A simple method to distinguish two commercially valuable eel species in Japan <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">A. anguilla</Emphasis> using polymerase chain reaction strategy with a species-specific primer

In order to distinguish the two eel species Anguilla japonica and A. anguilla irrespective of unprocessed and processed samples, a quick, convenient method using polymerase chain reaction (PCR) with a species-specific primer was established. The comparison of partial sequences of the mitochondrial 16S ribosomal RNA gene between A. japonica and A. anguilla facilitated designing of a primer pair common to both A. japonica and A. anguilla and a primer specific to A. japonica. PCR was carried out with the three primers for total 110 specimens of A. japonica and A. anguilla. PCR products with the species-specific primer showed two bands for A. japonica and one band for A. anguilla in an agarose gel electrophoresis. This analytical procedure required only a short period and is more convenient than PCR-restriction fragment length polymorphism, one of the standard methods employed for fish species identification.     

Species identification method for <Emphasis Type="Italic">Scombrops boops</Emphasis> and <Emphasis Type="Italic">Scombrops gilberti</Emphasis> based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

ABSTRACT:   Gnomefish Scombrops boops and Scombrops gilberti are commercially important fishes in Japan, but these species are often confused in the markets because of their morphological similarity. To identify these two species, we performed nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis on 16S ribosomal RNA (rRNA) gene and the control region in mitochondrial DNA. Five and 12 nucleotide substitutions were observed between species in the 777-bp 16S rRNA gene and 471-bp control region, respectively. Diagnostic restriction sites for discriminating between S. boops and S. gilberti were found in the 16S rRNA gene, but not in the control region. Polymerase chain reaction (PCR)–RFLP analysis using two enzymes, Eco NI and Mva I, clearly discriminated between S. boops and S. gilberti identified by meristic characters. The PCR–RFLP analysis identified most of the 168 Scombrops young caught in the coastal waters of the Izu and Miura peninsulas as S. boops , suggesting that S. gilberti juveniles are rare in this area.     

Occurrence of estuarine and sea eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and a migrating silver eel <Emphasis Type="Italic">Anguilla anguilla</Emphasis> in the Tokyo Bay area,Japan

Otolith microchemical analyses of the strontium (Sr) and calcium (Ca) concentrations in the eels Anguilla japonica and A. anguilla caught in Tokyo Bay were undertaken to reconstruct the eels’ migratory histories. A. japonica in the yellow stage (immature stage) were caught in a bay without any adjacent rivers or streams. A. anguilla was in the silver stage (early maturing stage), and the eel was confirmed to have just begun spawning migration to the open ocean from Tokyo Bay based on the otolith Sr:Ca ratios, which showed a typical catadromous life history with low Sr:Ca ratio values throughout the eel’s life after recruitment. The mean Sr:Ca ratios in A. japonica from the elver mark to the otolith edge indicated the eels belonged to several general categories of migratory histories, including sea eels (average Sr:Ca ratio ≥6.0 × 10⁻³) and estuarine eels (average Sr:Ca ratio 2.5 to 6.0 × 10⁻³) based on the criteria reported previously in A. japonica. All eels had a certain freshwater life period, although the period was highly variable among fish. These results indicate that A. japonica has a flexible pattern of migration, with the ability to adapt to various habitats and salinities.     

Development of a quantification assay for the cysts of the toxic dinoflagellate <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> using real-time polymerase chain reaction

ABSTRACT:   The cysts of toxic dinoflagellate Alexandrium tamarense are the seed population for the bloom responsible for paralytic shellfish poisoning (PSP). However, it is impossible to identify the Alexandrium spp. cyst on the basis of morphological features. In this study, we prepared A. tamarense cysts by sexual conjugation in laboratory conditions and developed an efficient DNA extraction method for polymerase chain reaction (PCR) assay. Using the A. tamarense cysts, we established the identification and quantification method showing the species specificity and the high sensistivity for A. tamarense cysts using real-time PCR. This assay was also able to detect and quantify the A. tamarense cysts accurately when mixed with excess cysts of A. catenella (Whedon and Kofoid) Balech prepared by conjugation experiment.     

