Cryopreservation of sperm from the marine shrimp Sicyonia ingentis

Sperm from a marine shrimp, Sicyonia ingentis, were frozen to -196 degrees C using a variety of cooling rates and cryoprotectants. A cooling rate of 1 degree C/min resulted in minimal cell breakage. Sperm samples were frozen in solutions of known membrane stabilizers--trehalose, sucrose, proline, and glycerol. These compounds were somewhat effective but a dramatic increase in sperm viability was seen when DMSO was present in the freezing medium. Sperm viability was assessed using the in vitro acrosome reaction technique of Griffin et al. (1987). The highest sperm survival (56%) was obtained with samples frozen at 1 degrees C/min in a 5% (v/v) DMSO solution. No decrease in viability was seen in sperm samples stored in liquid nitrogen (-196 degrees C) for 1 month.     

Vibrio parahaemolyticus and V. harveyi cause detachment of the epithelium from the midgut trunk of the penaeid shrimp Sicyonia ingentis

Shrimp Sicyonia ingentis were either injected with Vibrio parahaemolyticus (10(4) CFU) or V. harveyi (10(6) CFU) or immersed in ASW containing either species at 10(5) CFU ml(-1). These densities were shown in preliminary experiments to kill approximately half the population by 7 d. On Day 7, surviving shrimp were classified as either diseased or apparently healthy, and their midgut trunks (MGT) were examined by light and electron microscopy. All shrimp immersed in ASW containing either species of Vibrio showed detachment of the epithelium in the MGT. In shrimp injected with either species of Vibrio, epithelial detachment was common in diseased shrimp but not in apparently healthy animals. Experiments with live shrimp were supported by in vitro experiments where MGTs were removed, tied off at both ends, and injected with either pathogenic bacteria (V. parahaemolyticus or V. harveyi), non-pathogenic bacteria (Bacillus subtilis or Escherichia coli), or ASW. After 2 h incubations in ASW at 15 degrees C, the MGTs were processed and examined. The epithelium consistently detached from isolated MGTs injected with either species of Vibrio, but not from MGTs injected with non-pathogenic bacteria or ASW. Because the MGT epithelium secretes the peritrophic membrane, loss of the epithelium eliminates 2 layers that may restrict penetration of ingested pathogens into the shrimp body and may disrupt the osmoregulatory function of the MGT. A second finding was that fixed, large-granule hemocytes associated with the basal lamina degranulated in the presence of the 2 species of Vibrio, but not with the non-pathogenic bacteria or ASW. These blood cells may help fight specific bacteria penetrating the MGT.     

Development of the mesendoderm in the dendrobranchiate shrimp Sicyonia ingentis

Dendrobranchiate shrimp embryos form a 4-cell stage that resembles spiralians in its cell contacts, but cleavage proceeds radially without any further evidence of spiralian character. The fates of Sicyonia ingentis mesendoblasts were followed by nuclear staining and confocal microscopy. The dorsal mesendoblast produced yolk-endoderm, which proliferated from the anterior of the embryo to cover the dorsal interior. The ventral mesendoblast divided into the primordial endoblast and a cell that further divided into the primordial mesoteloblast and the primordial germ cell. The primordial endoblast divided into left and right endoblasts, which then underwent two teloblastic divisions, leaving behind two pairs of smaller descendants and the larger endoblasts at the dorsal posterior. The endoblasts then paused in cell division while extensive morphogenesis occurred in the ectoderm to form the naupliar segments. Ectoteloblasts formed at the posterior. The primordial mesoteloblast underwent two asymmetric divisions, synchronously with the endoblasts, to form two small descendants and M2 at the ventral posterior. From 15 to 18h, M2 divided laterally then dorsal-ventrally to form four descendants. The results extend the cell lineage of S. ingentis from the egg to the nauplius larva, and demonstrate that endoderm forms dorsal to teloblastic mesoderm from an early stage.     

Hatching envelope formation in shrimp (Sicyonia ingentis) ova: Origin and sequential exocytosis of cortical vesicles

The ova of Sicyonia ingentis lack cortical vesicles at the time of spawning. Within 30 min post-spawning, two populations of cortical vesicles are organized in the ooplasm which, during cortical vesicle exocytosis (cortical reaction), successively release two morphologically different exudates. The first type of cortical vesicles (dense vesicles) appears to be derived from the Golgi complexes after spawning. The second type (the ring vesicles) is formed by the fusion of asternal elements which contain loosely packed ring-shaped structures that are present in the unactivated ova. During exocytosis of the dense vesicles an electron dense material is released which coalesces with the surface coat of the ovum to form a thin hatching envelope which eventually lifts from the ovum's surface. Subsequent to the formation of the thin hatching envelope, the ring vesicles undergo exocytosis resulting in an accumulation of ring-shaped structures in the perivitelline space. These structures coalesce and form an electron translucent layer on the inner surface of the thin elevated envelope to form the thickened hatching envelope. The formation of the cortical vesicles, their exocytosis and the elaboration of the hatching envelope are normally completed within 40-45 min after spawning.     

