Evaluation of biomarkers in oyster larvae in natural and polluted conditions

Crassostrea gigas D-shaped larvae were subjected to different conditions of temperature and salinity for 24 h and four biomarkers (acetylcholinesterase (AChE) activity, thiobarbituric acid reactive substances (TBARS) levels, glutathione S-transferase (GST) and catalase (CAT) activities) were measured. AChE activity decreased when salinity increased from 25 to 30 and 35 psu at 20 and 25 degrees C. Temperature did not seem to have an influence on AChE activity. TBARS levels increased as a function of salinity when the temperature was maintained at 20 degrees C, whereas at 25 degrees C no effect of salinity could be observed. Variations in GST and CAT activities were not significant with salinity and temperature except that catalase activity was higher at 25 degrees C than at 20 degrees C. Exposure experiments were conducted at 23 degrees C and 30 psu with carbofuran (100 and 1000 microg/l) and malathion (100 and 300 microg/l). There was an inhibition of AChE activity with carbofuran, and a toxic effect shown by an increase in TBARS levels counteracted by increases in GST and CAT activities which protected the larvae. When two pairs of adults producing larvae were taken into consideration, significant differences in biomarker levels were noted between the larval offspring of each pair. Malathion induced a decrease in AChE activity and an increase in CAT activity.     

An antagonistic 5-HT receptor system in the auricles of the systemic heart complex of Sepia officinalis L. (Cephalopoda) shows 5-HT1 and 5-HT4 subtype properties

In pharmacological bioassays on isolated ring-shaped auricle preparations of Sepia officinalis, the action of the specific 5-hydroxytryptamine (5-HT) agonists 8-OH-DPAT (5-HT1a), CP-93129 (5-HT1b), TFMPP (5-HT1b) and RS-67333 (5-HT4) on these autonomously contractile compartments was studied. 8-OH-DPAT and CP-93129 induced mainly positive effects on frequency and tone on the isotonically suspended auricles. The positive effect of 8-OH-DPAT on frequency was blocked by the specific 5-HT1a antagonist NAN-190. The 5-HT1b agonist TFMPP caused similar effects on tone and a positive impact on the auricular amplitude. The highly specific 5-HT4 agonist RS-67333 induced an effect opposite to the action of 5-HT1 agonists inducing mainly negative effects on frequency, amplitude and tone, causing a diastolic standstill at a concentration of 10(-6) M. These negative effects were blocked by the adenylyl cyclase inhibitor SQ-22,536 in the absence of a diastolic standstill. The opposing action of 5-HT1 and 5-HT4 agonists on auricular contractile activity suggests that an antagonistic 5-HT-receptor system exists within the auricular myocardial cells of S. officinalis, probably consisting of 5-HT1- and 5-HT4-like subtypes. The results also suggest that adenylyl cyclase acts as the intracellular target enzyme of both signal transduction mechanisms.     

A putative hyperglycemic factor from the cerebral ganglia of Otala lactea (Mollusca: Pulmonata)

Mantle tissue pieces from adult Otala lactea continuously synthesized glycogen over a 72-h incubation period. Acid-saline extract of the cerebral ganglia inhibited glycogen synthesis by mantle tissue in vitro. This effect was dose-dependent. The glycogen reduction factor from the cerebral ganglia was heat stable, protease sensitive, and relatively hydrophobic. The cerebral ganglia extract also stimulated mantle glycogen phosphorylase in vitro in a dose-dependent manner. The results suggest the presence of a hyperglycemic factor in the cerebral ganglia of Otala. The molecular weight of this factor, estimated by size-exclusion chromatography, was approximately 10,000. Mammalian glucagon had no significant effect on glycogen synthesis by the mantle pieces. Accepted: 17 January 2000     

Structure and function of invertebrate 5-HT receptors: a review

Over the last decade, knowledge of invertebrate serotonin receptors has expanded greatly. The first 5-HT receptor from Drosophila was cloned 10 years ago, and subsequently, 11 additional receptor genes have been cloned from Drosophila, molluscs (Lymnaea and Aplysia) and nematodes (Caenorhabditis and Ascaris). Information has also accumulated from physiological and biochemical studies that have used vertebrate serotonergic ligands to characterize endogenous invertebrate receptors. Although the endogenous receptors are often classified according to mammalian-based categories, in many cases the pharmacological properties of vertebrate and invertebrate receptors differ significantly and the actual identity of the latter is questionable. By providing information on the gene structure and amino acid sequence, molecular cloning studies offer a more definitive way to identify and classify invertebrate 5-HT receptors. This review summarizes information on the pharmacological and transductional properties of cloned invertebrate 5-HT receptors, and considers recent studies of endogenous receptors in the light of this new data.     

