Sperm-aggregating factor from Spisula oocytes

A membrane protein possessing sperm-aggregating activity was partially purified from Spisula oocyest. Spisula oocytes were incubated with three different media: A) 1 M urea, 5 mM EDTA, 10 mM Tris-HCI, pH 7.4, B) 1 M urea, 10 mM Tris-HCI, pH 7.4, and C) 5 mM EDTA in artificial sea water. Oocytes incubated in media A or B at 22°C were viable up to 15 min of treatment based on the trypan blue exclusion test. After this treatment period, oocyte viability gradually decreased as demonstrated by a progressive increase in the uptake of the dye. However, oocytes excluded the dye when incubated in medium C for 2 hr or longer. Oocytes incubated in medium A or B did not undergo germinal vesicle breakdown (GVBD) on exposure to sperm, while GVBD was induced on treatment with 70 mM KCI, suggesting removal or alteration of sperm receptors by the treatment. When sperm were incubated with oocyte extract prepared by treatment with medium A or B, they aggregated and formed clusters. The clusters remained unchanged for at least 1 hr at 22–24°C and sperm within the aggreates were motile. Extracts of Spisula oocytes showed species specificity by not agglutinating sperm of Arbacia punctulata, Asterias forbesi, ovalipes ocellatus, or Chaetopterus peramentaceus. The factor was puridied by ammonium sulfate fractionation (30% saturation) and by gel filtration on a Sephadex G 100 column. Four major protein peaks were eluted. Fraction comprising the second and third peaks possessed sperm-aggregating activity at an affective does od 2.5 μg of protein per ml. The factor is a heat-stable protein with an estimated molecular weight (mol wt) of 15 to 25 kdaltons.     

Formation and function of the polar body contractile ring in Spisula

Initial studies suggested that spatial organization of the putative polar body contractile ring was determined by the peripheral aster in Spisula [Biol. Bull. 205 (2003) 192]. Here we report detailed supporting observations, including testing of aster and ring function with inhibitors. The metaphase peripheral aster was confirmed to spread cortically in an umbrella-like pattern, with microtubule-poor center. The aster disassembled during anaphase, leaving the spindle docked at the F-actin-poor center of a newly generated cortical F-actin ring that closely approximated the aster in location, measured diameter range, and pattern. Cytochalasin D and latrunculin-B permitted all events except ring and polar body formation. Nocodazole disassembly or taxol stabilization of the peripheral aster produced poorly defined rings or bulging anaphase asters within the ring center, respectively, inhibiting polar body formation. Polar body extrusion occurred at the ring center, the diameter of which diminished. Ring contractility-previously assumed-was verified using blebbistatin, a myosin-II ATPase inhibitor that permitted ring assembly but blocked polar body extrusion. The data support the hypothesis that peripheral aster spreading, perhaps dynein-driven, is causally related to polar body contractile ring formation, with anaphase entry and aster disassembly also required for polar body biogenesis. Previously reported astral spreading during embryonic micromere formation suggests that related mechanisms are involved in asymmetric somatic cytokinesis.     

Synaptic structure in the visceral ganglion of the lamellibranch mollusc,Spisula solida

Summary An examination using the electron microscope was carried out on the visceral ganglion of the marine bivalve mollusc Spisula solida. A range of fixation, block staining and section staining technique was used to study the structure of chemical synapses. Phosphotungstic acid employed as a block stain specifically stained pre- and post-synaptic structures associated with the membrane at synapses as well as one class of granular vesicle. The specialised contacts were however shown to be rare and in many parts completely absent. Many axons, containing several types of vesicle, were shown to be varicose and it is proposed that they may function in a similar way to the unspecialised varicose terminals of vertebrate autonomic neurons. The role of membrane specialisations in intercellular adhesion is discussed. This study concludes that many synapses may be morphologically unidentified using present criteria.     

Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks

-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of -1,3-glucanase Lu and -1,3-glucanases with different action types—endo--1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo--1,3-glucanase from the terrestrial snail Eulota maakii (LII)—was performed. It was found that -1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of -1,3-glucanase from S. intermedius were more similar to those of the endo--1,3-glucanase from the marine mollusk (LIV) than exo--1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of -1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).     

The influence of dredge design on the catch of Callista chione (Linnaeus, 1758)

To evaluate a possible introduction of a new dredge in the fishery of Callista chione (Linnaeus, 1758), IPIMAR has conducted a study with the objective of comparing the efficiency of two dredges (traditional dredge and the new dredge design) and evaluating their impact on the benthic community. The experiments were carried out during March 1999 on the Southwest coast of Portugal, from a site off Troia. Three different tow durations of 5, 10 and 20 min were investigated. A total of 24 hauls were accomplished, 4 for each tow duration and dredge. The experiments were conducted by attaching a cover bag with a 20 mm mesh to the gear. After each haul, the catches in the bag and in the cover were sorted separately. All individuals retained were attributed scores on a scale of 1–4 in which 1 equates to good and 4 equates to dead. The results obtained showed that catches from the traditional dredge (TD) are composed of a great fraction of juveniles of C. chione, while in the new dredge (NDD) catches are composed, almost entirely, by individuals with a superior size to the minimum legal length (50 mm). This result indicates that the mesh of the bag of the TD used in the exploitation of this resource is not adequate. For the 3 different tow durations, the mean fishing yield obtained for the NDD was always superior to the TD, due to its greater efficiency in capture. The proportion of by-catch is significantly higher when the TD is used. For all 3 tow duration, the TD caused mortalities on the target species and on the macrobenthic community in the same order of magnitude as the NDD. Since the fishery of C. chione is managed by daily quotas per boat, when using the NDD the impact on the macrobenthic community is reduced by about 50% due to its greater efficiency of capture. Another advantage in the usage of the NDD relatively to the TD, is to allow the smallest individuals (independently of the species) to escape rapidly through the metallic bars on the grid, increasing their probability of survival.     

