MAPK signaling by the D quadrant embryonic organizer of the mollusc Ilyanassa obsoleta

Classical experiments performed on the embryo of the mollusc Ilyanassa obsoleta demonstrate that the 3D macromere acts as an embryonic organizer, by signaling to other cells and inducing them to assume the correct pattern of cell fates. We have discovered that MAP kinase signaling is activated in the cells that require the signal from 3D for normal differentiation. Preventing specification of the D quadrant lineage by removing the polar lobe disrupts the pattern of MAPK activation, as does ablation of the 3D macromere itself. Blocking MAPK activation with the MAP Kinase inhibitor U0126 produces larvae that differentiate the same limited complement of tissues as D quadrant deletions. Our results suggest that the MAP Kinase signaling cascade transduces the inductive signal from 3D and specifies cell fate among the cells that receive the signal.     

Programmed cell death in the apical ganglion during larval metamorphosis of the marine mollusc Ilyanassa obsoleta

The apical ganglion (AG) of larval caenogastropods, such as Ilyanassa obsoleta, houses a sensory organ, contains five serotonergic neurons, innervates the muscular and ciliary components of the velum, and sends neurites into a neuropil that lies atop the cerebral commissure. During metamorphosis, the AG is lost. This loss had been postulated to occur through some form of programmed cell death (PCD), but it is possible for cells within the AG to be respecified or to migrate into adjacent ganglia. Evidence from histological sections is supported by results from a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which indicate that cells of the AG degenerate by PCD. PCD occurs after metamorphic induction by serotonin or by inhibition of nitric oxide synthase (NOS) activity. Cellular degeneration and nuclear condensation and loss were observed within 12 h of metamorphic induction by NOS inhibition and occur before loss of the velar lobes, the ciliated tissue used for larval swimming and feeding. Velar disintegration happens more rapidly after metamorphic induction by serotonin than by 7-nitroindazole, a NOS inhibitor. Loss of the AG was complete by 72 h after induction. Spontaneous loss of the AG in older competent larvae may arise from a natural decrease in endogenous NOS activity, giving rise to the tendency of aging larvae to display spontaneous metamorphosis in culture.     

Selective grazing by the mud snail Ilyanassa obsoleta

Summary Mud snails (Ilyanassa obsoleta) starved for 48 h were allowed to feed on sediments in laboratory microcosms. Sediment cores sliced at 2 mm intervals were compared to snail stomach contents for per cent carbon and nitrogen, plant pigment contents and species composition of benthic diatoms. Concentrations of carbon, nitrogen, phaeopigments, phycocyanin and chlorophyll were enriched in the top 2 mm of the sediments compared to 7–10 mm depth by a factor of 2–10. In turn, these materials were 20–40 times more concentrated in snail guts than in the surface sediments. Snail feces were enriched for carbon and nitrogen by 5–7 times over the surface sediments. Bacterial chlorophyll peaked at about 3–4 mm in the sediments and was not detectable in the snail stomach contents. The C/N ratio of the snail stomach contents was only 6 compared to a ratio of 8.5 for their feces and 12 for the surface sediments.The percentage of migratory diatoms (e.g. Nitzschia and Navicula) decreased with depth where non-migratory species, such as Fragilaria pinnata, dominated. These migratory species were more common in the snails than in the sediments on which they were feeding.A comparison of daily ingestion rates to the animal's energy budget shows that this selective ingestion is sufficient to meet Ilyanassa's energy needs.     

Induction of metamorphosis decreases nitric oxide synthase gene expression in larvae of the marine mollusc Ilyanassa obsoleta (say)

Many marine organisms spend the early part of their lives as larvae suspended in the water column before metamorphosing into benthic reproductive adults. Metamorphosis does not occur until a larva has become competent to respond to appropriate stimuli and after a suitable habitat for the young juvenile has been encountered. The gaseous neurotransmitter nitric oxide is thought to be important in the regulation of metamorphosis by holding the organism in the larval state. We have investigated expression of the neuronal nitric oxide synthase (nNOS) gene in larval and metamorphosing individuals of the marine mud snail Ilyanassa obsoleta. Our results indicate that nNOS is expressed at constant levels throughout larval development. In contrast, expression of nNOS decreases markedly during the first 24 h of metamorphosis. Our observations support previous findings that demonstrate that nitric oxide is present in larvae though competence. The decrease in nNOS gene expression that occurs during metamorphosis corresponds with a previously described reduction in nNOS activity.     

