Energy metabolism of juvenile scallops Nodipecten subnodosus under acute increased temperature and low oxygen availability.

High temperature increases energy demand in ectotherms, limiting their physiological capability to cope with hypoxic events. The present study aimed to assess the metabolic tolerance of juvenile Nodipecten subnodosus scallops to acute hyperthermia combined with moderate hypoxia. A previous study showed that juveniles exhibited a high upper temperature limit (32 °C), but the responses of juveniles to combined hyperthermia and low dissolved oxygen are unknown. Scallops were exposed to control conditions (treatment C: 22 °C, ∼7.1 mg O2 L-1 or PO2 156.9 mmHg), acute hyperthermia under normoxia (treatment T: 30 °C, ∼6.0 mg O2 L-1 or PO2 150.9 mmHg) or acute hyperthermia plus hypoxia (treatment TH: 30 °C, ∼2.5 mg O2 L-1 or PO2 62.5 mmHg) for 18 h. In T, juveniles exhibited an enhanced oxygen consumption, together with a decrease in adenylate energy charge (AEC) and arginine phosphate (ArgP), and with no changes in metabolic enzyme activity in the muscle. In TH, scallops maintained similar AEC and ArgP levels in muscle as those observed in T treatment. This response occurred along with the accumulation of inosine monophosphate and hypoxanthine. Besides, reduced citrate synthase and pyruvate kinase activities, enhanced hexokinase activity, and a higher octopine dehydrogenase/lactate dehydrogenase ratio in the mantle indicated the onset of anaerobiosis in TH. These responses indicate that juvenile scallops showed tissue-specific compensatory responses regarding their energy balance under moderate hypoxia at high temperatures. Our results give an insight into the tolerance limit of this species to combined hyperthermia and hypoxia in its northern limit of distribution.

Biochemical composition and adenylate energy charge shifts in longfin yellowtail (Seriola rivoliana) embryos during development under different temperatures.

The longfin yellowtail Seriola rivoliana is an emerging species for aquaculture diversification worldwide and production relies on fertilized eggs from captive broodstock. Temperature is the main factor that influences the developmental process and success during fish ontogeny. However, the effects of temperature on the utilization of the main biochemical reserves and bioenergetics are scarcely investigated in fish, whereas protein, lipid and carbohydrate metabolism have critical roles in maintaining cellular energy homeostasis. In this context, we aimed to evaluate metabolic fuels (protein, lipids, triacylglicerides, carbohydrates), adenylic nucleotides and derivates (ATP, ADP, AMP, IMP), and the adenylate energy charge (AEC) during embryogenesis and in hatched larvae in S. rivoliana at different temperatures. For this purpose, fertilized eggs were incubated at six constant (20, 22, 24, 26, 28 and 30 °C) and two oscillating (21⇄29 °C) temperatures. Biochemical analyses were made at blastula, optic vesicles, neurula, prehatch and hatch periods. Results indicated that the developmental period had a major influence on the biochemical composition at any temperature regime tested during the incubation. Protein content decreased only at hatching mainly due to the loss of the chorion, total lipids tended to increase at the neurula period and variations in carbohydrates depended on the particular spawn analyzed. Triacylglicerides were a critical egg fuel during hatching. The high AEC during embryogenesis and even in hatched larvae suggested an optimal energy balance regulation. The lack of critical biochemical changes from different temperature regimes during embryo development confirmed that this species exhibits a high adaptive capacity in response to constant and fluctuating temperatures. However, the timing of hatching was the most critical period of development, where biochemical components and energy utilization significantly changed. The oscillating temperatures tested may have physiological advantages without detrimental energetic effects that will require further research on larval quality after hatching.

Dietary supplementation of marine yeast Yarrowia lipolytica modulates immune response in Litopenaeus vannamei.

