Snail Ilyanassa Obsoleta Research Articles

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.

Kinetic characterization of the inhibition of acyl coenzyme A:Steroid acyltransferases by tributyltin in the eastern mud snail ( Ilyanassa obsoleta)

Exposure to tributyltin (TBT) has been causally associated with the global occurrence of a pseudohermaphroditic condition called imposex in neogastropod species. TBT elevates free testosterone levels in these organisms, and this upsurge in testosterone may be involved in the development of imposex. We investigated the ability of TBT to inhibit acyl coenzyme A:testosterone acyltransferase (ATAT) activity as well as microsomal acyl-coenzyme A:17β-estradiol acyltransferase (AEAT) in a neogastropod, the eastern mud snail Ilyanassa obsoleta as a mechanism by which TBT elevates free testosterone. TBT significantly inhibited both ATAT and AEAT activities in vitro at toxicologically relevant in vivo concentrations. Kinetic analyses revealed that TBT is a competitive inhibitor of ATAT ( K i = ∼9 μM) and is a weaker, noncompetitive inhibitor of AEAT ( K i = ∼31 μM). ATAT and AEAT activities associated with different microsome preparations were significantly correlated, and 17β-estradiol competitively inhibited the fatty acid esterification of testosterone suggesting that one enzyme is responsible for biotransforming both testosterone and 17β-estradiol to their corresponding fatty acid esters. Overall, the results of this study supply the much-needed mechanistic support for the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting their major regulatory process for maintaining free testosterone homeostasis—the fatty acid esterification of testosterone.

Seasonal variation in the regulation of testosterone levels in the eastern mud snail (Ilyanassa obsoleta)

Abstract. Some gastropods appear to utilize a strategy for regulating testosterone levels that is atypical of such processes thus far identified in other metazoans. While most animals convert testosterone largely to polar derivatives that are readily eliminated from the organism, the mud snail Ilyanassa obsoleta converts testosterone to apolar testosterone‐fatty acid esters that are retained by the organism. In the present study, we tested the hypothesis that fatty acid esteri‐fication serves to store testosterone and that stored testosterone‐fatty acid ester pools vary through the reproductive cycle of the snail. This hypothesis was tested by 1) modulating total testosterone levels in individual snails and measuring the amount of the hormone stored as the fatty acid ester relative to the amount retained as free steroid and 2) measuring changes in esterified testosterone in a field population of snails during their reproductive cycle. Adult snails were experimentally manipulated to contain from ∼36–300% the level of total testosterone measured in unmanipulated individuals. The amount of testosterone‐fatty acid ester present in these organisms increased in direct proportion to the level of total testosterone, while, free testosterone levels remained relatively constant. These observations suggest that free testosterone levels are regulated in the snail by fatty acid esterification/de‐esterification processes. Among field sampled snails, testosterone existed predominantly in the free, non‐esterified form at the onset and end of the egg laying period. At other times, the majority of testosterone was sequestered as fatty acid esters. This study provides compelling evidence that free testosterone levels in the mud snail are regulated through fatty acid esterification/de‐esterification processes and this regulatory function contributes to seasonal fluctuations in free testosterone levels.

A bibliography of the marine mud snail Ilyanassa obsoleta

Summary A bibliography of the marine mud snail Ilyanassa obsoleta has been compiled and organized under biological subtopics, dissertations, and gene sequences.

Infrapopulation Sizes of Co-Occurring Trematodes in the Snail Ilyanassa obsoleta

Infrapopulation Sizes of Co-Occurring Trematodes in the Snail Ilyanassa obsoleta

Regulation of metamorphosis in ascidians involves NO/cGMP signaling and HSP90.

