round) vesicles, occasional, large lipid-like vesicles, and scattered mitochondria; the second type contains 25–31 axonemes (peripherally arranged, interspersed with microtubules), occasional mitochondria and extensive cytoplasm. Results obtained for P. mirabilis from testis material are essentially as observed in Z. dufresnei, although the euspermatozoan acrosome still has to achieve its compressed transverse profile. Observations on paraspermatozoa were limited by fixation quality of available (testis) tissues, but these cells are similar to the first type of Zidona paraspermatozoa. Although most of the euspermatozoal features are also observed in many neotaenioglossans and neogastropods, the U-shaped outer layer of each mitochondrial element has not previously been reported and may prove a diagnostic feature of the Volutidae, the subfamily Zidoniinae or possibly only the Zidonini (in which Z. dufresnei and P. mirabilis are currently placed).     

Consumers of sea urchins,<Emphasis Type="Italic"> Paracentrotus lividus</Emphasis> and<Emphasis Type="Italic"> Arbacia lixula</Emphasis>, in shallow Mediterranean rocky reefs

Underwater observations on fish and asteroid consumers (i.e. predators and scavengers) of sea urchins, Paracentrotus lividus and Arbacia lixula, were carried out at several locations in shallow Mediterranean rocky reefs. Observations conducted in the marine reserve of Torre Guaceto (Adriatic Sea) revealed that sparid fishes, Diplodus sargus and D. vulgaris, are the main fish predators of small (<1 cm in test diameter) and medium (1–4 cm) sea urchins, whereas the labrids Coris julis and Thalassoma pavo preyed only upon small sea urchins. Large D. sargus were able to prey upon small and medium, and occasionally large (>4 cm) sea urchins, whereas medium and small Diplodus preyed mainly upon small sea urchins. The number of sea urchins preyed upon by fishes was negatively related to sea urchin size for both species. P. lividus appeared to be subject to higher predation levels than A. lixula. The scavenger guild comprised 11 fish species, with D. sargus, D. vulgaris, Coris julis and Chromis chromis accounting for about 80% of scavenger fishes. Observations performed at several locations in the Mediterranean on the predatory asteroid Marthasterias glacialis revealed that only 3% of the detected individuals were preying upon sea urchins. Due to the importance of sea urchins for assemblage structure and functioning of Mediterranean rocky reef ecosystems, these results may have also important implications for management of fishing activities.Communicated by H.-D. Franke     

Evolutionary modification of T-brain (tbr) expression patterns in sand dollar

The sand dollars are a group of irregular echinoids that diverged from other regular sea urchins approximately 200 million years ago. We isolated two orthologs of T-brain (tbr), Smtbr and Pjtbr, from the indirect developing sand dollar Scaphechinus mirabilis and the direct developing sand dollar Peronella japonica, respectively. The expression patterns of Smtbr and Pjtbr during early development were examined by whole mount in situ hybridization. The expression of Smtbr was first detected in micromere descendants in early blastula stage, similar to tbr expression in regular sea urchins. However, unlike in regular sea urchin, Smtbr expression in middle blastula stage was detected in micromere-descendent cells and a subset of macromere-descendant cells. At gastrula stage, expression of Smtbr was detected in part of the archenteron as well as primary mesenchyme cells. A similar pattern of tbr expression was observed in early Peronella embryos. A comparison of tbr expression patterns between sand dollars and other echinoderm species suggested that broader expression in the endomesoderm is an ancestral character of echinoderms. In addition to the endomesoderm, Pjtbr expression was detected in the apical organ, the animal-most part of the ectoderm.     

Resistance of the heart sea urchin Echinocardium cordatum (Echinoidea: Spatangoida) to extreme environmental changes

The ability to survive under extreme environmental conditions was studied in the adults of the heart sea urchin Echinocardium cordatum (Pennant). At seawater temperatures of 13.3 to 14.8°C and salinity of 33.2–33.4‰, being devoid of the possibility to burrow into the sand or eat, some sea urchins died on day 5 and all individuals had perished by the end of day 8. At a temperature of 19°C, the salinity tolerance range of adults was limited to 33–28‰. Only 30 to 20% of sea urchins transferred to a solid substrate survived for 7 days at a salinity of 33 to 24‰, but all of them perished toward the end of day 8.     

Ultrastructural differentiations in the developing follicle cortex of Locusta migratoria,with special reference to vitelline membrane formation

Summary Electron microscopic studies on developing follicles of Locusta migratoria show the vitelline membrane to be composed of two ultrastructurally distinguishable components: The vitelline membrane bodies (VMBs) and, in addition, fine granular material, cementing the VMBs together. VMBs form first in the oocyte-near zone within the oocyte-follicle cell space. Subsequently, the second vitelline membrane substance is secreted between the VMBs through apical protrusions of the follicle cells. The possible origin of the VMBs is discussed.Yolk uptake in Locusta seems to occur predominantly by pinocytosis. During oocyte development the oocyte membrane is enlarged by numerous microvilli and folds. In addition pinocytotic vesicles are pinched off. It is supposed that the latter loose their coat and eventually transform into large proteid yolk spheres.This work was supported by the Volkswagenstiftung, HannoverI wish to thank Prof. Dr. H. Emmerich, Techn. Hochschule Darmstadt, for valuable discussions