The immunostimulatory potential of the marine yeast Yarrowia lipolytica (D1 and N6 strains) administered orally was evaluated in the white shrimp Litopenaeus vannamei. Yeasts and commercial glucans were mixed with a commercial feed to formulate diets with a 1.1% concentration of immunostimulants. The shrimp were fed daily for a period of 21 days. Weekly determinations were performed for immunological parameters in hemolymph, such as total hemocyte count (THC), lysozyme activity (LYZ), prophenoloxidase activity, antioxidant enzymatic activities (superoxide dismutase [SOD], catalase [CAT], and peroxidases), and bactericidal activity against Vibrio parahaemolyticus. Expression profiles of penaeidin (PEN), lysozyme (LYZ), and prophenoloxidase (proPO) immune genes were evaluated in hemocytes. In general, an increase in the immune parameters was observed in shrimp fed yeast diet compared to glucan and the control diets. Yarrowia lipolytica, especially strain N6, provided maximum immunostimulatory effects evidenced by the increase of immune parameters (THC, LYZ, SOD, CAT) and gene expression profile. In conclusion, this study demonstrated that Y. lipolytica had immunostimulatory effects and increased bactericidal activity in L. vannamei hemocytes against V. parahaemolyticus. These findings open the path for the potential application of Y. lipolytica-based immunostimulant for shrimp aquaculture.

Litopenaeus vannamei oxygen consumption and HSP gene expression at cyclic conditions of hyperthermia and hypoxia.

Although Litopenaeus vannamei is a widely studied species, the information on how the organisms respond to natural daily variations of environmental conditions such as temperature and dissolved oxygen, and how such conditions alter the physiological responses, is scarce. In the present work, the strategies used by shrimps to cope with temperature and dissolved oxygen fluctuations during 24 days were investigated through the evaluation of oxygen consumption and heat shock proteins (HSP) gene expression. During daily fluctuations, no change in oxygen consumption in the short-term, but a significant increase in the long-term during hyperthermia conditions was registered, whereas a significant decrease during hypoxia was observed during all the bioassay. On the other hand, HSP70 and HSP90 gene expression increased in gills under thermal stress but was down-regulated under hypoxia, in both the short- and the long-term. This study highlights that to counteract environmental variations of temperature and dissolved oxygen, the shrimps use molecular compensatory mechanisms (HSP gene expression) that are different to those used under constant hypoxic conditions, suggesting that hypoxia can compromise physiological cytoprotection.

Pathological changes by Eosinophilic Rickettsia-like organism in Japanese oyster, Crassostrea gigas.

Rickettsia infection in adult Japanese oysters (Crassostrea gigas) was observed in 2015 at San Ignacio Lagoon, Baja California Sur, Mexico and characterized using molecular tools. In the present study, the degree of infection by Eosinophilic Rickettsia-Like Organism (E-RLO), characterized by intracellular inclusions in gill, mantle, labial palps, digestive tract and gonadal ducts, was evaluated using histological methods and was associated with visible injuries on body surface, such as blisters, shell damage and necrosis. Most of the oysters (92.2%) had some type of observable symptoms; 90.2% of all oysters had blisters in the mantle, and all oysters presented with E-RLO in the epithelia of at least one of the analyzed tissues (gills, labial palps, mantle, gonadal ducts and digestive tract). The highest intensity of infection (percent of E-RLO coverage) was observed in the labial palps (18.6%) and the lowest in digestive tract (2.6% of the examined tissue). Oysters with external injuries had higher infection intensities than the oysters without external injuries. Considering the clinical signs (observable symptoms and histological findings), we propose three infective stages of E-RLO disease development, a scale that could be used for early detection.

Transcriptomic information from Pacific white shrimp (Litopenaeus vannamei) ovary and eyestalk, and expression patterns for genes putatively involved in the reproductive process.

