The Annual Reproductive Cycle, Proximate Composition, Fatty Acid and Amino Acid Content of Pacific Oyster, Crassostrea gigas (Magallana gigas), in Gadeok-do, Korea.

Environmental factors impact oyster growth, condition, and gonadal development, which is linked to gamete characteristics observed through histology. The reproductive cycle of bivalves is related to energy storage and utilization. Therefore, in this study, the year-round growth change and gonadal development of oysters were observed using histological analysis, and the biochemical composition changes were confirmed. The oysters used in this study are being nurtured in Gadeok-do, and 40 oysters were randomly sampled monthly from March 2021 to February 2022. Result of histological analysis of gonads, oysters were showed early development from December to February, late development from March and April, mature and ripe from May to July, spawned from August to October, and spent from November to December. Condition index values of oysters decreased in summer and autumn and increased again when entered the spent after spawning. The protein content of oysters was high in May, the maturity period, and the lipid content decreased during the spawning period. In addition, EPA and DHA, the major fatty acids of oysters, were low during the spawning period and high during the maturation period. As a result, this study suggested a close relationship between changes in oyster growth, biochemical composition, and the reproductive cycle.

Effects on the survival rates, hematological parameters, and neurotransmitters in olive flounders, Paralichthys olivaceus, reared in bio-floc and seawater by Streptococcus iniae challenge.

Bacterial infections cause huge losses to aquaculture globally, and increased antibiotic resistance means that alternative methods of reducing mortality from bacterial diseases are required. We compared the resistance of Juvenile olive flounders, Paralichthys olivaceus, to Streptococcus iniae between those reared in biofloc and seawater conditions for ten months. Experimental fish were challenged with S. iniae at concentrations of 0, 3.36 × 106, 3.36 × 107, 3.36 × 108, and 3.36 × 109 colony forming units (CFU)/g fish for 96 h to evaluate the difference in S. iniae susceptibility of flounders reared in biofloc and seawater. The 96 h lethal concentration 50% (LC50) of fish injected with S. iniae was 2.41 × 109 CFU/g fish in biofloc and 1.51 × 108 CFU/g fish in seawater. Hematological parameters such as hemoglobin and hematocrit significantly decreased when fish were challenged by S. iniae. Plasma components such as calcium, glucose, cholesterol, total protein, GOT, GPT, and ALP were significantly altered by S. iniae infection and acetylcholinesterase activity was significantly inhibited. These results indicate that S. iniae infection affects the survival rates, hematological parameters, and neurotransmitter levels of flounders reared in biofloc and seawater, and that S. iniae susceptibility was higher in flounders reared in seawater than those reared in biofloc.

Salinity-mediated changes in hematological parameters, stress, antioxidant responses, and acetylcholinesterase of juvenile olive flounders (Paralichthys olivaceus).

The purpose of this study was to confirm the limit of salinity tolerance in juvenile olive flounders (Paralichthys olivaceus) by changes in blood parameters, AChE, antioxidant and stress responses. The P. olivaceus (mean weight 38.8 ± 4.2 g and mean length 16.4 ± 1.2 cm) were exposed to different concentrations of salinity (seawater, 16, 8, 4, 2, and 0 psu) for 2 weeks. Plasma osmotic pressure was significantly decreased in the P. olivaceus at 0 psu. Hematological parameters such as hematocrit and hemoglobin were significantly decreased in the P. olivaceus at low salinity. Plasma components also changed significantly in the low salinity environment. As a stress indicator, cortisol was significantly increased at low salinity. SOD and GST antioxidant responses, were significantly increased. GSH level in the liver was significantly increased, whereas a significant decrease was observed in the gill GSH level. AChE was significantly increased in P. olivaceus at low salinity. The results of this study indicate that exposure to salinities lower than 8 psu leads to changes in hematological parameters, neurotransmitter, antioxidant and stress responses of P. olivaceus.

Toxic effects of waterborne ammonia exposure on hematological parameters, oxidative stress and stress indicators of juvenile hybrid grouper, Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀.

