Metabolic adjustments in the oyster Crassostrea gigas according to oxygen level and temperature

The purpose of this study was to examine the responses of the oyster Crassostrea gigas to oxygen levels at subcellular and whole organism levels. Two experiments were carried out. The first experiment was designed to measure the clearance and oxygen consumption rates of oysters exposed at different concentrations of oxygen at 15, 20 and 25°C for 20 h. The goal of this first part was to estimate the hypoxic threshold for oysters below which their metabolism shifts towards anaerobiosis, by estimating the oxygen critical point (PcO₂) at 15, 20 and 25°C. The second experiment was carried out to evaluate the metabolic adaptations to hypoxia for 20 days at three temperatures: 12, 15 and 20°C. The metabolic pathways were characterized by the measurement of the enzymes pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK), the alanine and succinate content and the adenylate energy charge. Respiratory chain functioning was estimated by the measurement of the activity of the electron transport system (ETS). The values of PcO₂ were 3.02±0.15, 3.43±0.20 and 3.28±0.24 mg O₂ l^-1 at 15, 20 and 25°C, respectively. In whole oysters, hypoxia involved the inhibition of PK whatever the temperature, but PEPCK was not stimulated. Succinate accumulated significantly only at 12°C and alanine at 12 and 15°C. A negative relationship between the PK activity and the alanine content was only found in hypoxic oysters. Finally, hypoxia increased significantly the activity of ETS. With high PcO₂ values, the metabolic depression occurred quickly, showing that oysters had a low capacity to regulate their respiration when oxygen availability is reduced, particularly in the summer.     

Metabolic response to starvation in late pregnant rats. II. Fetal response

Effect of prolonged maternal fasting on the fetal liver and heart glycogen and triglyceride content and on concentration of glucose, urea, uric acid and alpha amino-nitrogen in the amniotic fluid has been studied in rats. The animals were divided into four groups: fed (control), fasted for one day (from 20 to 21 day of pregnancy), fasted for two days (from 19 to 21 day) and fasted for three days (from 18 to 21 day). Maternal fasting for two and three days resulted in reduction in fetal growth. The fetal liver glycogen content was reduced already after one day of fasting, stabilized after two days and then further decreased after three days. The fetal heart glycogen content was reduced only after three days of fasting. The fetal liver triglyceride content increased gradually during the first two days of fasting and then stabilized. The content of triglycerides in the heart was elevated after two and three days of food deprivation. The amniotic fluid glucose concentration decreased after one day of fasting and then stabilized. Fasting did not effect the concentration of the nitrogenous compounds in the amniotic fluid. It is concluded that maternal fasting affects markedly metabolism of energy substrates stored in the fetal liver and the heart and the composition of the amniotic fluid.     

Metabolic response to starvation in late pregnant rats. I. Maternal response

The aim of the present study was to examine effect of prolonged fasting on muscle glycogen and triglyceride concentration as well as on non-protein nitrogen excretion with urine in late pregnant rats. They were divided into four groups: I--fed, pregnant for 21 days, II--fasted for one day (from 20 to 21 day of pregnancy), III--fasted for two days (from 19 to 21 day) and IV--fasted for three days (from 18 to 21 day). The concentration of glycogen and triglycerides was determined in the following tissues: the white and red layers of the vastus lateralis, the soleus, the diaphragm, the heart and the liver. The urine was collected in each group 24 h (from 20 to 21 day). It has been found that concentration of glycogen in the leg muscles is reduced by about 50% and in the diaphragm by 75% already after 24 h fasting and then remains stable. The concentration of glycogen in the heart increases after one day of fasting and then returns to the control value. The effect of fasting on the concentration of triglycerides in the tissues depends on a tissue studied. It decreases gradually in the white vastus, and in the soleus only on the third day. It is elevated during the first two days of fasting in the red vastus, diaphragm and liver and returns to the control level on the third day. The fasting doubled the concentration of triglycerides in the heart. The urinary urea, creatinine, and uric acid excretion decreases and ammonia excretion increases during fasting. The results obtained indicate that the late gestation does not alter response of muscle glycogen metabolism to fasting as compared to the male rats. It does effect metabolism of triglycerides.     

Quantitative trait locus analysis of stage-specific inbreeding depression in the Pacific oyster Crassostrea gigas