An unusual cytoplasmic organelle in oocytes of Ilyanassa obsoleta

An unusual organelle in the oocytes of the marine mud snail Ilyanassa obsoleta is described and called a polymerosome, because of its several components. Polymerosomes appear at the beginning of vitellogenesis and persist, in part, through postvitellogenesis. The persistent part is similar to an organelle that has been described by other workers in the vegetal region and the polar lobe of the Ilyanassa egg.     

Trematode accumulation by the estuarine gastropod Ilyanassa obsoleta.

The accumulation of larval trematodes by Ilyanassa obsoleta (Gastropoda) at 2 estuarine sites in Delaware was studied. Initial infection status of snails was assayed by looking for shed cercariae. Native snails (most already infected) were deployed at sites A and B, and sentinel snails (putatively uninfected) were deployed at site B. All were individually marked and, if found, reassessed for infection after being free 1-3 summers. Himasthla quissetensis, Lepocreadium setiferoides, Zoogonus rubellus, Austrobilharzia variglandis, and Gynaecotyla adunca infections were observed in recovered snails. At site A, in 1993, 62 natives were recovered. Among the 26 initially testing uninfected, 15 had infections at recovery. Of 36 that released cercariae, 26 had the infection initially indicated. Of sentinels released at site B in 1996, 3 of 16 had infections when recollected. One probably was infected before transplant; at least 2 were infected at site B during 1996-1997. Among site B natives, 26 were later examined by dissection in 1996; 22 had the infection status initially revealed. Some site B natives (n = 35) escaped recapture in 1996 but were found in 1997, or 1998, or both, and were reexamined, most by cercarial release. The same cercariae were produced by 30. Among natives (both sites, n = 123), 27.6% exhibited some difference in infection status compared to the initial assay. This probably overestimates changes. Some differences were real but most can be discounted as cases where initial infection status was misrepresented.     

Infrapopulation sizes of co-occurring trematodes in the snail Ilyanassa obsoleta

This study addresses the infrapopulation sizes of 2 larval trematode species Himasthla quissetensis and Zoogonus rubellus as they co-occur within their estuarine snail host Ilyanassa obsoleta. Rediae of H. quissetensis and sporocysts of Z rubellus were counted in snails singly infected with each parasite and in snails infected with both. Comparisons of the counts indicate that infrapopulations of H. quissetensis were unaffected by co-occurrence with Z rubellus. However, Z. rubellus infrapopulations were reduced when co-occurring with H. quissetensis. It is proposed that this situation does not result from an interspecific interaction between parasite species. Although this double infection is relatively frequent in certain snail populations, it is contended that these trematode species do not co-occur often enough to evolve responses to one another. However, the host environment must be encountered in each life cycle, and both trematode species must be adapted to use it. On this basis, whatever happens when these 2 species occupy the same host is based on adaptations of the parasites to the host. It is proposed that these parasites are adapted to self-limit their infrapopulations in the snail host. They can, thus, preserve and use the host for many years and thereby enhance total cercarial transmission (fitness). Infrapopulation sizes would be determined by host resource levels, which, among other factors, would be influenced by the presence of multiple parasite species. In single infections, by far the most common situation, host resource levels would be set by the nutritional status or age (size) of the host (or both). The reduced infrapopulation sizes of Z rubellus on co-occurrence suggest that this trematode is more sensitive to host resource levels than is H. quissetensis.     

Regulation of Protein Synthesis in Embryos of Ilyanassa obsoleta

Much of the protein synthesis during early development in Ilyanassaresults from the translation of oogenetic mRNA. We show thatmicrotubule proteins are products of this translation, and thattheir synthesis is subject to translation level regulation.We also show that translation level regulation is involved inthe function of the polar lobe by making comparisons of theelectrophoretic patterns of synthesis of ¹⁴C labeled proteinsof normal embryos with the patterns of synthesis of ³H labeledproteins of embryos from which the polar lobes had been removedat the trefoil stage. Controls utilizing biochemical and morphologicalmarkers were performed to assure that normal and delobed embryoswere developing at equivalent rates. The expression of significantdifferences in the patterns of protein synthesis were foundbetween normal and delobed embryos, and these differences werenot dependent upon concomitant RNA synthesis. These differenceswere observable as early as after only 24 hours of development,although organogenesis does not begin until much later in development.Therefore, the observed differences probably reflect determinativeevents. The results support the hypothesis that the developmentaldeterminants of the polar lobe may include specific, preformedmRNA sequences, or specific regulators of translation.     