Juvenile hybrid grouper, Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀ (mean weight: 26.5 ±â€¯2.8 g, mean length: 11.8 ±â€¯1.3 cm) were exposed to different, sub-lethal levels of waterborne ammonia (0, 1, 2, 4, and 8 mg NH4+/L) for 2 weeks. We assessed the hematological parameters, antioxidant enzymes, and stress responses of juvenile hybrid grouper after 1 week and after 2 weeks. Hematological parameters such as hemoglobin and hematocrit levels, were significantly decreased by ammonia exposure. Plasma components such as the magnesium and total protein contents, and the glutamic oxaloacetic transaminase and glutamic pyruvic transaminase activities were significantly altered by ammonia exposure; however, no changes in the magnesium levels were detected. Antioxidant responses, such as superoxide dismutase and glutathione S-transferase activities, were also significantly affected by ammonia exposure. Stress indicator levels, i.e., plasma cholesterol and heat shock protein 70 levels, were significantly increased by ammonia exposure. The results of this study indicated that ammonia exposure has toxic effects on juvenile hybrid grouper and affects their hematological parameters, antioxidant enzymes, and stress responses.

Effects on hematological parameters, antioxidant and immune responses, AChE, and stress indicators of olive flounders, Paralichthys olivaceus, raised in bio-floc and seawater challenged by Edwardsiella tarda.

Studies on the resistance of fish raised in bio-floc systems against bacterial infection are limited. We aimed to evaluate the changes in hematological parameters, antioxidant and immune responses, stress indicators, and acetylcholinesterase (AChE) in olive flounder, Paralichthys olivaceus, raised in bio-floc and seawater for 10 months and, then, infected with Edwardsiella tarda at concentrations of 0 (control), 6.61 × 104, 6.61 × 105, 6.61 × 106, and 6.61 × 107 CFU/g fish for 7 days. The lethal concentration 50% was 4.32 × 107 in bio-floc and 3.11 × 106 in seawater. Hematological parameters were significantly decreased by E. tarda challenge, and plasma components were significantly changed. The superoxide dismutase, catalase, and glutathione-S-transferase activities, as antioxidant responses, were significantly increased after infection, whereas the reduced glutathione level was significantly decreased. The lysozyme activity was significantly increased and the AChE level was significantly decreased after infection. Cortisol and HSP 70, as stress indicators, were also significantly increased. The results indicate that E. tarda infection affected various physiological factors in P. olivaceus. Additionally, P. olivaceus raised in seawater were more susceptible to E. tarda infection than those raised in bio-floc.

Toxic effects of waterborne nitrite exposure on antioxidant responses, acetylcholinesterase inhibition, and immune responses in olive flounders, Paralichthys olivaceus, reared in bio-floc and seawater.

Paralichthys olivaceus (mean weight, 280.1 ± 10.5 g; mean length, 28.37 ± 2.3 cm) was reared in bio-floc and seawater for 6 months to determine the toxic effects of waterborne nitrite exposure (0, 25, 50, 100, and 200 mg/L) for 1 week, compared to those observed with bio-floc and seawater only. The effects on antioxidant activity, immune responses, and acetylcholinesterase activity were measured. Following nitrite exposure, superoxide dismutase activity in the liver and gills was significantly elevated and catalase activity was significantly increased, except for in the gills of P. olivaceus reared in bio-floc. Further, glutathione S-transferase activity was significantly elevated in the liver and gills, and glutathione was significantly lower. Meanwhile, acetylcholinesterase activity in the liver and gills was significantly inhibited and plasma lysozyme activity and immunoglobulin M were considerably elevated.

Hematological parameters and antioxidant responses in olive flounder Paralichthys olivaceus in biofloc depend on water temperature.

The purpose of this study was to ascertain the optimum water temperature for breeding juvenile Paralichthys olivaceus in biofloc. Hemoglobin and hematocrit were significantly decreased when the temperature was higher than 28 °C. Plasma calcium, glucose, cholesterol, glutamic-oxaloacetic transaminase, and glutamic pyruvic transaminase were significantly elevated at high temperatures, whereas total protein was substantially lower. Superoxide dismutase and glutathione-S-transferase activities in the liver and gills were significantly elevated at high temperatures, whereas glutathione was significantly lower. This indicates that temperatures greater than 26 °C induced hematological changes and oxidative stress in the juvenile P. olivaceus in biofloc. We ascribe these changes to thermal stress.

Effects of waterborne nitrite on hematological parameters and stress indicators in olive flounders, Paralichthys olivaceus, raised in bio-floc and seawater.