Treatment of larvae of the ascidians Boltenia villosa (Family: Pyuridae) and Cnemidocarpa finmarkiensis (Family: Styelidae) with drugs that inhibit the function of the molecular chaperone HSP90 increased the frequency of tail resorption, the primary morphogenetic event of metamorphosis. If treatment was initiated at hatching, metamorphic events subsequent to tail resorption failed to occur, indicating an ongoing role for HSP90 during morphogenesis. Removal of tails from heads of mature, but not newly hatched larvae, induced metamorphosis of the head. Decapitation experiments indicate that the capacity of tails to shorten in response to inhibition of HSP90 function requires communication with heads. To identify candidate proteins with which HSP90 may interact to regulate metamorphosis, we noted that in mammalian cells, nitric oxide synthase (NOS) interacts with HSP90 and its activity is sensitive to drugs that inhibit HSP90 function. In addition, nitric oxide (NO) signaling in the marine snail Ilyanassa obsoleta is an important regulator of metamorphosis. Inhibition of NOS activity in these ascidian larvae with L-NAME increased the frequency of metamorphosis, consistent with a putative interaction of NOS and HSP90. NOS is present in tail muscle cells, implicating them as targets for the drug treatments, consistent with the decapitation experiments. Inhibition of soluble guanylyl cyclase, the most common effector of NO signaling, also increased the frequency of metamorphosis. In contrast to treatment with anti-HSP90 drugs, metamorphosis induced with L-NAME or ODQ was complete. The results presented suggest that an HSP90-dependent, NO-based regulatory mechanism localized in tails represses ascidian metamorphosis. We discuss these results in relation to the induction of ascidian metamorphosis by several unrelated agents.

Biotransformation and Disposition of Testosterone in the Eastern Mud Snail Ilyanassa obsoleta

Elevated testosterone levels have been reported to be associated with imposex (pseudohermaphroditism), the superimposition of male characteristics such as a penis and vas deferens on female gonachoristic snails. Tributyltin (TBT), a marine biocide in anti-fouling paints, is a known causal agent of imposex. Evidence suggests that imposex is elicited by TBT-mediated changes in the biotransformation and disposition of testosterone. To identify potential targets of TBT in gastropod species susceptible to imposex, biotransformation and disposition of testosterone in normal individuals must first be characterized. Nonimposex mud snail, Ilyanassa obsoleta, readily extracted [14C]testosterone, added to aqueous media, and converted the testosterone to at least five apolar conjugates designated AP1 through AP5. All were retained by the organisms. No significant amount of [14C]testosterone was retained or eliminated as polar metabolites. Following enzymatic hydrolysis of the most abundant metabolite (AP1), free fatty acids and [14C]testosterone were liberated. Furthermore, AP1 was produced when homogenized snail tissue was incubated with [14C]testosterone and oleoyl coenzyme A or palmitoyl coenzyme A. These results indicate that AP1, which represents over 70% of the testosterone biotransformation products, is a fatty acid ester of testosterone. Apolar metabolites AP2–AP5 might represent testosterone derivatives that are multiply conjugated to fatty acid molecules. Fatty acid conjugates of testosterone have not been previously described in the gastropods. The esterification of testosterone to fatty acids might be a mechanism where by steroid titers are regulated and could represent a target of TBT toxicity.

CONSUMPTION OF ULVA LACTUCA (CHLOROPHYTA) BY THE OMNIVOROUS MUD SNAIL ILYANASSA OBSOLETA (SAY)

Marine invertebrate grazing on temperate macroalgae may exert a significant “top‐down” control on macroalgal biomass. We conducted two laboratory experiments to test (1) if consumption by the omnivorous mud snail Ilyanassa obsoleta (Say) on the macroalga Ulva lactuca Linnaeus was a function of food quality (nitrogen content) and (2) if grazing on benthic macroalgae occurred at significant rates in the presence of alternative food sources in the sediment (detritus, larvae, benthic microalgae). Grazing rates were higher for N‐enriched macroalgae; however, all snails lost weight when grazing on macroalgae alone, indicating that U. lactuca was a poor food source. The presence of sediment from two sites, a sandy lagoon and an adjacent organic‐rich muddy tidal creek, did not affect consumption of macroalgae in microcosm experiments, and the grazing snails were capable of significantly reducing macroalgal biomass associated with both sediment types. Grazing rates by this omnivore were as high as 10.83 mg wet weight·individuals−1·d−1 and were similar to those recorded for herbivorous species. In situ loss rates calculated from average grazing rates per individual and snail abundances (up to 3.5 g dry weight·m−2·d−1) also were comparable with those calculated for herbivorous species. This level of grazing could remove up to 88% of new macroalgal growth at the lagoon site where the N supply was relatively low but had a much smaller effect (18% of new growth) at the high‐nutrient creek site. Snails facilitated macroalgal growth at both sites by increasing tissue N content by 40%–80%. Consumption and digestion of macroalgae aided in the recycling of nutrients temporarily bound in the algae and resulted in enrichment of surficial sediments. Increased N sequestration in the sediments also was associated with an interruption of snail burrowing behavior due to persistent anoxia in sediments rich in decaying algal material. Our data suggest that in shallow lagoons where mud snails and benthic macroalgae coexist, grazing may influence N retention in macroalgal biomass.

Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta.