The increased use of massive sequencing technologies has enabled the identification of several genes known to be involved in different mechanisms associated with reproduction that so far have only been studied in vertebrates and other model invertebrate species. In order to further investigate the genes involved in Litopenaeus vannamei reproduction, cDNA and SSH libraries derived from female eyestalk and gonad were produced, allowing the identification of expressed sequences tags (ESTs) that potentially have a role in the regulation of gonadal maturation. In the present study, different transcripts involved in reproduction were identified and a number of them were characterized as full-length. These transcripts were evaluated in males and females in order to establish their tissue expression profiles during developmental stages (juvenile, subadult and adult), and in the case of females, their possible association with gonad maturation was assessed through expression analysis of vitellogenin. The results indicated that the expression of vitellogenin receptor (vtgr) and minichromosome maintenance (mcm) family members in the female gonad suggest an important role during previtellogenesis. Additionally, the expression profiles of genes such as famet, igfbp and gpcr in brain tissues suggest an interaction between the insulin/insulin-like growth factor signaling pathway (IIS) and methyl farnesoate (MF) biosynthesis for control of reproduction. Furthermore, the specific expression pattern of farnesoic acid O-methyltransferase suggests that final synthesis of MF is carried out in different target tissues, where it is regulated by esterase enzymes under a tissue-specific hormonal control. Finally, the presence of a vertebrate type steroid receptor in hepatopancreas and intestine besides being highly expressed in female gonads, suggest a role of that receptor during sexual maturation.

The influence of dietary supplementation of arachidonic acid on prostaglandin production and oxidative stress in the Pacific oyster Crassostrea gigas.

In a previous study, dietary supplementation with arachidonic acid (ARA) to oysters Crassostrea gigas increased haemocyte numbers, phagocytosis, and production of reactive oxygen species level (ROS) by haemocytes (Delaporte et al., 2006). To assess if the observed stimulation of these cellular responses resulted from changes of ARA-related prostaglandin (PG) production, we analysed prostaglandin E2 metabolite (PGEM) content on the same oysters fed three levels of ARA. Dietary supply of polyunsaturated fatty acids (PUFA) could also induce an oxidative stress that could similarly increase cellular responses; therefore, two indicators of oxidative stress were analysed: peroxidation level and antioxidant defence status. Together the observed positive correlation between ARA and PGEM levels and the absence of lipid peroxidation and antioxidant activity changes supports the hypothesis of an immune stimulation via PG synthesis. Although ARA proportion in oyster tissues increased by up to 7-fold in response to ARA dietary supplementation, peroxidation index did not change because of a compensatory decrease in n-3 fatty acid proportion, mainly 22:6n-3. To further confirm the involvement of PG in the changes of haemocyte count, phagocytosis and ROS production upon ARA supplementation, it would be interesting to test cyclooxygenase and lipooxygenase inhibitors in similar experiments.

Specific regulations of gill membrane fatty acids in response to environmental variability reveal fitness differences between two suspension-feeding bivalves (Nodipecten subnodosus and Spondylus crassisquama).

Bivalves' physiological functions (i.e. growth, reproduction) are influenced by environmental variability that can be concomitant with trophic resource variations in terms of quality and quantity. Among the essential molecules that bivalves need to acquire from their diet to maintain physiological functions, fatty acids (FAs) such as polyunsaturated fatty acids (e.g. 20:4n-6 (arachidonic acid), 20:5n-3 (eicosapentaenoic acid) and 22:6n-3 (docosahexaenoic acid)) have been described to play a critical role. The present study examined the FA composition of gill membrane lipids of two bivalve species, Nodipecten subnodosus and Spondylus crassisquama, sampled in a coastal lagoon of the Northeastern Pacific (Ojo de Liebre, Mexico), at two contrasting locations (inner versus outer part of the lagoon) and at two different periods (February and August 2016). Spatiotemporal variations showed that FA composition of gill membrane lipids was highly correlated to FA composition of reserve lipids from digestive gland. This highlights the marked impact of the diet on FA composition of gill membranes. Interestingly, both species presented differences in the seasonal accumulations of plasmalogens and of particular FA that are not found in their diet (e.g. non-methylene interrupted FA, 22:4n-9trans, 20:1n-11), suggesting specific regulations of FA incorporation and lipid class composition in gill membranes to maintain optimal membrane function in their specific and changing environment. This study highlights the importance to characterize the spatial and temporal variability of food resources in order to apprehend the physiological consequences of environmental variability, as well as species differential regulation capacities in a changing world.

