Surf clam

Thurberg, F.P., W.D. Cable, M.A. Dawson, J.R. Maclnnes, and D.R. Wenzloff. 1975. Respiratory response of larval, juvenile, and adult surf clams, Spisula solidissima to silver. Pages 41-52 in J.J. Cech, Jr., D.W. Bridges and D.B. Horton (eds.). Respiration of Marine Organisms. TRIGOM Publ., South Portland, ME. 

Nelson, D.A., J.E. Miller, and A. Calabrese. 1988. Effect of heavy metals on bay scallops, surf clams, and blue mussels in acute and long-term exposures. Arch. Environ. Contam. Toxicol. 17 595-600. 

Hughes, S.E. "Abundance, Quality and Production Fishing Studies on the Surf Clam, Spisula polynyma, in the S.E. 

Maoka, T., Fujiwara, Y., Hashimoto, K., and Akimoto, N. 2007. Characterization of fucoxanthin and fucoxanthinol esters in the Chinese Surf Clam, Mactra chinensis. 

Jorgensen, K., Scanlon, S., and Jensen, L.B. 2005. Diarrhetic shellfish poisoning toxin esters in Danish blue mussels and surf clams. FoodAdd Contam 22(8), 743-751. 

The soft-shell clam (Mya arenaria) burrows deep into the sand, extending its long siphons up in the water. Unlike some other species, the soft-shell clam s siphons will not completely retract into its shells when threatened by predators. The Florida coquina (Donax variabilis), whose siphons are shorter, can pull its siphons inside its shells and close them tightly. Because its siphons are short, this clam cannot burrow very deep into the sand. The Atlantic surf clam (Spisula solidissi-ma) has heavy, thick-lipped shells that are visible at low tide. The hard-shell clam (Mercenaria mercenaria), whose shell is colored purple on the inside, buries itself just below the surface of sand. Jackknife clams (Ensis directus), often found in mudflats, are speedy bivalves that can burrow through the sand faster than most predators can dig them out. 

Blue mussel Soft-shell clam Blue mussel Horse mussel Sea scallop Razor clam Dungeness crab Northern anchovy Blue crab Rock crab Stone crab Spiny lobster Maori scallop Greenshell mussel Pacific oyster New Zealand cockle Chilean oyster Tuata surf clam Mediterranean mussel Sea scallop... 

Like the gastric proteases, digestive cysteine or thiol proteases are active at acidic pH and inactive at basic pH. They are important components of the hepatopancreas of many marine crustaceans, and are responsible for over 90% of the protease activity in the hepatopancreas in short-finned squid (Jllex illecebrosus) (Raksakulthai and Haard, 2001). Cathepsin B is one example of a marine-derived digestive thiol protease. Only a few marine sources have been identified for cathepsin B, including surf clam (Spisula solidissimd), horse clam (Tresus capax), and mussel (Perna perna L.) (Simpson, 2000). 

Quahog, southern, Mercenaria campechiensis, 868 Rangia cuneata 105, 149, 661 Rangia spp., 105, 149, 661 Scrobicularia spp., 767 Softshell clam, Mya arenaria, 148, 149, 182, 558, 595, 867, 868 Strophitis spp., 54 Surf clam, Spisula solidissima, 774 Tapes decussatus, 43 Tellina tenuis, 188... 

Methods for Studying in Vitro Assembly of Male Pronuclei Using Oocyte Extracts from Marine Invertebrates Sea Urchins and Surf Clams... 

Shortly after fertilization, the sperm nuclear envelope is rapidly broken down inside the egg cytoplasm. In vivo, removal of the sperm nuclear envelope has been documented by electron microscopy in both the sea urchin (Longo and Anderson, 1968 Longo, 1976) and the surf clam (Longo and Anderson, 1970). These studies have shown that breakdown of these envelopes is incomplete. A small portion of the nuclear envelope associated with other material appears to... 

Egg extracts developed to date are not capable of disassembling sperm nuclear membranes. For example, sea urchin sperm heads isolated by sonication do not decondense in egg extracts. This may be due to plasma membrane enveloping isolated sperm heads. Thus, artificial permeabilization or solubilization of the sperm head membranes prior to incubation in egg extract is required. This may be achieved by treatment of intact sperm heads with lysolecithin or nonionic detergents such as Triton X-100 (TX-lOO). Procedures for demembranating sea urchin and surf clam sperm nuclei are described in this chapter. 

An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after ultracentrifugation of crude lysates and MVs can be recovered from the pellets. Both cytosolic extracts and MVs can be stored frozen without detectable loss of envelope assembly activity. They can also be manipulated easily by chemical or enzymatic treatments. Such manipulations have enabled the identification of distinct steps of male pronuclear formation and of factors required for each of these steps, notably in Xenopus (Lohka and Masui, 1984 Wilson and Newport, 1988 Vigers and Lohka, 1 1 Boman et al., 1992) and the sea urchin (Cameron and Poccia, 1994 Collas and Poccia, 1995a,b Collas etal., 1995). Studies in the sea urchin and surf clam have indicated that decondensation of sperm chromatin in vitro meets several criteria established by microinjection of sperm nuclei into living eggs (Cothren and Poccia, 1993) and by electron microscopy observations of normal pronuclear formation in vivo (Longo and Anderson, 19( 1970). 

