Assessing the end-point temperature of heated fish and shellfish meats

ABSTRACT: Attempts have been made to assess the end-point temperature (EPT) of heated fish and shellfish meats by using the coagulation method together with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and enzyme activity determination. Unfrozen and frozen fish and shellfish meats were heat-treated at different selected temperatures with 0, 15 or 30 min holding times. Proteins were extracted with NaCl solution. The coagulation method was able to determine EPT of heated fish and shellfish meats between 60 and 67°C. SDS-PAGE patterns of the filtrates from heated meats were closely related to the results of the coagulation method and enzyme activity determination. Two proteins responsible for producing coagulum of fish meat extracts seem to be lactate dehydrogenase (LDH) and glyceraldehyde phosphate dehydrogenase (GAPDH). End-point temperatures determined by these methods were not significantly different between unfrozen and frozen samples. On the contrary, a highly thermostable protein with a molecular mass of 35 kDa was detected in heated shellfish meats up to 108°C. In scallop adductor muscle, this highly thermostable protein was found to be the tropomyosin subunit from its amino acid composition and their partial sequences. Tropomyosin could be used as an EPT indicator up to 108°C for heated shellfish meats.     

Complete mitochondrial DNA sequence,gene organization and genetic variation of control regions in <Emphasis Type="Italic">Parargyrops edita</Emphasis>

ABSTRACT:   The complete nucleotide sequence of the mitochondrial genome of Parargyrops edita was determined by gene amplification using long polymerase chain reaction and direct sequencing techniques. The genome with 16 640 bp contained 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes). The content and order of genes was identical to those in typical teleosts. The major non-coding region of 964 bp in length with several conserved sequence features were identified as the control region (CR). Both COI and ND4 began with a GTG start codon, which is unusual in other fishes. The genetic variation of the CR from 27 wild samples was evaluated. A total of 536 bp consensus sequence of CR was aligned and 26 unique haplotypes were identified. The haplotype diversity (h) was estimated to be 0.997 (standard deviation [SD] 0.011) and nucleotide diversity ( π ) was 0.014 (SD 0.008) for the sample collected from coastal waters of Guangdong Province. It showed a very high level of genetic variation in the sample and also suggested that mtDNA CR sequence analysis can be use to evaluate the genetic diversity and genetic structure of P. edita .     

Early development of the silver catfish Rhamdia quelen (Quoy & Gaimard, 1824) (Pisces:Heptapteridae) from the São Francisco River Basin, Brazil

The silver catfish, Rhamdia quelen , is endemic to North, Central and South America with high aquaculture potential and wide acceptance in the market. Breeder fish were subjected to induced reproduction through hypophysation using a crude common carp pituitary extract. Egg characteristics, oocyte surface ultrastructure and histology of larval ontogenesis until whole yolk resorption were described for the first time for this species. Oocytes and semen were obtained by manual extrusion, and fertilization was conducted using the dry method. After fertilization, eggs were kept in incubators at 24 °C. The embryonic development was monitored using a stereomicroscope every 10 min until hatching. To analyse the larval development, larvae samples were collected from incubators daily until the fifth day, fixed in Bouin's fluid and subjected to routine histological techniques. The oocyte extrusion occurred 8 h after the second hormone dose at 26 °C. The oocytes were spherical, non-adhesive and yellow, with a diameter of 1471.75±47.63 μm. Scanning electron microscopy revealed a thin jelly coat covering the zona radiata in the animal pole around the micropyle. The blastopore closure occurred within 8 h after fertilization, and the fertilization rate was 79.9±5.2% at 24 °C. Embryonic development was completed within 25 h 30 min after fertilization. The complete resorption of the yolk and the formation of the digestive system organs and the mouth opening occurred on the fifth day, indicating a need for exogenous feeding. The results of this study provide information important for improvement in R. quelen culture and management.     