Metabolic responses of adult lion's paw scallops Nodipecten subnodosus exposed to acute hyperthermia in relation to seasonal reproductive effort.

In marine ectotherms, reproduction is an energetically expensive process that affects their thermal window tolerance. For most species, the impacts of hyperthermia during gametogenesis have still not been addressed. Our aim was to assess the metabolic response of adult Nodipecten subnodosus scallops to thermal challenges at early development (spring) and advanced gonad maturation (summer). Scallops collected in both seasons were exposed to acute hyperthermia (26 and 30 °C, 24 h), maintaining a group of scallops at acclimation temperature (22 °C) as a control condition. During the summer, relatively low activity of hexokinase (HK), as well as low levels of ATP and GTP were found in the adductor muscle, suggesting a shift in energy investment for reproduction, although arginine phosphate (ArgP) levels were higher in summer scallops. Hyperthermia (30 °C) induced an increased energy expenditure reflected by a transitory enhanced oxygen consumption (VO2) and relatively high activities of HK and arginine kinase (AK). Moreover, a slight decrease in adenylic energy charge (AEC) was partially compensated by a decrease in ArgP. An increase in nucleotide by-products inosine monophosphate (IMP) and hypoxanthine (HX) indicated a thermal stress at 30 °C. Some of the responses to acute hyperthermia were more pronounced at advanced maturation stages (summer scallops), indicating a possible lack of energy balance, with possible implications in animals challenged to global warming scenario.

Contrasting reproductive health of female clams Megapitaria squalida from two nearby metal-polluted sites in the Gulf of California: Potential effects of copper, lead, and cobalt.

We studied the effects of chronic exposure to metals on energy reserves and reproduction in the clam Megapitaria squalida in two nearby populations exposed to different levels of pollution from mining operations in the Gulf of California, Mexico. Female M. squalida from San Lucas beach had good reproductive health status, whereas Santa Rosalia specimens consistently showed low energy reserves, massive oocyte resorption throughout the year, high frequencies of undifferentiated individuals, low proportions of ripe and spawning organisms, smaller and fewer oocytes per follicle, and significantly lower follicular areas. Ovarian levels of Co, Cu, Pb, Mn, and Zn were consistently higher in clams from Santa Rosalia. The poor reproductive health of clams inhabiting this site may be attributed to their long-term exposure to high Co, Cu, and Pb concentrations, as these have been shown to cause toxicity and reproductive impairments in other marine organisms.

Modulation of stress response and productive performance of Litopenaeus vannamei through diet.

The high tolerance of Litopenaeus vannamei to a wide range of salinity (1-50 psu) makes this species an excellent candidate for culture under low salinity, decreasing shrimp epidemics and water pollution in some coastal areas. However, salinity levels outside the optimal range could impose several physiological constraints that would in turn affect growth and survival, particularly in the presence of additional stressors (e.g. high densities, handling practices, and hypoxia). Despite shrimp susceptibility to individual stressors has been widely addressed, information regarding response to chronic and acute stressors combined and its relation to diet is scarce. Thus, the aim of our study was to determine the effect of diet on the susceptibility to chronic (low salinity) and acute (hypoxia and escape response) stressors in terms of culture performance and physiological indicators. We evaluated overall performance during culture of L. vannamei at low salinity (6 psu), fed with an experimental diet with low protein and high carbohydrate content (26% protein and 6% fish meal plus probiotic mixture) and compared to a commercial formula with high protein and low carbohydrate content (40% crude protein and 20% fish meal without probiotic mixture). At the end of the rearing experiment, shrimp were exposed to two types of acute stress, hypoxia and escape. Biochemical (hemocyanin, total proteins, glucose, and lactate) and bioenergetic (adenylic energy charge and arginine phosphate levels) variables were measured to assess chronic stress response (salinity) and acute stress response (hypoxia or escape). The experimental diet resulted in higher muscle energy status that was not affected by low salinity, although lipid levels were lower under this condition. This diet partially counteracted the low performance at low salinity and promoted greater protein efficiency. Hypoxia induced strong hyperglycemic and lactate increase as response, whereas escape response was characterized by a depletion of arginine phosphate levels, with a stronger decrease in shrimp fed experimental diet, due to the high initial level of this reserve. Some data (glucose levels in hemolymph and lipids in hepatopancreas) suggest that shrimp under chronic stress conditions (low salinity and high densities) present a low ability to respond to subsequent acute stressors such as hypoxia or escape. This work indicates that diet can increase the energy status of shrimp, enabling them to overcome potential multifactorial stressors, which are common in farming systems.