Inbreeding depression and genetic load have been widely observed, but their genetic basis and effects on fitness during the life cycle remain poorly understood, especially for marine animals with high fecundity and high, early mortality (type-III survivorship). A high load of recessive mutations was previously inferred for the Pacific oyster Crassostrea gigas, from massive distortions of zygotic, marker segregation ratios in F(2) families. However, the number, genomic location, and stage-specific onset of mutations affecting viability have not been thoroughly investigated. Here, we again report massive distortions of microsatellite-marker segregation ratios in two F(2) hybrid families, but we now locate the causative deleterious mutations, using a quantitative trait locus (QTL) interval-mapping model, and we characterize their mode of gene action. We find 14-15 viability QTL (vQTL) in the two families. Genotypic frequencies at vQTL generally suggest selection against recessive or partially recessive alleles, supporting the dominance theory of inbreeding depression. No epistasis was detected among vQTL, so unlinked vQTL presumably have independent effects on survival. For the first time, we track segregation ratios of vQTL-linked markers through the life cycle, to determine their stage-specific expression. Almost all vQTL are absent in the earliest life stages examined, confirming zygotic viability selection; vQTL are predominantly expressed before the juvenile stage (90%), mostly at metamorphosis (50%). We estimate that, altogether, selection on vQTL caused 96% mortality in these families, accounting for nearly all of the actual mortality. Thus, genetic load causes substantial mortality in inbred Pacific oysters, particularly during metamorphosis, a critical developmental transition warranting further investigation.     

Metabolic response to short term starvation in non-pregnant and late pregnant cafeteria-obese rats

We have determined the blood metabolite responses to a 24-h starvation period of cafeteria obese rats, in both non-pregnant and late pregnant states. In the fed condition the concentrations of glucose, lactate, pyruvate, glycerol and urea do not differ when compared in control and obese rats, but acetoacetate and 3-hydroxybutyrate levels are higher in the obese group. The overall response of the cafeteria-obese rats to starving seems characterized by decreased rates of glucose and amino-acids utilization, substituted by a more intense utilization of lipid fuels, with excess ketone bodies production and increased utilization of the mobilized glycerol. What we observed in the obese pregnant response to starvation can be summarized as the additional or superimposed effects of excess fat reserves. In the obese pregnant starved rats a less severe hypoglycaemia, lower levels of glycerol (as a consequence of increased utilization), reduced urea levels, and increased acetoacetate and 3-hydroxybutyrate levels were observed. It can be assumed that the pregnant obese rat response to starvation is related to the size of the fat deposits: the more obese, the more hyperketonaemia and less hypoglycaemia, and even diminished rates of amino-acid utilization, as indicated by a lower levels, when compared to the lean pregnant.     

Metabolic response to short periods of starvation in hypo- and hyper-thyroid male rats.

1) Thyroidectomized rats were fed with a low iodine diet, injected daily with 0, 0.1, 1.8 or 25 microgram of L-thyroxine/100 g body wt., and compared with intact controls. 2) Plasma protein-bound iodine was decreased in the rats given the 0 and 0.1 microgram doses, unchanged in those given the 1.8 microgram doses, unchanged in those given the 1.8 microgram dose increased in those given the 25 microgram one. 3) The liver content of DNA-P, phospholipid-P, proteins and fatty acids was decreased in the rats that did not receive thyroxine, practically recuperated in those receiving 0.1 microgram and normal in those given 1.8 or 25 microgram of thyroxine. 4) 3 h of starvation produced a reduction in the liver content of total fatty acids that disappeared after 24 h. 5) When fed, liver glycogen concentration was low in the rats given 25 microgram of thyroxine. 6) With starvation, the fall in liver glycogen and blood glucose, and the rise in liver acetyl-CoA and citrate and blood glycerol concentrations were faster in the thyroidectomized rats that did not receive thyroxine than in the other groups. 7) The rise in plasma free fatty acid and blood ketone bodies concentrations were similar in all the groups, the greater level of the first parameter being observed after 6 h of starvation in the rats given 25 microgram of thyroxine and in the second one after 24 h in the rats given either 0.1, 1.8 or 25 microgram of thyroxine. 8) The rapid decrease in the availability of carbohydrate stores with starvation in the thyroidectomized rats could be responsible for their fast call for lipid utilization. The slower response to fasting in the hyperthyroid animals is probably a consequence of their reduced amount of endogenous substrates to be mobilized.     

Ontogeny and water temperature influences the antiviral response of the Pacific oyster,Crassostrea gigas

Disease is caused by a complex interaction between the pathogen, environment, and the physiological status of the host. Determining how host ontogeny interacts with water temperature to influence the antiviral response of the Pacific oysters, Crassostrea gigas, is a major goal in understanding why juvenile Pacific oysters are dying during summer as a result of the global emergence of a new genotype of the Ostreid herpesvirus, termed OsHV-1 μvar. We measured the effect of temperature (12 vs 22 °C) on the antiviral response of adult and juvenile C. gigas injected with poly I:C. Poly I:C up-regulated the expression of numerous immune genes, including TLR, MyD88, IκB-1, Rel, IRF, MDA5, STING, SOC, PKR, Viperin and Mpeg1. At 22 °C, these immune genes showed significant up-regulation in juvenile and adult oysters, but the majority of these genes were up-regulated 12 h post-injection for juveniles compared to 26 h for adults. At 12 °C, the response of these genes was completely inhibited in juveniles and delayed in adults. Temperature and age had no effect on hemolymph antiviral activity against herpes simplex virus (HSV-1). These results suggest that oysters rely on a cellular response to minimise viral replication, involving recognition of virus-associated molecular patterns to induce host cells into an antiviral state, as opposed to producing broad-spectrum antiviral compounds. This cellular response, measured by antiviral gene expression of circulating hemocytes, was influenced by temperature and oyster age. We speculate whether the vigorous antiviral response of juveniles at 22 °C results in an immune-mediated disorder causing mortality.     