Identification of stimuli and input pathways mediating food-attraction conditioning in the snail, Helix

Snails become conditioned by a single feeding episode to locate foods which they were unable to locate prior to feeding. To identify which of the different stimulus parameters of the food mediate learning, snails were presented with isolated stimulus components during feeding and re-tested the next day for their ability to locate the food. None of the individual components was sufficient to promote conditioning. Odor combined with a bulk stimulus conditioned the animals, as indicated by their subsequently locating the food. Elimination of the olfactory sensory inputs from the anterior and/or posterior tentacles prior to conditioning revealed that the acquisition of the olfactory memory requires olfactory stimulation of the sensory epithelia on the anterior tentacles. Recall of memory during olfactory orientation requires functional epithelia on the posterior tentacles, which suggests that the same odor is processed by different input pathways under different situations. Animals with the olfactory epithelia functional on the same side during conditioning and food searching were able to locate the conditioned food. Animals with different epithelia functional during conditioning and food searching failed, which suggests that olfactory memory is stored within one side of the nervous system and cannot be accessed from the contralateral side. Accepted: 28 November 1997     

Long-term affected flat oyster (Ostrea edulis) haemocytes show differential gene expression profiles from naïve oysters in response to Bonamia ostreae

European flat oyster (Ostrea edulis) production has suffered a severe decline due to bonamiosis. The responsible parasite enters in oyster haemocytes, causing an acute inflammatory response frequently leading to death. We used an immune-enriched oligo-microarray to understand the haemocyte response to Bonamia ostreae by comparing expression profiles between naïve (NS) and long-term affected (AS) populations along a time series (1 d, 30 d, 90 d). AS showed a much higher response just after challenge, which might be indicative of selection for resistance. No regulated genes were detected at 30?d in both populations while a notable reactivation was observed at 90 d, suggesting parasite latency during infection. Genes related to extracellular matrix and protease inhibitors, up-regulated in AS, and those related to histones, down-regulated in NS, might play an important role along the infection. Twenty-four candidate genes related to resistance should be further validated for selection programs aimed to control bonamiosis.     

Ultrastructural localization of esterase and acid phosphatase in digestive gland cells of fed and starvedCepaea nemoralis (L.) (Mollusca: Helicidae)

Summary The ultrastructural localizations of thiolacetic acid esterase, indoxyl acetate esterase and acid -glycerophosphatase have been studied in the digestive gland cells of fed and starvedCepaea nemoralis. In fed snails the major localization of all three enzymes was in the green granule vacuoles of digestive cells. In addition, the cytoplasm of calcium cells and the Golgi apparatus and GERL (?) of all cell types were acid phosphatase positive. Many digestive cells of starved snails showed a similar enzyme distribution to that found in fed snails but other digestive cells showed a very high cytoplasmic activity of all three enzymes. It is suggested that these cells are in the process of autolysis. New light is also thrown on the process by which food is transported from the digestive gland lumen to the phagosomes of digestive cells.     

Cryopreservation of Crassostrea gigas vesicular cells: viability and metabolic activity

Cryopreservation is widely used for long-term conservation of various tissues, embryos or gametes. However, few studies have described cryopreservation of invertebrate primary cell cultures and more particularly of marine invertebrate somatic cells. This technique would however be of great interest to facilitate the study of various metabolic processes which vary seasonally. The aim of the present study was to develop a protocol for cryopreservation of Crassostrea gigas vesicular cells. Different parameters were adjusted to improve recovery of cells after freezing. The most efficient cryoprotectant agent was a mix of Me(2)SO, glycerol, and ethylene glycol (4% each). The optimal cooling rate was -1 degrees Cmin(-1) down to -70 degrees C before transfer into liquid nitrogen. In these conditions the percentage of viable cells reached 70% of the control. The glucose metabolism of thawed cells was evaluated using radioactive glucose as a tracer. Immediately after thawing, glucose uptake involving membrane transporters was greatly reduced (24% of control) whereas glucose incorporation into glycogen was less affected (68% of control).     

Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia)

The aim of this study is to describe the early stages of spermatogenesis of the Pacific oyster Crassostrea gigas using both light and electron microscopy. The gonad is formed by gonadal tubules invaginated in a connective tissue constituting a storage tissue. Myoepithelial cells surround each gonadal tubule and are associated with an acellular matrix delimiting the outer part of the tubule, the inner part is composed by intragonadal somatic cells associated with germinal lineage. Two types of spermatogonia are identified, where type I spermatogonia (Spg I) are large, scarce and pale cells leaned against the base of the tubule (nuclear diameter: 5.5+/-0.5 microm). Type II spermatogonia (Spg II) are clustered and dark cells which appear smaller than type I (nuclear diameter: 4.3+/-0.3 microm). The aspect of nuage-like material in cytoplasm is described from pale spermatogonia to primary spermatocytes (nuclear diameter: pachytene 3.6+/-0.3 microm, diplotene 3.4+/-0.3 microm), while no structure related to a chromatoid body was observed in oyster spermatocytes and spermatids.