This chapter describes and discusses methods used in our laboratory to investigate each step of male pronuclear formation in a sea urchin egg cytoplasmic extract. When available, procedures previously reported for surf clam male pro-nuclear assembly in vitro are also indicated. These protocols may be applicable to other marine invertebrates as well. 

Surf Clam. Testes are removed from ripe males and held on ice. Sperm are released by mincing and collected dry. To our knowledge, storage of intact sperm has not been reported. 

A cytosolic extract essentially free of MVs can be prepared from the S o extract (Collas and Poccia, 1995a). Cytosolic extracts support sperm chromatin decondensation, histone phosphorylation (Green et ai, 1995), and sperm lamin removal, but not nuclear envelope assembly (Collas et ai, 1995). To our knowledge, no such extracts have been investigated for the surf clam. 

Fixing nuclei in the extract allows convenient observation of a great number of nuclei at many closely spaced time points during pronuclear formation. In the sea urchin, an aliquot of extract can be fixed by adding an equal volume of 7% paraformaldehyde in lysis buffer. A 3-/U.I sample is then placed on a slide as above. In the surf clam (Longo et ai, 1994), the extract is similarly fixed in 3% paraformaldehyde or glutaraldehyde for 1 hr the nuclei are pelleted and washed in PBS and 10 p.1 is mixed with an equal volume of glycerol, sealed with nail polish between a slide and cover slip and observed by fluorescence microscopy. 

The formation of a continuous nuclear envelope as a result of vesicle fusion can be conveniently monitored by the exclusion of a 150-kDa, FTTC-labeled dextran from nuclei (Newmeyer et ai, 1986 Newport and Dunphy, 1992 Cameron and Poccia, 1994 Collas and Poccia, 1995a). FITC-dextran exclusion from nuclei in vitro has been reported in sea urchins but not in surf clams. 

Sea urchin male pronuclei formed in vitro under the conditions described in this chapter remain small ( 4 /tm in diameter) and do not contain a lamina. Pronuclear swelling is promoted only if extra ATP is added to the egg extract. Nuclear swelling has been shown to be associated with, and dependent on, assembly of a nuclear lamina from a cytosolic pool of soluble lamins (Collas et al., 1995). More recent unpublished observations have shown that B-type lamins are also associated with a minor fraction of MV2i3 vesicles. The contribution of these vesicle-associated lamins to the nuclear lamina is under investigation. In the surf clam, sperm chromatin decondensation and pronuclear expansion are continuous processes that occur in parallel with nuclear envelope and lamina assembly in 65-min-activated egg extracts (Longo et al., 1994). Manipulations of this in vitro system to control each of these processes have not been reported. 

Surf Clam. Lamin assembly in clam male pronuclei occurs almost invariably in extracts made from 65-min-activated eggs (Longo et ai, 1994). For chromatin decondensation and nuclear envelope assembly, no exogenous nucleotides are required. These are presumably supplied by the cytoplasmic extract. Approximately half of the decondensed sperm nuclei in unactivated or 15-min-activated extracts assemble variable deposits of lamin. 

Surf Clam. Longo et al. (1994) have examined the Spisula lamin L67 in male pronuclei assembled in vitro. Nuclei are fixed in 3% paraformaldehyde in PBS for 1 hr and washed in PBS as described earlier. Fixed and washed nuclei are applied to a poly(L-lysine)-coated cover slip. Nuclei are reacted for 30 min with a 1 100 dilution of the anti-Spisula L67 Pab227 antibody. Cover slips are washed extensively in PBS, incubated for 30 min in a 1 20 dilution of FITC-conjugated antirabbit antibody, washed in PBS, and mounted in glycerol. 

All steps of male pronuclear formation in vitro can also be visualized by electron microscopy. Procedures have been well established in the frog system (see, for example, Vigers and Lohka, 1991). In the surf clam, in v/fro-assembled nuclei have also been examined by electron microscopy as described in Longo et al. (1994). In the sea urchin, a method for preparing nuclei for electron microscopy has been reported (Collas and Poccia, 1995a). A recent procedure for preparing swollen male pronuclei is described below. 

The procedures reported here have been used to investigate the development of male pronuclei in cell-free systems of sea urchins and, to a lesser extent, surf clams. Pronuclear formation in vitro is a slower process than that in vivo. This property has been used advantageously to examine the steps of pronuclear formation. Each step can be easily manipulated, but the methods described here may require some adjustments for other organisms. Sea urchin and surf clam male pronuclei formed in vitro are virtually complete, with decondensed chromatin, nuclear envelopes, pores, and lamina. 

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