Predation on glass eels of Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> in the Tone River Estuary,Japan

Aquaculture of Japanese eel Anguilla japonica relies upon the natural recruitment of their glass eels (juveniles); however, predation that could influence glass eel recruitment remains unknown. In the present study, we aimed to elucidate the proportion of predation on A. japonica glass eels through stomach content analysis of predatory fishes collected in the estuary region of the Tone River system and its vicinity in Japan. Species of the predated glass eels were identified by DNA barcoding. A total of 270 predatory fishes of 15 taxa was collected over 2 years. The overall proportion of predation on glass eels, genetically identified as Japanese eel, was 0.7%, but this rose to 2.0% when data were limited to fishes caught during the peak months of glass eel recruitment. A glass eel was found in the stomach contents of a channel catfish Ictalurus punctatus, an invasive species in this river system, and a blackfin sea bass Lateolabrax latus. These fishes are therefore considered potential predators of A. japonica glass eels. However, as the proportion of predation was low, and the glass eels represented only small proportions of predator stomach contents, further investigation is needed for a better understanding of predation on A. japonica glass eels, and its effects on the early life stages of this endangered species.     

Anosmic Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> can no longer detect magnetic fields

ABSTRACT:   Magnetosensitivity in anosmic Japanese eel Anguilla japonica was examined by conditioning and electrocardiography. Five eels from a freshwater farm in Kagoshima, Japan, were made anosmic by injection of boiling petroleum jelly (130–140°C) into the nasal cavity, a technique widely used in studies on olfaction and olfaction-related behavior of eels. Another five eels were not injected with boiling petroleum jelly and served as controls. Conditioning consisted of exposure of eels to an imposed magnetic field of 192 473 nT. After complete conditioning of the eels, the imposed magnetic field was reduced from 192 473 nT to 12 663 nT during the tests. The control eels showed a significant conditioned response (i.e. slowing of the heart beat) to the magnetic field after one set of conditioning runs. The anosmic eels did not respond to the magnetic field, presumably due to damage of the olfactory organ from the jelly injection. The results suggest that Japanese eels have a magnetosensitive organ in or around the nares. At 1 month after the anosmia treatment, there was no visible trauma to the olfactory lamellae of anosmic eels.     

Comparative fishing trials for <Emphasis Type="Italic">Charybdis japonica</Emphasis> using collapsible box-shaped and dome-shaped pots

ABSTRACT:   Comparative fishing trials were undertaken with two commercial collapsible pots for the crab Charybdis japonica . One pot was box-shaped with two slit entrances at the ends, while the other was dome-shaped with two open funnel entrances. Pots were placed at the bottom of a pond that is connected to Kagoshima Bay. Fishing trials were carried out using a 1-day soaking time; the bait used was mackerel. The box-shaped pot had higher catches of crabs and non-target organisms, but the number of large crabs (carapace width >8 cm) was almost half as many as that caught with the dome-shaped pot. The catch of non-target species was also greatly reduced in the dome-shaped pot, and fish caught were mostly enmeshed in the netting. The reduction in catches of small crabs and non-target organisms was due to the nature of the dome-shaped pot's larger mesh size and open entrances, which allowed for escape. The dome-shaped pot seemed more efficient for harvesting commercial size crabs, while the box-shaped pot seemed better for removing all crab sizes.     

Effects of environmental variables on recruitment of <Emphasis Type="Italic">Anguilla japonica</Emphasis> glass eels in the Yangtze Estuary,China

The recruitment pattern of Anguilla japonica glass eels into the Yangtze Estuary, China was studied during five successive migration seasons (2012–2016). The variation in daily catch was analyzed in relation to environmental factors that affect inshore migration. Two large migration waves of glass eels were observed in each recruitment season: one in the winter (January/February) and the other in the spring (March/April). A generalized additive model indicated that daily changes in glass eel catch were partially affected by local water temperature and tidal range. Water temperature seemed to have a threshold effect in winter, and there were optimal temperature ranges of 6–8 °C and approximately 10.5–12.0 °C associated with different recruitment waves to the Yangtze Estuary. The influence of tides was complex, with an overall positive correlation. The annual glass eel catch fluctuated greatly, but generally did not show a significant downward trend. To achieve sustainable use of glass eels, it is proposed that relevant management and conservation activities be reinforced.     