Paralytic shellfish poisoning toxin analysis of the genus <Emphasis Type="Italic">Alexandrium</Emphasis> (Dinophyceae) occurring in Korean coastal waters

ABSTRACT:   Paralytic shellfish poisoning (PSP) toxins produced by Alexandrium isolates from Korea were analyzed by high-pressure liquid chromography. Species designation of the regional isolates was determined by morphological criteria and ribotyping inferred from sequences of the 28S rDNA D1-D2 region. Toxin analysis performed at the exponential growth phase, revealed that the two strains of A. fraterculus were non-toxic, while the strains of A. tamarense and A. catenella were toxic. Toxic isolates DPC7 and DPC8 of A. catenella produced GTX1, 2, 3, 4, 5, dcGTX2, 3, C1, 2, neoSTX and STX with trace or non-detectable levels of C3 and C4, while isolates UL7, KDW981, SJW97043, SJW97046, KJC97111 and KJC97112 of A. tamarense produced GTX1, 2, 3, 4, dcGTX3, C1, 2, neoSTX with trace or non-detectable levels of C3, 4, dcSTX and STX, and no GTX5 and dcGTX2. The major toxins produced by A. catenella were C1 +2, and those of A. tamarense were C1 +2 and GTX4 in most of the isolates. A. tamarense strains other than SJW97046 produced a relatively high proportion of carbamate toxins, reflecting the high toxicity scores of shellfish intoxication in sampled coastal areas. Two representative toxic isolates, A. tamarense SJW97043 and A. catenella DPC7, were cultured for 30 days in batch mode and subjected to toxin analysis at 5-day intervals. Comparison of toxin productivity in terms of total toxin content, toxin components, and their variations with culture age revealed marked differences between the two strains.     

Changes in purine content of tilapia surimi products during processing

ABSTRACT:   Changes in purine-related compounds of tilapia surimi product during processing were investigated. The washing step could result in about 60% decrease of total purine content in tilapia mince during processing. The main released purine substance was inosine monophosphate. The major reducing effect was conducted in the first 10 min during washing. No significant changes were observed after washing for 20 and 30 min. The lowest total purine content of tilapia surimi product was obtained with repeating the washing step twice. Thus, this procedure could reduce the purine content of tilapia mince from a high purine content level to a middle level. The gel strength of tilapia surimi product increased with increasing washing duration within 30 min. However, tilapia surimi product with a middle purine content and acceptable gel strength might be produced by washing twice in 10 min during processing.     

Roles of water quality and disinfectant application on inactivation of fish pathogenic Streptococcus agalactiae with povidone iodine,quaternary ammonium compounds and glutaraldehyde

Streptococcosis is an important bacterial disease in Nile tilapia causing severe economic losses to tilapia aquaculture worldwide. The effects of water quality (low‐ [LS] and high‐level [HS] soiling, to mimic clean or dirty surface conditions and temperatures) and disinfectant application (diluted concentrations and exposure time) were characterized on the inactivation of Streptococcus agalactiae isolated from diseased tilapia. Five isolates were tested against three commercial disinfectant products with the main ingredients being povidone iodine (Anidine 100?; AD), benzalkonium chloride (Better BKC 80%?; BKC 80), and a mixture of quaternary ammonium compounds and glutaraldehyde (Chloraldehyde?; CR). CR demonstrated highest efficacy to S. agalactiae inactivation, followed by BKC 80 and AD, respectively. Higher‐level soiling, low temperature, diluted concentrations and short exposure time all decreased the disinfectant efficacy. CR and BKC 80 provided more than 5‐log inactivation at 1‐min exposure at 20°C under HS conditions, and also with ten‐fold‐diluted concentrations at 60‐min exposure time at 30°C. However, AD required 10‐min exposure to effectively remove bacteria under LS conditions at 30°C. The results could facilitate aquaculture management planning that leads to operating cost reductions and improvements in biosecurity.     

