Characterizing the metabolic actions of natural stresses in the California red abalone, <Emphasis Type="Italic">Haliotis rufescens</Emphasis> using <Superscript>1</Superscript>H NMR metabolomics

Withering syndrome in California red abalone (Haliotis rufescens) is caused by the Rickettsiales-like prokaryote (WS-RLP) Candidatus Xenohaliotis californiensis. WS-RLP infection is not sufficient to cause withering syndrome, and for reasons not yet well understood additional stressors such as elevated water temperature appear to influence disease development. Using nuclear magnetic resonance (NMR) based metabolomics, we have investigated the influence of food availability, temperature, and bacterial infection, both individually and in combination, on the metabolic status of the red abalone. Food limitation caused dramatic reductions in all observed classes of foot muscle metabolites, while at the same time metabolite levels within the digestive gland were preserved or increased. We also found that food limitation in combination with elevated temperature led to greater metabolic perturbations in both tissue types than those observed under food limitation alone. WS-RLP infection and food-limitation resulted in many of the same metabolic changes within the tissues studied, although the effects of infection were less severe. We observed increased levels of homarine in the digestive gland of both food-limited and WS-RLP-infected animals, yet only observed increased homarine levels in the foot muscle of infected abalone. These results further support the recently established glucose-to-homarine ratio in foot muscle as a potential marker for differentiating WS-RLP-infected animals from those of both healthy and food limited abalone. Furthermore, we found that the NMR metabolic data correlates well with histological measurements supporting the use of the metabolomics approach for characterizing both normal and pathological events in marine species, particularly during periods of environmentally relevant stress.     

Parasites of three commercially exploited bivalve mollusc species of the estuarine region of the Cachoeira river (Ilhéus, Bahia, Brazil)

This paper reports the parasites found in three commercially exploited bivalve molluscs (Mytella guyanensis, Anomalocardia brasiliana and Iphigenia brasiliana) of an estuarine region of Ilhéus, south of Bahia, Brazil (14°48′23′′S; 39°02′47′′W). Samples of 20 individuals of each species were collected fortnightly from August 2005 to August 2006. A total of 1480 individuals was collected and processed by standard histologic techniques; the histologic sections were stained with Harris haematoxylin and eosin and examined with light microscope. The water temperature in the study area varied from 24 to 30.5 °C and the salinity from 0 to 23 ppt. Remarkable differences were found in the parasitic community between the three mollusc species involved in the study, which occupied different habitats in the estuarine region of the Cachoeira river. The following parasites were found: intracellular rickettsia-like colonies in digestive epithelia; intracellular gregarine Nematopsis sp. in gills, mantle, gonad, digestive gland and foot muscle; sporocysts of a Bucephalidae trematode in gonads, mantle, gills, digestive gland and foot; unidentified digenetic metacercariae in digestive gland and gonad; metacestodes of Tylocephalum sp. in connective tissue in the digestive gland and in gonad; and an unidentified metazoan in mantle and intestinal lumen. No significant temporal variation in the prevalence of any parasite was detected, which could be due to the narrow temperature range of the region and the absence of patterns of salinity and rainfall variation through the year. The infestation by sporocyst was the only pathological threat detected for the studied populations because of its potential for host castration. The low infection intensity and/or prevalence of the other parasites and the lack of obvious lesions suggest that there is no other serious pathological risk for the studied mollusc populations.     

Estimating recent growth in the cuttlefish Sepia officinalis: are nucleic acid-based indicators for growth and condition the method of choice?

A laboratory calibration study was undertaken with juvenile Sepia officinalis (80-85 g initial wet weight) to investigate the effects of different food rations and different starving intervals on RNA/dry weight (DW) ratios and RNA/DNA ratios in cephalopod mantle muscle at two different temperatures. The digestive gland index was also used as an additional indicator of recent growth. High food rations and low temperature went along with high RNA/DW ratios and high RNA/DNA ratios. Starving resulted in a linear decline in growth performance and a concomitant decrease in RNA/DW and RNA/DNA ratio, with RNA/DNA ratios representing the growth data better. RNA/DNA ratios decreased faster at higher temperatures. A fluorimetric assay for nucleic acid analysis was optimized for cephalopod mantle tissues and yielded reproducible RNA/DNA ratios with a relative variance below 10%. Thus, it may be possible to use this estimator of recently encountered feeding regime for the evaluation of mortality rates of early teuthid paralarvae to eventually support stock management. Also, log relative digestive gland weight showed a strong relationship with starving time, but, surprisingly, not with temperature. Data from the two temperatures analyzed could be combined to form a common regression line of relative digestive gland index with starving time. This indicator for recent growth might be especially suitable for large specimens with a well-developed digestive gland.     