Gastrointestinal and metabolic effects of feeding schedule on voluntary feed intake and growth of European eel, <Emphasis Type="Italic">Anguilla anguilla</Emphasis>

Gastrointestinal and metabolic influences on short- and medium-term control of voluntary feed intake of European eel were investigated for groups of fish fed at different feeding schedules: 1 meal 2 days⁻¹, 1 meal day⁻¹, 2 meals day⁻¹ and continuous feeding for 12 h and 24 h daily. For fish fed daily, the feeding schedule had no effect on feed intake, metabolism, growth, size variation and body composition. Only fish fed once every 2 days could not completely compensate by increasing their average meal size and showed a reduced feed intake, metabolism and growth. Stomach capacity did not limit meal size and feed intake as large differences in appetite were found for eels with empty stomachs. Feed intake was related to metabolism as the levels of O₂ consumption and NH₄ excretion just before the meal were correlated to average meal size, suggesting that rate (and duration) of metabolism is sensed and is providing information about the metabolic consequences of feed intake and feed processing. Meal size is based on the (expected) scope for metabolism and eels seem to strive to an average daily feed intake, associated with their developmental state (growth potential). Plasticity in this feed intake behaviour, expressed by individual variation in feed intake and day-to-day fluctuations in individual and group intake, is regarded as adaptive on different time scales.     

Genetic comparison between torafugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and its closely related species karasu <Emphasis Type="Italic">Takifugu chinensis</Emphasis>

Abstract:   Sequence analyses of mitochondrial (mt) and nuclear genes were performed for genetic comparison between two Takifugu pufferfish species: torafugu T. rubripes and karasu T.  chinensis . With a sequence coverage of 20% in mtDNA, 640, 308, 344, 522 and 697 bp encoding mt 16S ribosomal RNA (rRNA), adenosine triphosphatase 6 ( ATPase 6 ), nicotinamide adenine dinucleotide dehydrogenase subunit 4 ( ND4 ), ND5 and cytochrome b (cyt b ), respectively, among 24 wild torafugu, 24 wild karasu and six hybrid-like samples, 15% of the torafugu identified by external color patterns showed nucleotide sequences consistent with karasu. Meanwhile, sequences of 60% karasu were consistent with those registered for torafugu (AJ421455). As for the hybrid-like samples, two possessed karasu-specific sequences in some base positions while torafugu-specific sequences in others. The remaining hybrid-like samples possessed torafugu-specific sequences. On the other hand, the mt control region did not show such type of consistency. Analysis of nuclear melanocortin receptor genes ( MC1R , MC4R ) among 54 samples showed 99–100% inter- and intraspecific sequence identity. Partial nuclear 18S  rRNA, complete internal transcribed spacer 1 ( ITS1 ), partial 5.8S  rRNA and ITS2 genes showed similar levels of identity, indicating a very low level of variation in their respective gene fragments between the two Takifugu species.     

Utilization of terrestrial organic matter by the bivalve <Emphasis Type="Italic">Corbicula japonica</Emphasis> estimated from stable isotope analysis

ABSTRACT:   Carbon and nitrogen isotopic ratios in tissue of the bivalve corbicula ( Corbicula japonica ) and particulate organic matter (POM) were measured along a salinity gradient in the Kushida Estuary, Japan. The bivalve exhibited a gradual isotopic enrichment from the uppermost estuarine site (δ¹³C = −24.8‰ and δ¹⁵N = 8.6‰) to the marine site (δ¹³C = −16.1‰ and δ¹⁵N = 11.8‰). Using the concentration-weighted mixing model, the bivalves' food source is estimated from the isotope values for the bivalves and POM from terrestrial plants, marine phytoplankton and benthic microalgae. The results indicated that the contributions of benthic micro algae and phytoplankton were small, while terrestrial particulate matter is significantly important for the corbicula diet, although the contribution varies among sampling sites.     

Estimation of gonadal status of Japanese female eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> by a single injection of salmon pituitary extract

ABSTRACT:   Cultivated eels often either lack a part of the ovary (abnormal) or do not have any gonadal tissue (completely sterile). These abnormalities may cause a failure of induction of sexual maturation of female eels. In order to effectively induce sexual maturation and obtain good quality eggs, female eels having normal ovaries should be selected before induction of maturation. The present study examined effects of injection of salmon pituitary extract (SPE) on serum estradiol-17β levels to estimate gonadal status of sexually immature Japanese eels. At the initial stage (just prior to SPE injection), serum estradiol-17β levels were low and no significant difference was observed among eels possessing the different gonad types (normal, abnormal, and sterile), suggesting that gonadal status cannot be estimated by the measurement of serum estradiol-17β levels. A single injection of SPE significantly increased serum estradiol-17β levels in eels possessing normal and abnormal ovaries, but not in sterile eels. Serum estradiol-17β levels significantly increased from 24 h and were high on day 3. These data indicate that eels possessing ovaries can be distinguished from sterile eels by measurement of serum estradiol-17β between 1 and 3 days after a single injection of SPE.     