Transcriptomic analysis of Pacific white shrimp (Litopenaeus vannamei, Boone 1931) in response to acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), caused by marine bacteria Vibrio Parahaemolyticus, is a huge problem in shrimp farms. The V. parahaemolyticus infecting material is contained in a plasmid which encodes for the lethal toxins PirABVp, whose primary target tissue is the hepatopancreas, causing sloughing of epithelial cells, necrosis, and massive hemocyte infiltration. To get a better understanding of the hepatopancreas response during AHPND, juvenile shrimp Litopenaeus vannamei were infected by immersion with V. parahaemolyticus. We performed transcriptomic mRNA sequencing of infected shrimp hepatopancreas, at 24 hours post-infection, to identify novel differentially expressed genes a total of 174,098 transcripts were examined of which 915 transcripts were found differentially expressed after comparative transcriptomic analysis: 442 up-regulated and 473 down-regulated transcripts. Gene Ontology term enrichment analysis for up-regulated transcripts includes metabolic process, regulation of programmed cell death, carbohydrate metabolic process, and biological adhesion, whereas for down-regulated transcripts include, microtubule-based process, cell activation, and chitin metabolic process. The analysis of protein- protein network between up and down-regulated genes indicates that the first gene interactions are connected to oxidation-processes and sarcomere organization. Additionally, protein-protein networks analysis identified 20-top highly connected hub nodes. Based on their immunological or metabolic function, ten candidate transcripts were selected to measure their mRNA relative expression levels in AHPND infected shrimp hepatopancreas by RT-qPCR. Our results indicate a close connection between the immune and metabolism systems during AHPND infection. Our RNA-Seq and RT-qPCR data provide the possible immunological and physiological scenario as well as the molecular pathways that take place in the shrimp hepatopancreas in response to an infectious disease.

Trophic ecology of suspension-feeding bivalves inhabiting a north-eastern Pacific coastal lagoon: Comparison of different biomarkers.

Understanding the nature and origins of food sources supporting coastal lagoon-inhabiting organisms is necessary to evaluate the ecological status of such ecosystems. The trophic ecology of a bivalve species Spondylus crassisquama was studied in the Ojo de Liebre lagoon (Baja California, Mexico), combining stable isotope (SI), fatty acid (FA) and sterol analyses along a transect under oceanic influences. The second objective of the study was to investigate if sterol compositions give complementary information to those obtained from FA and SI. Temporal and spatial patterns of the three biomarkers in bivalve tissues suggest oceanic inputs at the mouth of the lagoon, while the inner station was characterized by a contribution of local sources including an important role of micro heterotrophs. This study revealed that the association of lipid biomarkers provide higher taxonomic resolution of the spatiotemporal heterogeneity in nutrient sources influencing the trophic functioning of a large coastal lagoon.

A novel CHH gene from the Pacific white shrimp Litopenaeus vannamei was characterized and found highly expressed in gut and less in eyestalk and other extra-eyestalk tissues.