Effect of ammonia on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalone Haliotis diversicolor supertexta held in 30 parts/per thousand seawater and 26 degrees C were injected with TSB-grown Vibrio parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then placed in water containing different concentrations of ammonia-N (un-ionized plus ionized ammonia) at 0.01 mg l(-1) (control), 1.12, 3.22, 5.24 and 10.18 mg l(-1). Mortality of abalone increased directly with ambient ammonia-N concentration. After 12 h, the mortality of V. parahaemolyticus-injected abalone held in 3.22 mg l(-1) ammonia-N was significantly higher than those placed in 1.12 mg l(-1) ammonia-N and the control solution. In another experiment, the abalone which had been exposed to control, 1.08, 3.16, 5.37 and 10.34 mg l(-1) ammonia-N for 24, 72 and 120 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), and phagocytic activity and clearance efficiency to V. parahaemolyticus. The abalone when exposed to 3.16 mg l(-1) ammonia-N had decreased THC after 72 h, and decreased phenoloxidase activity, phagocytic activity and clearance efficiency after 24 h. However, the abalone when exposed to 3.16 mg l(-1) ammonia-N had increased respiratory burst after 24 h. The immune parameters except superoxide anion seemed to be suppressed in a dose-dependent fashion after 24 h. It is concluded that ammonia caused a depression in immune parameters and an increase in mortality of H. diversicolor supertexta from V. parahaemolyticus infection.     

First report of invertebrate Mx: cloning, characterization and expression analysis of Mx cDNA in disk abalone (Haliotis discus discus)

Myxovirus resistance (Mx) protein is one of the most studied antiviral proteins. It is induced by the type I interferon system (IFN alpha/beta) in various vertebrates, but its expression has not been identified or characterized in mollusks or other multi-cellular invertebrates to date. In this study, we isolated the Mx gene from a disk abalone (Haliotis discus discus) normalized cDNA library. Mx cDNA was sequenced, cloned and compared to other known Mx proteins. The full-length 1664 bp of abalone Mx cDNA contained a 1533-bp open reading frame that codes for 511 amino acids. Within the coding sequence of abalone Mx, characteristic features were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif and a dynamin family signature. In addition, leucine residues in the C-terminal region displayed a special leucine domain at L(468), L(475), L(489) and L(510), suggesting that abalone Mx may have a similar oligomerization function as other leucine zipper motifs. Abalone Mx protein exhibited 44% amino acid similarity with channel catfish Mx1, rainbow trout Mx2 and Atlantic halibut Mx. Abalones were injected intramuscularly with the known IFN inducer poly I:C and RT-PCR was performed for Mx mRNA analysis. The results showed enhanced Mx expression in abalone gill and digestive tissues 24h as well as 48 h after injection of poly I:C. Mx mRNA was expressed in gill, digestive gland, mantle and foot tissues in healthy abalone, suggesting that the basal level of Mx expressed is tissue-specific. There is no known Mx protein closely related to abalone Mx according to phylogenetic analysis. Abalone Mx may have diverged from a common gene ancestor of fish and mammalian Mx proteins, since abalone Mx showed high similarity in terms of conserved tripartite GTP-binding, dynamin family signature motifs and poly I:C enhancement of Mx mRNA expression.     

Digestive enzymes in juvenile green abalone,Haliotis fulgens,fed natural food

Enzymes responsible for the digestion of food protein by juvenile green abalone (Haliotis fulgens) were studied when fed algae or a sea grass (Phyllospadix torreyi) naturally occurring in the habitat. The effect of food on the composition and activity of the enzymes was also evaluated. Acid, serine proteinases and aminopeptidases, as confirmed by pH profile of activity, specific inhibition and synthetic substrate hydrolysis were found in the digestive organs of juvenile green abalone. Algae and sea grass differentially affected the digestive system in abalone.     

Metallothioneins and trace elements in digestive gland,gills, kidney and gonads of Octopus vulgaris

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (µg g^− 1, dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation.     

The effect of cold-acclimation on energy strategies of Apodemus draco in Hengduan Mountain region

Environmental factors play an important role in the seasonal adaptation of body mass and thermogenesis in small mammals. The purpose of the present study was to test the hypothesis that ambient temperature triggers adjustments in body mass, body temperature, energy intake, digestible energy intake, metabolic energy intake, and the length and weight of the digestive tract, in Apodemus draco during 42 days of cold exposure. Body mass and body temperature of the cold-acclimated group decreased during the first 28 days and then stabilized at the lower levels. After 14 days of cold acclimation, the body mass of the cold-exposed group was significantly lower and the energy intake, digestible energy intake, and metabolic energy intake were significantly elevated relative to control animals. The differences were maximal after 21 days. The length and weight of the digestive tract (both wet and dry mass of the stomach, small intestine, large intestine, and cecum) changed significantly in response to decreasing temperature. During cold exposure, A. draco was able to maintain physiological functions and reduce its absolute energy demands by reducing the body mass, increasing energy intake, and adjusting the length of the digestive tract.     