Mesendoderm cells induce oriented cell division and invagination in the marine shrimp Sicyonia ingentis

The mesendoderm (ME) cells are the two most vegetal blastomeres in the early developing embryo of the marine shrimp Sicyonia ingentis. These two cells enter mitotic arrest for three cycles after the 5th cell cycle (32-cell stage) and ingress into the blastocoel at the 6th cycle (62-cell stage). Circumjacent to the ingressing ME cells are nine presumptive naupliar mesoderm (PNM) cells that exhibit a predictable pattern of spindle orientation into the blastopore, followed by invagination. We examined the role of ME cells and PNM cells in gastrulation using blastomere recombinations and confocal microscopy. Removal of ME progenitors prevented gastrulation. Removal of any other blastomeres, including PNM progenitors, did not interfere with normal invagination. Altered spindle orientations occurred in blastomeres that had direct contact with one of the ME cells; one spindle pole localized to the cytoplasmic region closest to ME cell contact. In recombined embryos, this resulted in an extension of the region of ME-embryo contact. Our results show that ME cells direct the spindle orientations of their adjacent cells and are consistent with a mechanism of oriented cell division being a responsible force for archenteron elongation.     

Development of an anti-vitellin ELISA for the assessment of reproduction in the ridgeback shrimp, Sicyonia ingentis

To investigate the reproductive regulation of the ridgeback shrimp, Sicyonia ingentis, vitellin (Vn) synthesis was studied. Using gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Vn was found to have a molecular mass of 322 kDa and to be composed of three subunits of 182, 91 and 85 kDa. Purified Vn was used to prepare anti-Vn antiserum, which was used to develop an enzyme-linked immunosorbent assay (ELISA) with a dynamic range of 0.3-300 ng. The ELISA was used to measure hemolymph levels of yolk proteins. The mean hemolymph concentrations in fresh caught animals ranged from 256 (+/-36.6 S.E.M.) to 1073 (+/-87.6 S.E.M.) mg/ml in stage 2 and 4 animals, respectively. The ELISA was also used to determine the effects of steroid hormone injections in adult non-reproductive female shrimp. One milligram injections of progesterone, 17alpha-hydroxyprogesterone or 17beta-estradiol were administered for three consecutive days to individual females. There were no changes in hemolymph vitellogenin levels during the successive 7-day period following the first injection of any steroid.     

Cleavage and gastrulation in the shrimp Sicyonia ingentis: invagination is accompanied by oriented cell division.

Embryos of the penaeoidean shrimp Sicyonia ingentis were examined at intervals during cleavage and gastrulation using antibodies to beta-tubulin and DNA and laser scanning confocal microscopy. Cleavage occurred in a regular pattern within four domains corresponding to the 4-cell-stage blastomeres and resulted in two interlocking bands of cells, each with similar spindle orientations, around a central blastocoel. Right-left asymmetry was evident at the 32-cell-stage, and mirror-image embryos occurred in a 50:50 ratio. Gastrulation was initiated by invagination into the blastocoel at the 62-cell-stage of two mesendoderm cells, which arrested at the 32-cell-stage. Further invagination and expansion of the archenteron during gastrulation was accompanied by rapid and oriented cell division. The archenteron was composed of presumptive naupliar mesoderm and the blastopore was located at the site of the future anus of the nauplius larva. In order to trace cell lineages and determine axial relationships, single 2- and 4-cell-stage blastomeres were microinjected with rhodamine-dextran. The results showed that the mesendoderm cells which initiated gastrulation were derived from the vegetal 2-cell-stage blastomere, which could be distinguished by its slightly larger size and the location of the polar bodies. The mesendoderm cells descended from a single vegetal blastomere of the 4-cell-stage. This investigation provides the first evidence for oriented cell division during gastrulation in a simple invertebrate system. Oriented cell division has previously been discounted as a potential morphogenetic force, and may be a common mechanism of invagination in embryos that begin gastrulation with a relatively small number of cells.     