The crustacean hyperglycemic hormone (CHH) family is an important group of neuropeptides involved in controlling growth, reproduction, and stress response in decapod species. In this study, a new gene containing 4 exons-3 introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions was isolated from Litopenaeus vannamei. Two full length transcripts of this CHH were isolated from eyestalk and pericardial tissue of males and females using rapid amplification of cDNA ends (RACE). Transcripts sequences were 1578bp in length in males pericardial tissues and in males and females eyestalk with 100% identity, but the transcript isolated from females pericardial tissues was shorter (974bp). The differences in transcripts length is a result of two polyadenylation sites present in the 3'UTR resulting in two transcription termination signals. Transcript sequences encoded one unique protein that can be classified as type I CHH subfamily because of the 4 exons and 3 introns structure, although the CPRP region is not-well conserved and there is no amidation in the C-terminal of the deduced amino acid sequence. Furthermore, there is a glycine inserted in the mature peptide not at position 12 as in type II CHHs but after amino acid 31 and the phylogenetic analysis did not group the peptide within type I, but closer to type II CHHs. We demonstrated by endpoint-PCR, qPCR, and in situ hybridization (ISH), that this gene is expressed in neuroendocrine organs known to express CHHs in penaeid shrimp, including X-organ and optic nerve in eyestalk, supraesophageal ganglion (SoG), but it is also expressed in other organs as gill, gut, pericardial cavity, as well as in terminal ampoule or spermatophore and vas deferens of males.

Seasonal variations of biochemical, pigment, fatty acid, and sterol compositions in female Crassostrea corteziensis oysters in relation to the reproductive cycle.

Wild female Crassostrea corteziensis oyster (n=245) were analyzed over one year to understand the main ecophysiological events associated to gonad development. Different indicators (mainly biochemical) were analyzed to infer: i) utilization and accumulation of energy reserves (e.g. neutral lipids, carbohydrates, proteins; vitellogenin), ii) membrane components provided by the diet as essential nutrients and indicative of cell proliferation (e.g. highly unsaturated fatty acids linked to phospholipids, sterols), iii) indicators of food availability (chlorophyll a in water, pigments in tissues, specific fatty acids and sterols), iv) gonad development (e.g. gonad coverage area, vitellin). A PCA analysis was applied to 269 measured variables. The first PC (PC1) was composed of total carbohydrate and lipid concentration, percentage of esterified sterols, fatty acids specific of diatoms; 16:1n-7/16:0, 20:5n-3 in neutral lipids with positive loadings and non methylene-interrupted fatty acids (NMI) in neutral lipids with negative loadings. The second PC (PC2) was composed of 18:4n-3 in lipid reserves and the concentration of zeaxanthin, a pigment typical of cyanobacteria with positive loadings and the proportion of 20:4n-6 in polar lipids with negative loading. The third PC (PC3) was composed of gonad coverage area (GCA) and the concentration of vitellin. Variation in GCA confirms that gonad development began in April with an extended period of spawning and rematuration from April to November. The PCA further shows that a second period of minimal maturation from November to March corresponds to the accumulation of reserves (PC1) together with an initial high availability of food (PC2) at the beginning of this period. These two periods are in accordance with the classical periods of allocation of energy to reserves followed by gonad development reported for several mollusks.

Effect of temperature on lipids, proteins, and carbohydrates levels during development from egg extrusion to juvenile stage of Cherax quadricarinatus (Decapoda: Parastacidae).

The influence of temperature on biochemical composition, survival and duration of development of Cherax quadricarinatus from egg extrusion to juvenile was analyzed. Berried females were individually subjected to each of 22, 25, 28 and 31 degrees C (n=5 per temperature). Egg samples were obtained every 3 days from egg extrusion to juvenile stage for biochemical analysis. Duration of development and survival decreased with increasing temperature. At 22 and 25 degrees C half of the initial lipid content was consumed during development. At 28 and 31 degrees C, 80% of the initial amount of lipids was consumed. For proteins, depletion rate was significantly lower at 25 degrees C (36% of the initial amount) than at 22, 28 and 31 degrees C (61-65% of the initial amount). For carbohydrates, a significant consumption was observed only at 22 degrees C. Total energy consumption was lower at 22 and 25 degrees C than at 28 and 31 degrees C. We conclude that 22-25 degrees C is the optimal temperature range for C. quadricarinatus egg incubation, although 25 degrees C might be better in terms of development duration in terms of survival, energy cost and protein consumption.