The biology and metabolism ofOrchomene plebs (Hurley 1965) (Amphipoda: Gammaridea) from McMurdo sound,Ross Sea,Antarctic

Summary O. plebs (Hurley 1965) is a dominant bottom species of amphipoda, occuring in the investigated area at 10 to 760 m. Population structure and sex proportions depend upon the depth and the season of the year of sampling. Development of eggs occurs in winter; the young hatch in spring; the number of eggs produced is proportional to the volume of females. A relationship has been established between length and wet/dry body weight ofO. plebs. The species is necrophagus, long enduring without food. At -1.8°C retention of food in the gut may last from several hours to several days. Heart beat frequency oscillates between 40–80 beats/min at -1.8°C and depends on temperature. A temperature near 8°C is lethal. Oxygen consumption ofO. plebs depends on their developmental stage and sex and on trophic conditions. The level of metabolism of starved animals can be half that of fed animals. The greatest differences in oxygen consumption occur at-1.8°C, the ambient temperature. Oxygen consumption as a reaction to higher temperatures is different for starved and fed animals.O. plebs are hypostenothermic organisms; that is, they can adapt to the lowest temperature occuring in the marine environment.In memory of Mary Alice McWhinnie (1922–1980)     

Cold tolerance of the Australian spur-throated locust, Austracris guttulosa

The cold tolerance of overwintering adult Spur-throated locusts, Austracris guttulosa, was examined using measures of supercooling point relative to gender, environmental acclimation and feeding state as well as mortality for a range of sub-zero temperature exposure treatments. Freezing was lethal and supercooling points ranged from -6 to -12.8°C, but were statistically independent of fresh mass, body water content, acclimation, and/or gut content in fed and starved individuals. A significant interaction effect of gender and feeding status showed that the larger bodied females had decreased supercooling capacity with increased food material in the digestive tract. Post-freezing dissections revealed differences in the amount of freshly consumed and retained food material in the digestive tract between fed and starved individuals of each gender, which could explain this effect based on inoculation of ice crystallisation by food particles. Above supercooling temperatures, neither gender nor the rate of cooling had a significant effect on mortality. When cooled from 25°C at 0.1 or 0.5°Cmin(-1) to a range of experimental minimum temperatures held for 3h, survival was ~74% to -7°C, but declined sharply to ~37% when cooled to -8°C or lower. Although the laboratory experiments reported here suggest that A. guttulosa is not freeze tolerant and unable to rapidly cold harden, exposure to typical cold and frosty nights that very rarely reach below -8°C as a night minimum in the field would be unlikely to cause mortality in the vast majority of overwintering aggregations.     

Effect of heavy metals (Cu,Cd and Pb) on aspartate and alanine aminotransferase in Ruditapes philippinarum (Mollusca: Bivalvia)

The accumulation of cadmium, copper and lead and their effects on aspartate and alanine aminotransferases in digestive gland, gills, foot and soft body in the clam Ruditapes philippinarum were examined. The animals were exposed to different concentrations: Cd (200–600 μg·l⁻¹), Pb (350–700 μg·l⁻¹) and Cu (10–20 μg·l⁻¹) for 7 days. The highest concentrations were found in digestive gland for cadmium and copper, and in gills for lead, and the lowest values were observed in the foot. Aspartate aminotransferase activity (AST), in general, was not inhibited by cadmium, lead or copper during the exposure. Only in clams exposed to cadmium (600 μg·l⁻¹, 7 days) and copper (20 μg·l⁻¹, 5 days) were observed significant differences (P<0.05) in foot and gills, respectively, with respect to control. In the case of alanine aminotransferase activity (ALT), significant differences were observed for cadmium and lead in treated animals with respect to control. With regard to copper, a decrease in ALT was observed in gills and foot exposed to 20 μg·l⁻¹. A significant correlation (P<0.05) was observed between ALT and metal accumulation for cadmium, copper and lead in gills. In the case of soft body, only cadmium and lead showed a significant correlation. In summary, R. philippinarum can be considered a bioindicator species for cadmium and lead accumulation and ALT could be useful as biomarker of sublethal stress for these metals in soft tissues and gills. Only gills can be considered an adequate target tissue for copper.     