Effect of sodium dodecyl sulfate on polar lobe formation and function in Ilyanassa obsoleta embryos

Polar lobes, anucleate vegetal pole protrusions formed by Ilyanassa obsoleta embryos, serve as a mechanism for shunting morphogenetic determinants to one cell during the first two cleavages. Polar lobe material becomes segregated in the CD cell during first cleavage and in the D cell during second cleavage, resulting in a very unequal four-cell stage. Larval structures including external shell, foot, operculum, statocysts, and eyes develop only when polar lobe material is present. Treatment with the anionic detergent sodium dodecyl sulfate (SDS) before and during the first cleavage inhibited polar lobe formation and equalized cleavage, as the lobe material was distributed to two cells. No polar lobes formed during second clevage in SDS-equalized embryos, and the four-cell stage consisted of four equal cells with reduced cell contacts. SDS inrreversibly inhibited polar lobe formation without affecting cytokinesis. Although 27% of the larvae from SDS-equalized embryos had one or more lobe-dependent structures duplicated, morphogenesis was impaired: more than 40% of such larvae failed to form shell and/or statocysts. When cells were separated after equalized first cleavage and raised as pairs, the pairs of resulting larvae duplicated lobe-dependent structures with the same frequency as whole equalized embryos. Possible explanations for impaired morphogenesis in SDS-treated embryos are discussed.     

Membrane potential of fertilized eggs of Ilyanassa obsoleta during polar lobe formation and cytokinesis

The membrane potential of fertilized eggs of Ilyanassa obsoleta does not change significantly during the cell shape changes involved in formation and resorption of the third polar lobe and in cytokinesis. The membrane potential is predominantly K⁺-dependent.     

Malate dehydrogenase isozymes of the marine snail,Ilyanassa obsoleta

Malate dehydrogenase (MDH) exists in several isozymic forms in the marine snail, Ilyanassa obsoleta. The isozymes may be classified in two distinct groups, mitochondrial and supernatant. The kinetic properties of these two groups of MDH's resemble those reported for mammalian and avian MDH's except that the relative electrophoretic mobilities of the two groups of isozymes are reversed. The organs of I. obsoleta all contain the same MDH isozymes as revealed by starch gel electrophoresis of organ homogenates. However, quantitative differences in relative abundance were commonly observed, but these differences may be artifacts of preparation since they were not repeatable.All of the supernatant MDH isozymes were apparently of the same molecular weight and were all convertible to a single form by prolonged exposure to 2-mercaptoethanol. This conversion was reversible by removal of the mercaptoethanol. However, the mitochondrial MDH isozymes were not affected by mercaptoethanol. Comparisons of Ilyanassa and pig heart mitochondrial and supernatant MDH isozymes showed parallel responses to mercaptoethanol by the homologous preparations from the two species.     

Myosin and actin are necessary for polar lobe formation and resorption in Ilyanassa obsoleta embryos

 During the first mitotic divisions many spiralian embryos form a cytoplasmic protrusion at the vegetal pole called the polar lobe. In the gastropod Ilyanassa obsoleta the polar lobe is constricted by a contractile ring composed of filamentous actin, myosin, and associated proteins, similar to the contractile ring of the cleavage furrow. To resolve the role of myosin and actin in polar lobe formation and resorption, we have applied 2,3-butanedione monoxime and Latrunculin B at different stages of the first cleavage to inhibit myosin and F-actin, respectively. Our results show that myosin is important for both cytokinesis and polar lobe formation. Additionally, we have found that the resorption of the polar lobe is a two-step process: the first step is passive, driven by the tension of the actin-cortex and the second step is active, in which the ATP-hydrolysis of myosin/actin interaction supplies the force to complete the resorption of the polar lobe. We have summarized our results in a scheme of the first cleavage of Ilyanassa obsoleta. Received: 6 November 1997 / Accepted:15 March 1998     

Polysomes and protein synthesis during development of Ilyanassa obsoleta

Polysomes from various stages of development of Ilyanassa obsoleta have been isolated and their protein synthetic activity determined. Unfertilized eggs have a small population of polysomes active in protein synthesis. Fertilization induces an increase in polysomes resulting in an increase in protein synthesis. Approx. a 5-fold increase in polysomes occurs by the veliger larval stage.     