Spermiogenesis in the Marine Shrimp, Sicyonia ingentis

Spermiogenesis in the marine prawn Sicyonia ingentis was examined using transmission electron microscopy. The acrosomal vesicle, derived from the fusion of pro-acrosomal vesicles blebbed from the nuclear envelope, contains the membrane pouches, anterior granule and a spike. The anterior granule is formed from the coalescence of granular aggregates within the proacrosomal vesicles. Primordia underlying the apical acrosomal vesicle membrane polymerize to form a spike approximately 6 μm long. The convoluted pouch membranes arise from the posterior acrosomal vesicle membrane. Lateral and apical portions of the acrosomal vesicle are surrounded by a pentalaminar membrane comprised of the spermatid plasma membrane and the acrosomal vesicle membrane. Subacrosomal structures include the dense saucer plate, granular core and crystalline lattice. These components condense just posterior to the acrosomal vesicle and are separated from the chromatin by a nuclear plate.
The spermatid nucleus becomes surrounded by rough endoplasmic reticulum (RER) and membranous lamellar bodies. RER gives rise to smooth endoplasmic reticulum. These membrane systems degenerate, forming a band of reticular elements around the lateral and posterior portions of the nucleus. The nucleus undergoes condensation followed by decondensation with concomitant breakdown of the nuclear envelope. The resultant chromatin is fibrillar in appearance.     

Peritrophic membrane structure and formation in the larva of a moth, Heliothis

The peritrophic membrane (PM) in tobacco budworm larvae (Heliothis virescens, Lepidoptera: Noctuidae), is a continuous sac which encloses the food bolus in the midgut and hindgut. The PM is a single-walled structure 3-5 mum thick which is comprised of two main layers or laminae. The laminae may be fused into a single structure or remain separated by a space which may contain additional thin strands of matrix. Staining with an anti-PM antibody and wheat germ agglutinin (WGA) illustrate the laminar nature of the PM and suggest that protein and chitin have co-incident spatial distributions within the matrix. By transmission electron microscopy, the PM is composed of a loose network of fibrils and small granules, the only structural difference among laminae being a compaction of the matrix along the edges of the two limiting laminae facing the endoperitrophic and ectoperitrophic spaces. By scanning electron microscopy, the PM surface has a wrinkled, felt-like texture without pores or slits. Contrary to the classical view that lepidopterans are Type I insects with respect to PM formation in which the PM forms along the full length of the midgut, the PM in the tobacco budworm forms primarily from secretions of specialized midgut epithelial cells at the junction of the foregut and midgut. The secretory cells, their secretions and the nascent PM stain intensely with the anti-PM antibody but not with WGA suggesting that chitin is added more posteriorly. The PM may be supplemented by the addition of minor amounts of matrix material along the length of the midgut. PM synthesis begins during embryogenesis prior to the initiation of feeding. The PM in neonates is only about 0.1 mum thick but otherwise is structurally similar to that in older larvae.     

Structure of hematopoietic nodules in the ridgeback prawn,Sicyonia ingentis: Light and electron microscopic observations

The architecture and fine structure of the epigastric hematopoietic nodules of the ridgeback prawn, Sicyonia ingentis, are described. The nodules consist of a highly branched series of tubules that contain the maturing hemocytes within a connective tissue stroma. Hemocytes can exit the hematopoietic nodules by penetrating through fenestrations in the endothelial cell layer into the central hemal space or by migrating through the outer later of capsular cells and associated collagen fibrils. Four hemocyte categories were observed: agranular, small granule with cytoplasmic deposits, small granule without cytoplasmic deposits, and large granule hemocytes. This classification was based upon the presence, size, and type of cytoplasmic granules and the presence of cytoplasmic deposits. Only agranular cells and small granule hemocytes without cytoplasmic deposits appeared capable of division. Intermediate stages were observed between agranular hemocytes and small granule hemocytes with deposits and between small granule hemocytes without deposits and large granule hemocytes, suggesting existence of two distinct hemocyte lines.     

Extracellular Mg2+ induces an intracellular Ca2+ wave during oocyte activation in the marine shrimp Sicyonia ingentis.