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.

Effect of salinity reduction on oxygen consumption by larval estuarine invertebrates

Unpredictable events can cause rapid sizable changes in environmental conditions. Storm events are an example of an unpredictable event; in estuarine habitats, storms can bring about drastic changes in salinity levels within several hours. This study focused on the effect of salinity reduction on larval oxygen consumption. Two species of larval invertebrates were exposed to salinity reduction, the marine polychaete Arenicola cristata Stimpson and the mud snail Ilyanassa obsoleta (Say). In experimental treatments, salinity was reduced to 10 or 15‰; control individuals were maintained at 30‰. Salinity was reduced for 3 d when larvae were between the ages of 1 and 4 d, post-hatch. Oxygen consumption was the same among treatments during salinity reduction for I. obsoleta larvae. The oxygen consumption of A. cristata larvae was significantly lower at 10‰S compared with controls and with salinity reduction to 15‰; larvae eventually died at 10‰. After salinity reduction ceased, larvae exposed to a salinity reduction to 15‰ consumed more (A. cristata) or the same (I. obsoleta) amount of oxygen as individuals maintained at 30‰. We have shown in previous experiments that salinity reduction results in significant reductions in larval growth in A. cristata and I. obsoleta larvae as well as in changes in developmental rates of A. cristata (but not I. obsoleta) larvae. Taken in conjunction with the results of our present study on larval oxygen consumption, the results suggest that salinity reduction has a large impact on estuarine invertebrates.

Metamorphosis in the Marine Snail Ilyanassa obsoleta, Yes or NO?

Metamorphosis is a crucial life-history event that can change an organism's form, function, behavior, and ecological interactions. In the Mollusca, several neurotransmitters and neuromodulators play inductive or inhibitory roles in the pathways that govern larval metamorphosis. Nitric oxide (NO) has been implicated in developmental processes in vertebrates and arthropods, but not previously in molluscs. We determined that NO donors block pharmacologically induced metamorphosis in the mud snail Ilyanassa obsoleta, whereas injections of inhibitors of nitric oxide synthase (NOS) allow competent larvae to become juveniles. We describe a new developmental role for NO, as an endogenous inhibitor of molluscan metamorphosis.

Resuspension of postlarval soft-shell clams Mya arenaria through disturbance by the mud snail Ilyanassa obsoleta

Transport and mortality of newly settled post larvae potentially have a large influence on the population dynamics and adult distributions of coastal benthic species, including the soft-shell clam Mya arenaria. Post-settlement transport typically occurs when boundary shear stresses are high enough to resuspend the surface sediments in which the small clams reside. The objective of the present study was to examine the effect of disturbance by the mud snail Ilyanassa obsoleta on the hydrodynamic transport of recently settled M. arenaria. Laboratory flume experiments showed that disturbance by activities of I. obsoleta caused suspension of small clams (1.8 and 2.3 mm) at boundary shear velocities (1.0 and 1.3 cm s -1 ) that were too slow to suspend undisturbed clams. In shear velocities high enough to cause bulk sediment transport (1.4 and 2.0 cm s -1 ), more clams were suspended in the presence of snails than in their absence. Manipulative field experiments using cages to exclude snails demonstrated that abundances of juvenile M. arenaria (year- 1 recruits) were lower in sediments where snails were present than where snails were absent. These results suggest that biological disturbance, such as that imposed by activities of mobile, benthic deposit feeders, may play an important role in postlarval transport and, eventually, in the adult distributions of infaunal bivalves.

Shell differentiation and engrailed expression in the Ilyanassa embryo.

We have used in situ hybridization and immunocytochemistry to study the expression of the engrailed-related gene, Ily-en in embryos of the marine mud snail Ilyanassa obsoleta. We find that Ily-en is only expressed in shell gland cells. Only mRNAs localized in the shell gland hybridize to an antisense probe of the Ily-en homeobox. Similarly, only shell gland cells or shell-forming cells are stained by the monoclonal antibody 4D9, which was raised to the engrailed-class protein from Drosophila. Ilyanassa embryos made deficient in vegetal cytoplasm by removing the third polar lobe fail to differentiate an organized external shell. They do however make some randomly oriented internal shell fragments in which Ily-en is expressed. Because Ily-en is expressed in shell gland cells of both normal and lobeless embryos, we conclude that the determinant(s) required for Ily-en expression are not exclusively localized in the polar lobe.

Serotonin Injections Induce Metamorphosis in Larvae of the Gastropod Mollusc Ilyanassa obsoleta.