Complete mitochondrial DNA sequence of the Japanese flying fish <Emphasis Type="Italic">Cypselurus hiraii</Emphasis>

ABSTRACT:   In this study, to develop a technique that enables authentication of processed seafood, the complete nucleotide sequence of the mitochondrial genome for the Japanese flying fish Cypselurus hiraii was determined. Three segments spanning the entire genome were amplified using polymerase chain reaction, and products were subsequently used as templates for direct sequencing with 60 primers. The genome (16 528 base pairs) was found to contain the same 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as those found in other vertebrate mitochondrial genomes, with the gene order being identical to that typical of vertebrates. A major non-coding region between the tRNA^Pro and tRNA^Phe genes (868 base pairs) appears to be the control (D-loop) region, as it has several conserved blocks characteristic of control regions.     

Swimming physiology of European silver eels (<Emphasis Type="Italic">Anguilla anguilla</Emphasis> L.): energetic costs and effects on sexual maturation and reproduction

The European eel migrates 5,000–6,000 km to the Sargasso Sea to reproduce. Because they venture into the ocean in a pre-pubertal state and reproduce after swimming for months, a strong interaction between swimming and sexual maturation is expected. Many swimming trials have been performed in 22 swim tunnels to elucidate their performance and the impact on maturation. European eels are able to swim long distances at a cost of 10–12 mg fat/km which is 4–6 times more efficient than salmonids. The total energy costs of reproduction correspond to 67% of the fat stores. During long distance swimming, the body composition stays the same showing that energy consumption calculations cannot be based on fat alone but need to be compensated for protein oxidation. The optimal swimming speed is 0.61–0.67 m s⁻¹, which is ~60% higher than the generally assumed cruise speed of 0.4 m s⁻¹ and implies that female eels may reach the Sargasso Sea within 3.5 months instead of the assumed 6 months. Swimming trials showed lipid deposition and oocyte growth, which are the first steps of sexual maturation. To investigate effects of oceanic migration on maturation, we simulated group-wise migration in a large swim-gutter with seawater. These trials showed suppressed gonadotropin expression and vitellogenesis in females, while in contrast continued sexual maturation was observed in silver males. The induction of lipid deposition in the oocytes and the inhibition of vitellogenesis by swimming in females suggest a natural sequence of events quite different from artificial maturation protocols.     

Effect of salinity on occurrence of notochord deformities in Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> larvae

One of the difficulties to rear Anguilla japonica larvae is the frequent occurrence of notochord deformities. We tested the effect of salinity on the occurrence of the deformities, because we have been using 50 % diluted seawater (50 % SW) for glass eel production, on the basis of the fact that intermediate salinity saves energy due to lower cost for osmoregulation and contributes higher survival and growth rates. We reared 6-day-old larvae in 50 and 100 % SW for 85 days and observed their morphology. The occurrence rate of deformed larvae, including kyphosis and scoliosis, was significantly higher in 50 % SW (35.8 %) than in 100 % SW (25.4 %), while survival rate was significantly higher in 50 % SW (69.8 %) than in 100 % SW (32.3 %) and growth in 50 % SW (mean body depth: 7.9 ± 5.3 mm) was better than in 100 % SW (6.8 ± 4.6 mm). We speculate that the most of severely deformed larvae could not survive in the tougher condition of 100 % SW, showing the lower occurrence of deformed larvae. Eventually, the yield of normal larvae after 85 days rearing was 1.9-fold higher in 50 % SW than in 100 % SW, implying that the advantage of 50 % SW for rearing eel larvae outweighs the risk of deformities.     

Identification of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> and <Emphasis Type="Italic">Undaria undarioides</Emphasis> Laminariales,Phaeophyceae using mitochondrial 23S ribosomal DNA sequences

ABSTRACT:   Mitochondrial 23S ribosomal (r) DNA were sequenced from two Undaria species. The 23S rDNA from seven Undaria pinnatifida individuals were 2707 bp in length, whereas the 23S rDNA from four Undaria undarioides individuals were 2708–2709 bp in length. We found 15–20 nucleotide substitutions and 1–2 gaps between U. pinnatifida (the major haplotype) and U. undarioides . Based on the differences in sequences, we carried out PCR/RFLP analyses to distinguish between U. pinnatifida and U. undarioides , which showed different PCR/RFLP patterns upon Hin fI digestion. Sequence differences and PCR/RFLP analyses of mt 23S rDNA are useful for species identification of Undaria species .