Methods of microbial control in marine fish larval rearing: clay‐based turbidity and passive larval transfer

This study focused on methods to reduce bacterial loads in the larval culture tanks of California yellowtail (Seriola lalandi). We conducted two trials to evaluate methods to minimize bacterial loads in the larval rearing water. The first trial examined the use of bentonite clay as a turbidity agent to replace algae in a green water‐type environment. This trial consisted of three treatments: (1) clay with continuous feeding (CCO), (2) clay with batch feedings (CBA) and (3) algae paste with batch feedings (ALG). The results showed that both clay treatments had significantly fewer Vibrio colonies in the water column (CBA – 180 ± 78; CCO – 377 ± 120 CFU mL^?1) than the ALG treatment (5692 ± 2396 CFU mL^?1) after 14 days of culture. Survival was significantly higher in the CCO treatment (14.1 ± 2.6%) than either the CBA (2.3 ± 0.5%) or ALG treatments (2.8 ± 1.5%). The second trial attempted to limit bacterial loading in the larval culture tank by passively transferring the larvae into an adjacent, clean tank at 1, 5 and 9 days post hatch during the first 2 weeks of culture. The results from this trial showed that after 12 days of culture, water in the transfertank had fewer Vibrio colonies (1025 ± 541 CFU mL^?1) than the water in the control tanks (1962 ± 1415 CFU mL^?1). Also, survival was significantly higher among larvae that were transferred (43.9 ± 13.5%) than in the control tanks (23.1 ± 6.3%).     

Menthol as anaesthetic for lambari Astyanax altiparanae (Garutti & Britski 2000): attenuation of stress responses

The anaesthetic potential of menthol was evaluated in lambari Astyanax altiparanae by exposing fingerlings to concentrations 50, 100, 150, 200, 250 and 300 mg L^?1 and measuring the induction and recovery times to deep anaesthesia, the mortality rates during and 96 h after procedure and after 6 min of continuous exposure. The effect of menthol on stress responses were evaluated by comparing glucose and cortisol levels of juveniles subjected to anaesthesia (50 mg L^?1), stress (air exposure) or pre‐anaesthesia associated to stress. All concentrations induced deep anaesthesia within 0.5 to 1 min, with recovery between 1.83 and 4.16 min, without mortality during the induction or up to 96 h after exposure. Induction time decreased and recovery time increased linearly as the menthol concentration increased. Continuous exposure to 50, 100 and 150 mg L^?1 concentrations resulted in mortality rates of 0%, 20% and 80% respectively. Anaesthesia or air exposure increase blood glucose but prior anaesthesia with menthol suppressed the elevation of cortisol caused by stress. Menthol has an anaesthetic effect and attenuates the stress response in lambari and 50 mg L^?1 is the most effective concentration for inducing deep anaesthesia in 1.0 min, safe for up to 6 min exposure.     

Effect of short‐term treatment with potassium permanganate on stress markers and blood biochemistry in goldfish Carassius auratus

Potassium permanganate (PM) is a therapeutic agent in aquaculture with strong oxidant properties. In this study, the effects of potassium permanganate treatment (PMT) on stress response and blood biochemistry were investigated in goldfish Carassius auratus. Two triplicate groups of fish were exposed to either 10 mg L^?1 PM or clean water, over 30 min. Blood samples were collected at 0 and 0.5 h after exposure as well as 3 and 24 h. PMT caused significant increase in serum cortisol and glucose levels, 0.5 h post exposure, which stayed elevated until 24 h. Sodium decreased after exposure and did not return to initial levels. Chloride decreased at 0.5 and 3 h, but returned to initial levels at 24 h. Calcium decreased at 0.5 h and returned to initial at 3 h, but significantly increased at 24 h. Total protein, albumin and globulin levels increased at 3 h and stayed elevated until 24 h. Albumin:globulin ratio (A:G) levels decreased at 24 h post exposure. Results indicated that short‐term PMT caused stress response, hydromineral imbalance and possibly change in blood protein profile in goldfish, which lasted until 24 h post treatment.     

Development and application of fluorescence in situ hybridization (FISH) method for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella in cultured and natural seawater

ABSTRACT:   The toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and A. catenella (Whedon and Kofoid) Balech produce potent neurotoxins, such as saxitoxin and gonyautoxin and have been mainly responsible for paralytic shellfish poisoning (PSP) in Japan. To prevent a negative effect on the fishery industry, it is necessary to identify these toxic species precisely and rapidly before and during the bloom. In this paper, a rapid and simple protocol of a fluorescence in situ hybridization (FISH) method using ribosomal RNA (rRNA)-targeted probes has been established for identifying the cultured strains and natural cells of A. tamarense and A. catenella . Using the FISH method established in this study, it was possible to identify these toxic species species-specifically and rapidly, within 30 min. The procedure of detection constituted three steps: (i) fixation/dehydration; (ii) hybridization; and (iii) washing; this made the identification simple. Moreover, this method did not require either special techniques or equipment, and the cost for detection was low. The specificity, rapidity, and simplicity of the developed method suggest that it might be useful for routine monitoring of these toxic microalgae.     