Bath-applied serotonin (5-HT) induces competent larvae of the marine snail Ilyanassa obsoleta to metamorphose. Previously, the mode of action of 5-HT, whether as an external ligand or as an internal neurotransmitter, was unknown. Larvae were injected with 10-4 M 5-HT and other pharmacological agents to provide evidence that serotonergic neurons are necessary for metamorphosis in Ilyanassa larvae and that serotonin functions as a neurotransmitter or neuromodulator during this process. About 50% of 5-HT-injected animals metamorphose within 48 hours. Fluoxetine, a 5-HT reuptake inhibitor, and alpha-methyl-5-hydroxytryptamine (αm5HT), a 5-HT agonist, were also effective inducers of metamorphosis. Gramine (3-[dimethylaminomethyl]indole), a 5-HT antagonist, inhibited the inductive activity of 5-HT, while the amino acid gammaaminobutyric acid (GABA) resulted in rates of morphological restructuring similar to those of controls. Collectively, the results of our experiments support the idea that serotonergic neurons are active during larval metamorphosis of Ilyanassa and that 5-HT does not induce metamorphosis by binding to epidermal chemoreceptors.

Short-term fluctuations in salinity:effects on planktonic invertebrate larvae

Estuarine habitats are characterized by environmental changes on many time scales. While certain physical parameters change on a regular, predictable basis, rapid environmental fluctuatlons can also occur as a result of stochastic events. For example, rainstorms are frequent, unpredictable events that can cause dramatic changes in the estuarine environment, in short periods of time As a result of storm precipitation and terrestrial freshwater runoff, estuarine sallnity can drop in excess of 2 0 ? ~ in 6 h. Our research focuses on the effects of these storm-induced salinity fluctuations on estuanne embryos and larvae in terms of growth, mortality, rate of development, and activity. We studied 2 species of estuarine invertebrates: the embryos and larvae of the mud snail Ilyanassa obsoleta and the larvae of the marine polychaete Arenicola cristata. In both species, greater reductions in salinity resulted in smaller larvae. Both the duration of salinity reduction and the age of embryos and larvae when exposed to a sallnity reduction affected growth rates: longer and earlier (relative to embryonic or larval age) storms had a greater effect on growth rates, resulting in smaller larvae. Both species also exhibited changes in activity when exposed to salinity reductions: I. obsoleta larvae swam significantly less at 10'!:*), while A. cristata larvae displayed a delay in activities associated with the sequence of development from swimm~ng trochophore, to crawling larva, to settled metamorphic juvenile. Our results show that stochastic events may have a significant impact on the growth and development of pre-settlement estuarine invertebrates, implying ecological effects on recruitment success.

Sexual anomalies in the estuarine snail Ilyanassa obsoleta: imposex in females and associated phenomena in males.

The spatial distribution of the "imposex" phenomenon (the imposition of male characters on females) among populations of Ilyanassa obsoleta in southern Delaware, USA estuaries was studied. Imposex was most frequent among females in a segment of the Lewes and Rehoboth Canal (68%, n=376) and there was significant spatial heterogeneity of imposex frequencies (range=43-98%) even within this habitat. Two lines of evidence indicate that conditions which generated female imposex (organotin molecules in the water) also affected male sexuality: 1) healthy males normally lose the penis following reproduction but they tended to retain it where imposex-inducing conditions were strong; and 2) males parasitized by trematodes usually lose the penis but tended not to under strong imposex-inducing conditions. The altered biology associated with imposexinducing environments extends beyond female I. obsoleta to males and to other species. This must be recognized when studying organisms exposed to these conditions.

H1 histone subtypes and subtype synthesis switches of normal and delobed embryos of Ilyanassa obsoleta

Histone H1 subtype complexity and H1 histone subtype synthesis switches were characterized during the development of normal embryos of the mud snail Ilyanassa obsoleta. The effect of the removal of the third polar lobe on the normal H1 pattern of synthesis was then investigated in the delobed embryo to determine if classical polar lobe effects are accompanied by a perturbation of these patterns. SDS-gel electrophesis and fluorography of radiolabeled 5% perchloric acid-soluble nuclear extracts resolved six H1 proteins designated bands 1–6. Bands 1–5 migrate as a cluster of individual bands with similar mobilities. Band 6 has a substantially slower mobility. The synthesis of band 6 is predominant during the first 6 hr post-trefoil. During cleavage and gastrulation bands 1 and 2 are predominant while band 3, 4, and 5 become predominant during organogenesis. In addition, it has been found that removal of the polar lobe delays the off-switch of the early bands 6, 1, and 2 and the on-switch of the late bands 3, 4, and 5. This must result in a different H1 composition in the chromatin of the two embryo types. Cell number data of normal and delobed embryos reveal that the delay in subtype synthesis switching is not caused by an overall delay of cell division in the delobed embryo. However, the data indicate that a subpopulation of cells may not divide, or may divide late, in the delayed embryo. The data also suggest that the D cell lineage may be involved in the control of histone synthesis switching in the A, B, and C cell lineages.