The potential role of spherocrystals in the detoxification of essential trace metals following exposure to Cu and Zn in the fighting conch <Emphasis Type="Italic">Strombus</Emphasis> (<Emphasis Type="Italic">Lobatus</Emphasis>) <Emphasis Type="Italic">pugilis</Emphasis>

Crypt cells—one of the three cell types composing Strombidae digestive tubules—are characterized by the presence of numerous metal-containing phosphate granules termed spherocrystals. We explored the bioaccumulation and detoxification of metals in Strombidae by exposing wild fighting conch Strombus pugilis for 9 days to waterborne CuSO₄ and ZnSO₄. The total amount of Cu and Zn was determined in the digestive gland and in the rest of the body by Inductively Coupled Plasma (ICP) analyses. The digestive gland spherocrystal metal content was investigated based on the semi-quantitative energy dispersive X-ray (EDX) elemental analysis. ICP analyses of unexposed individuals revealed that 87.0?±?5.9% of the Zn is contained in the digestive gland, where its concentration is 36 times higher than in the rest of the body. Regarding Cu, 25.8?±?16.4% of the metal was located in the digestive gland of the control individuals, increasing to 61.5?±?16.4% in exposed individuals. Both Cu and Zn concentrations in the digestive gland increased after exposures, pointing to a potential role of this organ in the detoxification of these metals. EDX analysis of spherocrystals revealed the presence of Ca, Cl, Fe, K, Mg, P, and Zn in unexposed individuals. No difference was found in the relative proportion of Zn in spherocrystals of exposed versus control individuals. Contrastingly, copper was never detected in the spherocrystals from controls and Zn-exposed individuals, but the relative proportion of Cu in spherocrystals of Cu-exposed individuals varied from 0.3 to 5.7%. Our results show the direct role of spherocrystals in Cu detoxification.     

Digestive enzyme profiles reveal digestive capacity and potential energy sources in fed and starved spiny lobster (Jasus edwardsii) phyllosoma larvae

The impact of starvation on the digestive enzyme (protease, trypsin, lipase and amylase) activities of Stage I and IV Jasus edwardsii phyllosoma larvae was used to identify the nutrients metabolised or conserved during food deprivation, highlighting the most critical energy reserves. Protease activities increased significantly in both Stages I and IV phyllosoma, suggesting that protein catabolism provided energy during food deprivation. Lipase activity decreased significantly in starved Stages I and IV larvae indicating that lipid may be spared for fuelling later developmental moults. The use of protein, while sparing lipid, may provide immediate energy but not at the expense of long-term lipid energy stores which are known to be important during their lengthy larval phase. The preferential use of protein during short-term periods of starvation suggests that particular attention must be given to providing sufficient protein in artificial diets at all times. Amylase activity in starved Stage I larvae was lower than in fed animals, suggesting that the starved animals are not gaining sufficient carbohydrate. However, amylase activity was similar in fed and starved Stage IV larvae, possibly due to the catabolism of accumulated glycogen stores that were not sufficiently developed in Stage I animals.     

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.     

Metabolic and thermoregulatory effects of photoperiod and melatonin on Peromyscus maniculatus acclimatization

A photoperiod-related seasonal rhythm in active period (scotophase), metabolic rate and core temperature was documented for animals held at 21.0 +/- 0.1 degrees C ambient; animals that were habituated to long nights (10:14LD) had a greater metabolic reserve than those held in summer photoperiods (14:10LD). While relative weights of gonads and sex accessory tissues of mice show typical "winter" regression, interscapular brown adipose tissue mass was unaffected by photoperiod; moreover, IBAT beta adrenergic responses under "winter" photoperiods did not differ from "summer" photoperiods in the absence of cold stimulus. Thermogenic efficiency, measured as the increment of active temperature level achieved per increment of active period metabolic effort, was highest for animals exposed to short photoperiods. Thermal conductance was reduced in animals exposed to short (10:14LD) photoperiods. Heat conservation and thermogenic response capacity was enhanced by melatonin treatment and short photoperiod.     

Oxidative stress in digestive gland and gill of the brown mussel (Perna perna) exposed to air and re-submersed

Mussels Perna perna were exposed to air for 24 h showing a clear increase in the levels of lipid peroxidation and oxidative DNA damage, measured as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). The levels of lipid peroxidation increased both in the digestive gland and gills, while oxidative DNA damage increased only in the gills. After the 24 h of air exposure, mussels were re-submersed for a period of 3 h, leading values to return to a pre-aerial exposure levels. Control animals were kept immersed during the whole period. Several antioxidant and complementary enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), glutathione S-transferase (GST) and the levels of total glutathione (Total GSH) were assayed in a second set of experiments where one group of mussels were exposed to air for 18 h and other to 1 h re-submersion after 18 h aerial exposure. Only a 52% increase in the glutathione S-transferase activity was observed in the digestive gland, which remained elevated to about 40% after 1 h re-submersion, showing that defense systems can be modulated even during oxygen deprivation in P. perna. The DNA and lipid oxidative damage observed after aerial exposure indicates that mussels face an oxidative challenge, and are able to counteract such an “insult” as values of lipid peroxidation and DNA damage returned to control values after 3 h re-submersion.     

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis)

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.