Confirmation of yessotoxin production by the dinoflagellate <Emphasis Type="Italic">Protoceratium reticulatum</Emphasis> in Mutsu Bay

ABSTRACT:   Yessotoxin (YTX) is a shellfish toxin and its contamination in bivalves has seriously damaged shellfish industries. The biogenetic origin of YTX was identified as the dinoflagellate Protoceratium reticulatum (Claparède et Lachmann) Bütschli collected in New Zealand and Yamada Bay, Iwate in Japan. Scallops cultured in Mutsu Bay, Japan, were frequently contaminated with YTX, however, occurrence of P. reticulatum in this bay and YTX production by the local strains have not been investigated. Eight strains of P. reticulatum , isolated from the bay, were cultured in the laboratory, and analyzed by fluorometric high-pressure liquid chromatography and liquid chromatography/mass spectrometry methods for YTX production and composition. All strains tested were confirmed to produce YTX, and none of them produced known YTX analogs. Toxin amount and composition differed from strain to strain. This result is also confirmation of one of the biogenetic origins of YTX in Mutsu Bay.     

Efficacy of tricaine methanesulphonate and clove oil as anaesthetics for juvenile cobia Rachycentron canadum

Six experiments were designed to determine the optimal anaesthetic dosage of tricaine methanesulphonate (TMS) and clove oil that could be used safely on juvenile cobia Rachycentron canadum of two sizes [G1=4.9±0.8 g; G2=13.9±3.1 g]. We documented the stage of anaesthesia and the acute toxicity as 96 h LC₅₀ (lethal concentration 50% population) at various exposure times of the two anaesthetics. At 10 min induction time, the TMS 96 h LC₅₀ was 93.9 mg L⁻¹ in G1 and 97.0 mg L⁻¹ in G2. Compared with clove oil, the 96 h LC₅₀ was 60.0 mg L⁻¹ in G1 and 69.8 mg L⁻¹ in G2. The difference between the two groups (G1, G2) did not influence anaesthesia safety ( P >0.05). Rachycentron canadum achieved stage 3 anaesthesia more rapidly at a lower clove oil concentration level (40 mg L⁻¹, 10 min) than TMS (60 mg L⁻¹, 10 min), but the recovery period of clove oil, was significantly longer. Clove oil was the most effective in reducing the short-term stress induced by routine biometry (20 mg L⁻¹, 10 min) and also by transporting (1 mg L⁻¹, 8 h). Whereas, for long-term exposure, 40 mg L⁻¹ TMS was found to be safe.     

Pharmacokinetics of oxolinic acid in gilthead sea bream, Sparus aurata L.

This is the first study on the pharmacokinetic parameters of oxolinic acid (OA) in gilthead sea bream, Sparus aurata L. The kinetic profile of OA was studied after a single intravascular injection (20 mg kg⁻¹) in 100 g fish at 20 °C. The distribution half-life ( t _1/2α) and the elimination half-life ( t _1/2β) of the drug were found to be short (0.51 and 12.60 h, respectively). The drug penetration from the plasma to the tissues was adequate as the apparent volume of distribution of the drug at steady-state ( V _d(ss)) was found to be 2.11 L kg⁻¹. The mean residence time ( MRT ) of OA was short (14.25 h) and the total clearance rate ( Cl _T) of the drug was low (0.15 L kg⁻¹ h⁻¹). The bioavailability ( F %) of OA following oral administration (30 mg kg⁻¹) was also low (14%). Maximum values were observed for muscle at 0.5 h after injection, with levels declining as with subsequent sampling. At the first two time points (0.5 and 1 h) plasma levels of OA were higher than muscle, however, the reverse was evident for subsequent samples. Following oral administration, highest muscle levels were found at 16 h and, with the exception of the 24-h sampling, muscle OA concentrations were higher than plasma at all time points. The fast elimination of OA suggests short withdrawal times with reference to human consumption of treated fish.     