Disturbance, emigration, and refugia: How the mud snail, Ilyanassa obsoleta (Say), affects the habitat distribution of an epifaunal amphipod, Microdeutopus gryllotalpa (Costa)

In the presence of the mud snail Ilyanassa obsoleta (Say), the tubicolous amphipod Microdeutopus gryllotalpa (Costa) emigrates to snail-free sediments, as demonstrated in laboratory and field experiments. Emigration occurs predominantly in the dark when the amphipod is most active. Unlike crevices, the thickness of sediments in which the amphipod is established offers no protection from snail disturbance. Emigration is shown to be caused by the disturbance generated by the snail's plowing and burrowing across the sediment surface, and not a response to a reduction in the shared microfloral food supply. The crawling and burrowing of the smaller mud snail, Hydrobia totteni Morrison, does not disturb Microdeutopus, supporting the hypothesis that relative body sizes affects the ability of bioturbators/burrowers to disturb tube-dwellers. As the burrows of Microdeutopus extend only ≈2 cm below the sediment surface, thick mud layers do not offer any refuge from Ilyanassa. However, very small solid surfaces (≈1–2 mm in relief) to which the amphipods build tubes do provide some protection from Ilyanassa. In soft-sediment benthic communities, such small structures may provide significant refuge for small epifauna and shallow burrowing infauna escaping from small-scale, biogenic disturbance.

FORMATION, ORGANIZATION, AND COMPOSITION OF THE EGG CAPSULE OF THE MARINE GASTROPOD, ILYANASSA OBSOLETA.

Embryos of the marine mud snail Ilyanassa obsoleta undergo early development within an egg capsule. After about a week of encapsulation, embryos hatch by releasing a chemical substance that removes the plug found at the apex of a capsule. However, the mechanism of action of this hatching substance remains poorly understood. To study how the hatching substance functions, we examined the composition of the egg capsule, particularly the plug region, to determine what the "substrate" of the hatching substance might be. We have also examined the formation and organization of the egg capsule to determine the origin and identity of the regions of a capsule that the hatching substance must remove. The results show that the Ilyanassa egg capsule is organized into four layers, the outer three of which are composed of protein and carbohydrate. Portions of the two inner layers of the capsule wall extend into the capsule apex and form the plug, which is dissolved by the hatching substance. The isolated capsule plug region contains three major glycoproteins resolved on sodium dodecyl sulfate-polyacrylamide gels. Therefore, the hatching substance may be a protease similar in action to the enzymes released by many other embryos at hatching.

COMPETITIVE DISPLACEMENT OF NATIVE MUD SNAILS BY INTRODUCED PERIWINKLES IN THE NEW ENGLAND INTERTIDAL ZONE.

During the nineteenth century the mud snail Ilyanassa obsoleta was abundant on sand and mud flats, wood works, sea walls, salt marshes, eel grass beds, and cobble beaches in New England. With the exception of sand and mud flats, these habitats are now largely occupied by the introduced periwinkle, Littorina littorea. To determine whether Littorina competitively displaces Ilyanassa, an experimental study was conducted at a site in Barnstable Harbor, Massachusetts where the observed distributions overlapped by 3% by Morisita's index. Mark-recapture studies suggested that the distribution of Littorina was limited by an abiotic factor, currents, through which this species realized its fundamental niche. In contrast, density manipulations demonstrated that Ilyanassa emigrated from areas where Littorina exceeded densities of 2 to 5 per 0.25 m2. Littorina limited the upper and lower distribution of Ilyanassa and affected its microhabitat distribution in the mid intertidal zone. Habitat displacement was 70% for Ilyanassa, calculated as the difference between Ilyanassa's observed distribution and its distribution during littorinid removal experiments. The two species display reciprocal niche overlap with each possessing an exclusive region from which the other is physically restricted. The results suggest that the historical change in the distribution of Ilyanassa was due to competitive exclusion by introduced Littorina.