Cloning and mRNA expression of antioxidant enzymes in the Pacific oyster, Crassostrea gigas in response to cadmium exposure

Cadmium (Cd) is one of the most toxic heavy metal pollutants in the aquatic environment and can induce the formation of reactive oxygen species (ROS) that cause oxidative stress. In present study, we cloned catalase (CAT) and glutathione peroxidase (GPX) cDNA, and investigated its time- and dose-related effects of three Cd concentrations (0.01, 0.05 or 0.1 ppm) on mRNA levels of antioxidant enzymes (superoxide dismutase (SOD), CAT, GPX) in the gill and changes enzyme levels in the hemolymph of the Pacific oyster, Crassostrea gigas. The cDNA indentified encoded proteins of 516 and 244 amino acids corresponding to CAT and GPX, respectively. BLAST analysis from other species indicated that the residues essential to the enzymatic function of CAT and GPX proteins of C. gigas are highly conserved. Cd treatment significantly increased antioxidant enzyme mRNA expression in the gill in a time- and dose-dependent manner. The mRNA expression at 0.1 ppm Cd concentration increased up to 3 days (CAT, GPX) or 7 days (SOD) and then decreased by 7 days (CAT, GPX) or 11 days (SOD). Aspartate aminotransferase, alanine amintransferase and hydrogen peroxide (H(2)O(2)) concentrations levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results suggest that antioxidant enzymes play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.     

Metabolic response of cerambycid beetle (Morimus funereus) larvae to starvation and food quality

The response of xylophagous Morimus funereus larvae to a direct change of diet demonstrated that the larvae from nutrient-poor substrates, e.g. oak, are very sensitive to such a change. Depending on dietary protein quality and quantity, an increase of proteolytic activity, i.e. an intensified protein metabolism accompanied by changes in body mass gain, was observed. At the same time, amylolytic activity was usually decreased. In the larvae reared on Robert's diet, sensitivity to the switch in diet was lower at the level of proteolytic enzymes that remained at the control level, while amylolytic activity was elevated. If the switch to a new diet was preceded by 7-day-starvation that disturbed nutritional homeostasis, the response of the larvae was similar to that recorded upon a direct switch only after short-term feeding (24 h) upon starvation. Differences in the response to changes in the diet of the larvae from nature, those reared under laboratory conditions and those of different physiological status could be ascribed to plasticity in the expression of the genes coding for proteases and their isoenzymes, as well as to the multi-functionality of some neurosecretory neurons, synthetic products that participate in the regulation of digestive enzyme activities.     

Molecular cloning of calnexin and calreticulin in the Pacific oyster Crassostrea gigas and its expression in response to air exposure

Calnexin (CNX) and calreticulin (CRT) are endoplasmic reticulum (ER) chaperones. CNX is a type I transmembrane protein and CRT is a soluble CNX homologue. In the ER, CNX and CRT are important for Ca(2+) homeostasis and protein maturation. Here, we describe the full-length cDNA of the first mollusk CNX (cgCNX) and a second mollusk CRT (cgCRT) from the oyster Crassostrea gigas. CgCNX, containing 3255bp, was composed of a 1764bp open reading frame (ORF) that encodes a 588-amino acid protein. CgCRT, containing 1727bp, was composed of a 1242bp ORF that encodes a 414-amino acid protein. CgCNX and cgCRT contains an N-terminal 21- and 16-amino acid sequence, respectively, which is characteristic of a signal sequence. At the C-terminus, cgCRT also contains the KDEL (-Lys-Asp-Glu-Leu) peptide motif suggesting that cgCRT localizes in the ER. Northern blot analysis showed that both cgCNX and cgCRT mRNAs are induced by air exposure. The expression patterns of cgCNX mRNA differed from those of cgCRT during air exposure. This suggests that these two molecular chaperones have different roles in the response to air exposure.     

Susceptibility of juvenile Crassostrea gigas and resistance of Panope abrupta to Mikrocytos mackini

Samples from the field and laboratory exposure to Mikrocytos mackini (a tiny protistan parasite of unknown taxonomic affiliation) confirmed that juvenile Pacific oysters (Crassostrea gigas) are susceptible to infection and the resulting disease. In the laboratory bath exposure experiment, a prevalence of infection approaching 100% and mortalities were observed in the small oysters (about 18 mm in shell length). However, in the same laboratory exposure experiment, similar aged geoduck clams (Panope abrupta, about 8mm in shell length) were resistant to infection. The main route of infection in the oysters appeared to be via the digestive tract and possibly the gills where the parasite multiplied within host cells. Other tissues such as the adductor muscle and vesicular connective tissue were subsequently colonized. Although the infection resulted in the mortality of some oysters, others appeared to overcome the disease.     

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.     

Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure.

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.