In contrast to most systems in which oocyte activation is triggered by the fertilizing sperm, Sicyonia ingentis oocytes are activated by seawater Mg2+ during spawning. S. ingentis oocytes were spawned into Mg(2+)-free seawater and microinjected with the fluorescent Ca2+ indicator Fluo-3 to study the effects of added Mg2+ on intracellular Ca2+ levels. The Mg2+ induced a wave of fluorescence across the oocyte that traveled at a speed of 13 +/- 3 microns/sec. Extracellular Ca2+ was not required for induction of the wave. Treatment with Ca2+ ionophore in Mg(2+)-free medium or a localized injection (0.3% oocyte volume) of 3-5 microM Ca2+ also initiated the wave; injection of 250 mM Mg2+ (up to 1.5% oocyte volume) had no effect. Microinjection of 750 microM EGTA (final) suppressed the Mg(2+)-induced wave, while an identical concentration of EDTA had no inhibitory effect. Subsequent to the initial Mg(2+)-induced intracellular Ca2+ increase, a second Ca2+ increase was observed at approximately 15 min postspawning; the timing of this second increase appeared to be independent of when the Mg(2+)-induced wave was initiated, thus an event associated with spawning may be involved. While oocytes in normal seawater were monospermic, those in Mg(2+)-free seawater were polyspermic, suggesting a role for the Mg(2+)-induced Ca2+ wave in regulating sperm entry into the oocyte.     

Peritrophic membrane structure and formation of larvalTrichoplusia niwith an investigation on the secretion patterns of a PM mucin

Peritrophic membrane or matrix (PM) secretion and formation patterns were examined in the cabbage looper larvae (Trichoplusia ni[Hubner]) by transmission and scanning electron microscopy (SEM). PM first became visible in the lumen between tips of the microvilli and the stomodeal valves as a single layered fibrous structure that became more compact in appearance in the middle and posterior mesenteron. In the anterior mesenteron, nascent PM was visible within the brush border as a fibrous linear structure that contained both the major PM matrix protein, invertebrate intestinal mucin (IIM) and chitin-containing structures. Even though delamination events were confined to the anterior mesenteron, IIM was secreted by columnar epithelial cells throughout the length of the mesenteron. SEM of the midgut epithelium revealed PM covering individual epithelial cells.     

The Morphology and Physiology of the Acrosome Reaction in the Sperm of the Decapod, Sicyonia ingentis.

Sperm of the shrimp, Sicyonia ingentis , undergo a biphasic acrosome reaction consisting of acrosomal exocytosis and acrosomal filament formation. These events are temporally separated by 10–20 min, in vivo. Using egg water preparations the complete reaction can be induced, in vitro, albeit the temporal separation of the two phases is lengthened. External Ca⁺⁺ is required for the exocytotic phase, while a cytoplasmic acidification and K⁺ efflux are associated with polymerization of the acrosomal filament.     

Sperm of the Shrimp Sicyonia ingentis Undergo a Bi-Phasic Capacitation Accompanied by Morphological Changes

Sperm removed from seminal receptacles of female Sicyonia ingentis can be induced to undergo a bi-phasic acrosome reaction (AR), acrosomal exocytosis followed by filament formation, using egg water (EW). Sperm removed from males will not undergo any phase of the AR when incubated with EW, indicating that these sperm undergo a capacitation process after insemination. Freshly molted females (functional virgins) were placed in aquaria with males and monitored for copulation. Mated females were isolated and allowed to carry sperm for specific periods of time. At these time points, sperm were removed and assayed for the ability to undergo the AR using EW. The results indicate that sperm are competent to undergo acrosomal exocytosis after approximately 25 hr, while competency to form acrosomal filaments is not achieved until around 145 hr post-insemination. Morphological examination of sperm removed from males and sperm removed from females revealed dramatic differences. Microscopic evidence indicates that some of the morphological changes seen during capacitation are necessary for the successful completion of the AR.     

Development of cortical vesicles in Sicyonia ingentis ova: their heterogeneity and role in elaboration of the hatching envelope