Alcalase treatment for elimination of stickiness in pikeperch (Sander lucioperca L.) eggs under controlled conditions

Elimination of egg stickiness is an important factor in artificial reproduction of pikeperch (Sander lucioperca L.). This study was conducted to evaluate the effectiveness of Alcalase enzyme to remove the adhesive layer of pikeperch eggs. The eggs were treated with Alcalase at 0.5, 1.0, 1.5, 2.0 and 5.0 mL L^?1 or a milk/talc solution 2 min post insemination. Duration of exposure was 2 min in Alcalase and 60 min in milk/talc. The highest, albeit not significant, hatching rate (85.4%) was found with 1.5 mL L^?1 of Alcalase, but hatching rates were similar in 0.5, 1.0 and 2.0 mL L^?1. Hatching rates were significantly lower groups treated with 5.0 mL L^?1 Alcalase enzyme (56.4%) compared to groups treated with milk powder and talc (61.3%). Nominally complete removal of adhesiveness was observed in 1.5 and 2 mL L^?1. All Alcalase treatments led to significantly lower incubation duration compared with the traditional milk/talc treatment. The application of Alcalase successfully eliminated pikeperch egg stickiness in less time than with traditional milk/clay/talc methods.     

Cryopreservation of trochophore larvae from the hard clam Mercenaria mercenaria: Evaluation of the cryoprotectant toxicity,cooling rate and thawing temperature

Aquaculture of hard clams Mercenaria mercenaria is a $65 million industry along the east coast and Gulf of Mexico coast in the United States. The goal of this study was to develop a preliminary protocol to cryopreserve trochophore larvae of hard clams. The objectives were to evaluate the: 1) toxicity of cryoprotectants, including dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol and glycerol, at 5, 10, 15 and 20% for exposure time of 1, 15, 30, 45, 60 and 75 min; 2) effects of cooling rates (5, 10, 20 and 30°C/min for the first trial; and 1, 3 and 5°C/min for second trial from 4 to ?80°C), thawing temperature (30, 40 and 50°C) and their interactions on post‐thaw viability. A basic protocol was concluded as: 15‐hr trochophore larvae mixed with DMSO or propylene glycol (5, 10%), equilibrated for 15 min, cooled in a programmable freezer from 4 to ?80°C at a cooling rate of 5°C/min and thawed at 50°C for 6 s. With this protocol, the immediate post‐thaw trochophore survival was 23 ± 14%, and survival to D‐stage was 27 ± 14%. This is the first report on larval cryopreservation in the hard clam and would have application for genetic breeding and seed production.     

Developmental stages, larval and post-larval growth of angelwing clam Pholas orientalis

Angelwing clam were induced to spawn by thermal stimulation. Mature eggs measured 50 μm in diameter. Cell division occurred within 36 min after fertilization. Mobile trochophore larvae were seen after 12 h and larvae developed within 18 h. Reared on a diet of Isochrysis galbana the larvae reached the umbo stage in 6–7 days. On day 10 the foot could be seen and settlement occurred if a suitable substrate was present. The larvae completed metamorphosis into juveniles within 20 days after settling.     

The application of tannic acid to the elimination of egg stickiness at varied moments of the egg swelling process in pikeperch,Sander lucioperca (L.)

The aim of this article was to analyse the process of pikeperch, Sander lucioperca, egg swelling and to apply tannic acid to eliminate egg stickiness at different moments of the swelling process on artificially obtained eggs. The first experiment involved observation of egg swelling process and the second determined the effect of temperature (12, 14 and 16°C) on the egg swelling rate. The third experiment involved elimination of egg stickiness in a tannin solution (0.75 g L^?1) where eggs were submerged in a solution for 0.5, 1, 2 and 5 min – 5, 10, 15, 20, 25 and 30 min following gamete activation. The results indicate that the pikeperch egg swelling process lasts 30 min. It was found that the temperature did not affect the process duration. The results of the third experiment showed that the effectiveness of tannic acid application in egg stickiness elimination increases with time. The best result was obtained in groups of eggs submerged for 1 and 2 min (86.5% and 80.5% of larvae were obtained respectively) 30 min following the gamete activation. The results presented in this study for the first time indicate the possibility of highly effective procedure of egg stickiness elimination with tannic acid in pikeperch aquaculture.     