Effects of local anesthetics on cytokinesis and polar lobe formation in fertilized eggs of Ilyanassa obsoleta

Summary

We have examined the ability of fertilized eggs of Ilyanassa obsoleta to form polar lobe constrictions and undergo cytokinesis in the presence of several local anesthetics and compared these effects with those of drugs known to affect microtubules. Procaine, lidocaine (Xylocaine), mepivacaine, tetracaine, and dibucaine all delay the beginning of polar lobe constrictions at low concentrations and in the order of their lipid solubilities. All of the anesthetics are effective at lower concentrations in the absence of extracellular Ca²⁺. Procaine and tetracaine ‘lock’ cells for several hours halfway through the constriction of the polar lobe neck and prevent subsequent cytokinesis, effects similar to those of the microtubule agents, colchicine and nocodazole. Procaine has no effect on membrane potential, ψ_m, or on intracellular chloride activity, (Cl)_c, as determined with ion-selective microelectrodes. This suggests that procaine does not inhibit cellular shape changes by affecting the ionic activities of the predominant intracellular cation (K⁺) or anion (Cl^?).     

Fate maps of the first quartet micromeres in the gastropod Ilyanassa obsoleta.

Cell fate specification in the gastropod mollusc Ilyanassa obsoleta involves both cell autonomous and inductive mechanisms, which depend on determinants localized first in the polar lobe and then in the D quadrant of the embryo. A complete cell lineage is lacking for this embryo and is essential for a critical interpretation of previous experimental results and an analysis of the mechanisms at the molecular level. Lineages of the first quartet micromeres were followed using Lucifer Yellow dextran as a tracer. The tracer was injected into individual first quartet micromeres using iontophoresis and patterns of fluorescence were analyzed in the larva after 8 days of development. Fluorescence was limited to head structures, including eyes, tentacles and velum. Structures on the left side were derived from 1a and 1d micromeres; 1a gave rise to the left eye, including the lens. Right side structures were derived from the 1c micromere and 1b contributed to the apical plate between the eyes and symmetrically to both sides of the velum. First quartet lineage data are compared with results from previous cell ablation experiments and with lineage data from other species.     

Polyadenylation of nascent RNA during the embryogenesis of Ilyanassa obsoleta.

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated $\text{32}\text{64}$ cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

Serotonin and Nitric Oxide Regulate Metamorphosis in the Marine Snail Ilyanassa obsoleta

Several neuroactive compounds have been implicated as playingroles in the circuitry that controls larval metamorphosis inmarine molluscs. For the caenogastropod Ilyanassa obsoleta,results of neuroanatomical studies suggest that the productionof nitric oxide (NO) increases throughout the planktonic stageand that NO production is necessary for the maintenance of thelarval state, especially as it becomes metamorphically competent.Bath application or injection of exogenous serotonin (5HT) caninitiate metamorphosis in competent larvae, and exogenous NOcan inhibit such serotonergically-induced metamorphosis. Inhibitionof endogenous nitric oxide synthase (NOS) can also trigger larvalmetamorphosis. The production of endogenous NO appears to decreaseconcurrently with the initiation of metamorphosis, but the specificinteractions between serotonergic and nitrergic neurons areunknown. Evidence in support of NO acting to up-regulate theenzyme guanylyl cyclase (GC) is still equivocal. Thus, we donot yet know if NO exerts its effects through the actions ofcyclic 3',5'-guanosine monophosphate (cGMP) or by a cGMP-independentmechanism. The ubiquity of nitrergic signalling and its significancefor developing molluscan embryos and larvae are still the subjectof speculation and require further investigation.