In the marine shrimp Sicyonia ingentis, ova lack cortical vesicles at spawning. Previous ultrastructural studies suggested that two different populations of cortical vesicles (dense vesicles and the ring vesicles) appear within 30 min post-spawning. These vesicles undergo sequential exocytosis (exocytosis of the dense vesicles followed by exocytosis of the ring vesicles) that leads to the formation of a hatching envelope around the ovum (see Pillai and Clark: Tissue & Cell 20:941-52, 1988). In the present study, lectins were used as molecular probes to study the development of cortical vesicles subsequent to spawning and the role of these vesicles in formation and elaboration of the hatching envelope. Isolated envelopes were screened with 11 different lectins to determine what group(s) were specific to the envelope glycoconjugates; Concanavalin A (Con A), Griffonia simplicifolia (GS II), Lens culinaris (LCA), and wheat germ agglutinin (WGA) bound to the envelopes. FITC-lectin studies of sectioned ova (fixed at various time points after spawning) utilizing WGA and LCA showed different labelling patterns. Data obtained at the light microscopical level indicated that WGA was specific to the dense vesicles and the outer portion of the envelope, while LCA exhibited specificity for the ring vesicles and the inner portion of the envelope. At the ultrastructural level, gold-LCA labelling was seen associated with the cisternal elements (containing ring-shaped structures), ring vesicles, and the inner layer of the fully formed envelope. These data demonstrated that 1) the ring vesicles are formed by fusion of cisternal elements containing ring-shaped structures; 2) the two species of cortical vesicles are chemically heterogeneous; and 3) the components of each type of vesicle contribute to different integral parts (the outer and inner layers) of the hatching envelope.     

Clearance of bacteria injected into the hemolymph of the ridgeback prawn,Sicyonia ingentis (Crustacea: Decapoda): Role of hematopoietic tissue

In an earlier investigation, radiolabelled bacteria injected into the hemolymph of the shrimp Sicyonia ingentis were cleared rapidly from circulation. Most of the bacteria were localized in the gills, followed by other organs including the heart, abdominal musculature, and hematopoietic nodules (HPN). We determined the organ-specific effectiveness of bacterial clearance and found the HPN to be the most effective on a per gram basis. Light and electron microscopy were used to determine the mechanism of clearance in the HPN. The HPN are readily permeable to individual bacteria, which then are recognized and phagocytosed exclusively by small granule hemocytes. Within the first hour, the bacteria are degraded, leaving only whorls of membrane in the phagocytic vacuoles. Subsequently, the hemocytes that ingested bacteria lyse, as has been observed in vitro. These observations support earlier suggestions that hemocytes in the HPN are functionally mature and held in reserve until they are released into circulation. © 1996 Wiley-Liss, Inc.     

Observations on the process of wound repair in penaeid shrimp

The Petersen disk tag is a standard mark for penaeid shrimp, and attachment of the tag involves the insertion of a stainless steel pin through the shrimp's abdomen, resulting in a relatively large puncture wound. The wound healing process first observed at 24 hr post-tagging showed a pronounced hemocytic infiltration of the wound area. Hemocytes in contact with the pin became fusiform, began adhering to one another, and formed several concentric layers around the pin. Scattered foci of bacteria or nercrotic tissue in the vicinity of the would also became encapsulated by concentric layers of fusiform hemocytes, thereby forming nodules. Melanin appeared in association with the layers of hemocytes nearest the pin and in the nodules. Hemocytic infiltration was lollowed by the appearance of fibrocytes and the deposition of collagenlike fibers along the would channel 48 hr after wounding. Involution of epidermis and consequential cuticular involution into the would channel began at 96 hr after wounding. Complete epidermal and cuticular formation along the wound occurrd by 384 hr post-tagging.     

Preloading of micromolar intracellular Ca2+ during capacitation of Sicyonia ingentis sperm, and the role of the pHi decrease during the acrosome reaction.

In studying the mechanism controlling the sperm acrosome reaction (AR) in the marine shrimp Sicyonia ingentis, intracellular Ca2+ and pH were measured using the fluorescent indicators Fura-2 and Fluo-3 for Ca2+, and SNARF-1 for pH. Capacitated sperm possessed an apparent resting Ca2+ concentration of 1-2 microM which remained constant upon induction of the AR with egg water. Uncapacitated sperm had extremely low Ca2+ levels and did not respond to egg water. These results suggest that, while in other species the Ca2+ is elevated to micromolar levels during initiation of the AR, S. ingentis sperm are preloaded with Ca2+ during capacitation and the trigger for the AR is downstream of the Ca2+ increase. The notion that Ca2+ influx is not involved at the actual time of the AR in capacitated S. ingentis sperm is supported by the inability of Ca2+ ionophore A23187 to induce the AR and the ineffectiveness of Ca2+ channel antagonists to block egg water-induced AR. Measurements of capacitated sperm pH showed a significant decrease during the first 10-15 min of the AR, which did not correlate temporally to either acrosomal exocytosis (at 5 min post-induction) or filament formation (after 45 min). Inhibition of egg protease activity required for induction of filament formation did not inhibit the pH drop, indicating that intracellular acidification is not the final trigger for filament formation, although it may be required prior to action of the protease.