Suspension feeding of the ark shell <Emphasis Type="Italic">Scapharca subcrenata</Emphasis> as a function of environmental and biological variables

ABSTRACT:   To assess the roles of the ark shell Scapharca subcrenata in material cycling in the northern part of Ariake Sound (Mae-no-umi), Japan, filtration of the ark shell fed on small diatoms was examined as a function of environmental and biological variables. The clearance rate ( CR ) specific to the soft-body dry weight ( w ) of the animals (shell length, 8–26 mm) followed a power function of w with an exponent of −0.35. Over the range of 10–20°C, CR increased 2.7 times, and filtration did not occur below a salinity of 14 practical salinity unit. Neither food concentration (10–40 µg/L of chlorophyll- a ) nor weight-specific daily ration (0.5–6%/day in terms of ash-free dry weight) notably affected CR . Using this information on CR as well as data regarding abundance and size distribution, the population filtration rate was calculated to be 1.6 m³/m² per day in the ark-shell culturing ground of Mae-no-umi (mean water depth, ∼3 m), corresponding to the potential to locally process a volume of water equivalent to the water column within 2 days. Because the culturing ground accounts for 12% of Mae-no-umi (mean water depth, ∼8 m), the ark shell seems to play an important role in its material cycling.     

Effects of developmental stage, seawater concentration and rearing temperature on cryopreservation of Pacific oyster Crassostrea gigas larvae

ABSTRACT: For the development of a stepwise cryopreservation technique for larvae of the Pacific oyster Crassostrea gigas , various conditions were examined. Larvae at 9, 12, 15, 18 and 21 h after insemination were cooled at a rate of −1°C/min (seeding at −8°C for 15 min) and then plunged into liquid nitrogen at −35 or −40°C using 1.5 M dimethyl sulfoxide (DMSO) and 250 mM trehalose as cryoprotectants. Among these larvae, 15 h after insemination (the trochophore stage before formation of the shell gland) showed the highest motility and the best external appearance after thawing. Trochophore larvae were cryopreserved in preservation media containing different dilutions (1/4, 1/6, 1/8, 1/10 and 1/30) of seawater. Larvae preserved in the 1/4 seawater medium showed the highest appearance of shelled larvae 4 days after thawing. Trochophore larvae reared in seawater at 21, 25 or 29°C were cryopreserved for 8 months and then reared at 26°C after thawing. Larvae reared at 25°C showed the highest survival rate and normal larval ratio at day 6 after thawing, although larvae reared at 21°C showed the highest rates until day 4. One larva developed at 25°C succeeded to settle.     

Inhibitory effect of 2,4-dibromophenol and 2,4,6-tribromophenol on larval survival and metamorphosis of the sea urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis>

ABSTRACT:   As a possible factor leading to the low recruitment level of sea urchins in kelp forests, the inhibitory effect of 2,4-dibromophenol (DBP) and 2,4,6-tribromophenol (TBP) released from the large perennial brown algae Ecklonia kurome and Eisenia bicyclis on survival and metamorphosis of eight-armed larvae of the sea urchin Strongylocentrotus nudus was examined. The percentage of larvae that underwent metamorphosis in filtered sea water after 1 h exposure to one-half dilution of saturated dibromomethane solution (∼60 ppm) as a chemical inducer reached approximately 100% after 1 h, while that in filtered sea water containing 1 ppm TBP was reduced to 73%. This was further reduced to less than 40% in the presence of 10 and 20 ppm TBP after 2 h. In filtered sea water containing 1 and 10 ppm DBP, the proportion of metamorphosed larvae was reduced markedly to 43 and 5% after 2 h, respectively. All larvae exposed to 50 ppm TBP and to 20 and 50 ppm DBP died after 1 h. These findings suggest that DBP is more toxic than TBP for sea urchin larvae, strongly inhibiting their metamorphosis.