Etude exploratoire de la spermatogenèse induite par la chaleur chez l'escargot Helix aspersa en hibernation: Rôle du cerveau / An exploratory study on temperature-induced spermatogenesis in the hibernating snail Helix aspersa: the role of the brain

Summary

The male cells in the ovotestis of hibernating snails undergo multiplication when the temperature of the environment is raised from 5°C to 25°C. If the temperature is maintained at 25°C for 4 weeks the process of spermatogenesis is completed but the rate of spermatogenesis (DNA synthesis) starts decreasing from the 3rd week (Table 1; Fig. 1).

Brain ablation in hibernating snails maintained at 25°C causes a significant increase in DNA synthesis exclusively in male cells of the ovotestis. This suggests that the brain exerts an inhibitory influence on spermatogenic multiplication. This influence is effective only during the first and the fourth week of exposure of hibernating snails to 25°C (Fig. 1; Table 1) indicating the existence of an endogenous cyclical control. Spermiogenesis is, however, not affected by brain extirpation (Fig. 4 A,B,C).

When reimplanted in the head haemocoel the brain appears normal histologically (Fig. 5 A,B) and it reestablishes the inhibitory influence on DNA synthesis in the ovotestis only during the first day of temperature-induction. During the 1st, 3rd and 4th week the reimplanted brain, deprived of its neural connections, fails to exert its inhibitory influence suggesting that for this influence to function neural connections to the brain are essential. Surprisingly, the implanted brain seems to inhibit DNA synthesis during the 2nd week of temperature-induction (Table 1).

These experiments show that the brain control temperature-induced spermatogenic multiplication in the ovotestis in snails at the onset of hibernation and this control is exerted by one or more inhibitory factors originating from the brain which may function in concert or independently to produce the neuroendocrine effect. It seems therefore justified to consider spermatogenesis in hibernating snails as being neuroendocrinologically controlled.     

Regulated polyadenylation of clam maternal mRNAs in vitro

During meiotic maturation of Spisula oocytes, maternal mRNAs undergo changes in translation and in the length of their poly(A) tails. In general, those mRNAs that are translationally activated, i.e., unmasked become polyadenylated, while deactivated mRNAs lose their poly(A) tails. The activated class of mRNAs encode ribonucleotide reductase, cyclins A and B and histone H3, while the proteins that stop being made include tubulin and actin. Previously, we demonstrated that mRNA-specific unmasking can be brought about in vitro by preventing the interaction of protein(s) with central portions of the 3′ noncoding regions (masking regions) of ribonucle-otide reductase and cyclin A mRNAs. In this report, we show that clam egg extracts are capable of sequence-specific polyadenylation of added RNAs since the 3′ untranslated regions (UTRs) of ribonu-cleotide reductase and histone H3 mRNAs are polyadenylated, while that of actin mRNA is not. In contrast, oocyte extracts, as in vivo, are essentially devoid of polyadenylation activity. We present an initial characterisation of the cis-acting sequences in the 3′ UTR of ribonucleotide reductase mRNA required for polyadenylation. The results suggest that the sequences for cytoplasmic polyadenylation are more complex and extensive than those determined in vertebrates and that they may partly overlap with the masking regions. © 1993 Wiley-Liss, Inc.     

Development and characterization of 30 polymorphic microsatellite markers for the Atlantic surfclam, Spisula solidissima (Dillwyn, 1817)

Thirty polymorphic microsatellite markers were developed for the Atlantic surfclam, Spisula solidissima, from an enriched library and characterized in 24 clams from a wild population. The number of alleles ranged from 3 to 16 per locus. The expected and observed heterozygosities ranged from 0.1942 to 0.9238 and 0.0833 to 0.875 respectively. Six loci showed significant (P < 0.05 after Bonferroni correction) deviation from Hardy–Weinberg equilibrium, probably because of the presence of null alleles. Three primer pairs amplified duplicated loci with two involving tandem mini‐satellite repeats. Most of the microsatellite markers developed here should be useful for genetic studies in this species.     

Isolation and characterization of new microsatellite markers in the surfclam Mactromeris polynyma

We describe primers and amplification conditions for seven microsatellite loci that were developed and characterized in the commercially harvested surfclam Mactromeris polynyma. Five of these loci were polymorphic: we found 6 to 23 alleles per locus among 100 individuals from one population in Nova Scotia, with some large heterozygote deficits that may reflect unrecognized temporal genetic variation. We are using these markers to investigate temporal and spatial genetic structure in this species.     

Additive interactions between the moon snail Euspira heros and the sea star Asterias forbesi, two predators of the surfclam Spisula solidissima

A laboratory experiment was conducted to determine whether the sea star Asterias forbesi and the naticid gastropod Euspira heros feed on surfclams, Spisula solidissima, in an additive or non-additive manner. Predators were allowed to feed on clams with conspecifics and in the presence of the other predator species. Clam mortality (measured as the rate of decline of clam number) and predator feeding rates were noted. To determine the effects of temperature on interactions among the predators, the experiment was conducted at three different temperatures. At all temperatures, feeding rate of each predator was not affected by the presence of the other species, and clam mortality in the presence of both predators was predictable from mortality in the presence of a single predator species. These additive interactions are most likely a result of habitat partitioning between the predators, with naticid snails being infaunal and sea stars being epifaunal. Previous studies in a variety of systems show no clear pattern of occurrence of non-additive interactions. Relatively small differences in predator or prey behavior may be responsible for the presence or absence of non-additive interactions. Received: 6 August 1998 / Accepted: 25 January 1999