Juvenile olive flounders, Paralichthys olivaceus (mean weight 2.69 ±â€¯0.31 g), were raised in bio-floc and seawater for six months, these P. olivaceus (mean weight 280.1 ±â€¯10.5 g, mean length 28.37 ±â€¯2.3 cm) were exposed to different concentrations of waterborne nitrite (0, 25, 50, 100, and 200 mg NO2-/L) for 7 days. None of the P. olivaceus individuals exposed to bio-floc and seawater containing waterborne nitrite concentrations of 200 mg/L for 7 days survived. Hematological parameters (hemoglobin and hematocrit) were significantly reduced by nitrite exposure. Regarding plasma components, the concentrations of glucose, glutamic oxalate transaminase (GOT), and glutamic pyruvate transaminase (GPT) increased significantly in response to nitrite exposure, whereas cholesterol concentrations significantly decreased. Stress indicators, including concentrations of plasma glucose, cortisol, and liver and gill concentrations of heat shock protein 70 (HSP70) were significantly increased by nitrite exposure. The results of the study indicate that nitrite exposure affected the hematological parameters and stress indicators of P. olivaceus raised in bio-floc and seawater, and these changes were more prominent in the P. olivaceus raised in seawater than those raised in bio-floc.

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant.

Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis(TH),ice recrystallization inhibition(IRI),and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type.

Cloning, Expression, and Characterization of a Cold-Adapted Shikimate Kinase from the Psychrophilic Bacterium Colwellia psychrerythraea 34H.

Most cold-adapted enzymes possess higher Km and kcat values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near Km. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately 30°C and 40°C, respectively. The melting points were 33°C and 45°C for CpSK and EcSK, respectively. The ΔGH2O (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower Km for its substrate shikimate than most mesophilic SKs, and the kcat was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

Ice-Binding Protein Derived from Glaciozyma Can Improve the Viability of Cryopreserved Mammalian Cells.

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1°C/min in a -80°C freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

Ruegeria halocynthiae sp. nov., isolated from the sea squirt Halocynthia roretzi.

A Gram-negative, non-motile, rod-shaped bacterial strain, designated MA1-6T, was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea, and was characterized to determine its taxonomic position. Strain MA1-6T grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2-3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MA1-6T fell within the clade comprising Ruegeria species and exhibited 95.3-96.5 % similarity to the type strains of recognized Ruegeria species. Strain MA1-6T contained Q-10 as the predominant ubiquinone and C18:1ω7c as the major fatty acid, which is consistent with data for Ruegeria species. The major polar lipids detected in strain MA1-6T and Ruegeria atlantica KCTC 12424T were phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and an unidentified lipid. The DNA G+C content of strain MA1-6T was 58.6 mol%. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain MA1-6T can be distinguished from recognized Ruegeria species. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain MA1-6T is considered to represent a novel species of the genus Ruegeria, for which the name Ruegeria halocynthiae sp. nov. is proposed; the type strain is MA1-6T (=KCTC 23463T=CCUG 60744T).

Description of Litoreibacter meonggei sp. nov., isolated from the sea squirt Halocynthia roretzi, reclassification of Thalassobacter arenae as Litoreibacter arenae comb. nov. and emended description of the genus Litoreibacter Romanenko et al. 2011.

A Gram-negative, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated MA1-1(T), was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain MA1-1(T) is phylogenetically closely related to Litoreibacter species and to Thalassobacter arenae. It exhibited 16S rRNA gene sequence similarities of 97.3, 97.1 and 97.3% to the type strains of Litoreibacter albidus, Litoreibacter janthinus and T. arenae, respectively. Strain MA1-1(T) contained Q-10 as the predominant ubiquinone and C(18:1)ω7c as the predominant fatty acid. The polar lipid profile of strain MA1-1(T) was similar to those of the type strains of L. albidus and L. janthinus. T. arenae was found to be phylogenetically and chemotaxonomically more closely related to Litoreibacter species and strain MA1-1(T) than to Thalassobacter stenotrophicus, the type species of the genus Thalassobacter. The DNA G+C content of strain MA1-1(T) was 57.9 mol%, and DNA-DNA relatedness to the type strains of the two Litoreibacter species and T. arenae was 9-14%. Differential phenotypic properties, together with the observed phylogenetic and genetic distinctiveness, distinguished strain MA1-1(T) from the two Litoreibacter species and T. arenae. On the basis of the data presented, strain MA1-1(T) is considered to represent a novel species of the genus Litoreibacter, for which the name Litoreibacter meonggei sp. nov. is proposed. The type strain is MA1-1(T) ( = KCTC 23699(T)  = CCUG 61486(T)). In this study, it is also proposed that Thalassobacter arenae is reclassified as a member of the genus Litoreibacter, Litoreibacter arenae comb. nov. (type strain GA2-M15(T)  = DSM 19593(T)  = KACC 12675(T)). An emended description of the genus Litoreibacter is also presented.

Non-specific defensive factors of the Pacific oyster Crassostrea gigas against infection with Marteilioides chungmuensis: a flow-cytometric study.

In order to assess changes in the activity of immunecompetency present in Crassostrea gigas infected with Marteilioides chungmuensis (Protozoa), the total hemocyte counts (THC), hemocyte populations, hemocyte viability, and phagocytosis rate were measured in oysters using flow cytometry. THC were increased significantly in oysters infected with M. chungmuensis relative to the healthy appearing oysters (HAO) (P<0.05). Among the total hemocyte composition, granulocyte levels were significantly increased in infected oysters as compared with HAO (P<0.05). In addition, the hyalinocyte was reduced significantly (P<0.05). The hemocyte viability did not differ between infected oysters and HAO. However, the phagocytosis rate was significantly higher in infected oysters relative to HAO (P<0.05). The measurement of alterations in the activity of immunecompetency in oysters, which was conducted via flow cytometry in this study, might be a useful biomarker of the defense system for evaluating the effects of ovarian parasites of C. gigas.

Isolation and characterization of polymorphic microsatellite DNA markers in the sea squirt Halocynthia roretzi.

The sea squirt Halocynthia roretzi is an important marine food resource species that is found in the waters around Korea. We describe the isolation and characterization of 13 new polymorphic microsatellite loci in 96 sea squirt samples that were collected from the marine environment of Samcheok on the east coast of Korea. The number of alleles that were observed for each locus ranged from six to 32, and the value of expected and observed heterozygosities was 0.504-0.922 and 0.396-0.813, respectively. These markers will be useful tools for future population studies.

Generation of expressed sequence tags for immune gene discovery and marker development in the sea squirt, Halocynthia roretzi.

Expresssed sequence tag (EST) analysis was developed from three cDNA libraries constructed from cells of the digestive tract, gonad, and liver of sea squirt. Randomly selected cDNA clones were partially sequenced to generate a total of 922 ESTs, in which 687 unique ESTs were identified respectively. Results of BLASTX search showed that 612 ESTs (89%) have homology to genes of known function whereas 75 ESTs (11%) were unidentified or novel. Based on the major function of their encoded proteins, the identified clones were classified into ten broad categories. We also identified several kinds of immune-related genes as identifying novel genes. Sequence analysis of ESTs revealed the presence of microsatellite-containing genes that may be valuable for further gene mapping studies. The accumulation of a large number of identified cDNA clones is invaluable for the study of sea squirt genetics and developmental biology. Further studies using cDNA microarrays are needed to identify the differentially expressed transcripts after disease infection.

Identification of softness syndrome-associated candidate genes and DNA sequence variation in the sea squirt, Halocynthia roretzi.

The mortality of sea squirts, Halocynthia roretzi, with softness syndrome threatens the sea squirt aquaculture industry in Asian countries. The molecular approach to understanding the pathogenesis of softness syndrome began with differential gene expression analysis of tissues from normal and dying organisms. In the present study, we show that the expression of Halocynthia roretzi metalloproteinase (HrMMP) was significantly upregulated in the tissues of dying organisms through screening of differentially expressed genes, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR. HrMMP is composed of 482 amino acids, contains a conserved domain found in the astacin family, and has typical metalloproteinase activity. To discriminate between the differential expression of the HrMMP gene in normal and dying organisms, we cloned the HrMMP gene promoter and identified a polymorphism in the HrMMP promoter region that resulted in distinct polymorphisms (G/T) at position - 308 bp. These results suggest that organisms with the GT genotype may have more resistance to softness syndrome than those with the TT genotype. These findings suggest that the HrMMP promoter polymorphism may be associated with an increased risk of softness syndrome in cultivated sea squirts and should be evaluated as a candidate molecular marker for the selective breeding of softness syndrome-resistant sea squirts.