Biochemical properties of the sensitivity to GABA<Subscript>A</Subscript>ergic ligands,Cl<Superscript>−</Superscript>/HCO<Subscript>3</Subscript><Superscript>−</Superscript>-ATPase isolated from fish (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) olfactory mucosa and brain

This paper presents a comparative study of the roles of Cl^? and HCO₃ ^? in the functioning of the GABA_AR-associated Cl^?/HCO₃ ^?-ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl^?(15–30 mmol l^?1), whereas the enzyme from MBNs was not significantly affected by Cl^?. By contrast, HCO₃ ^?(15–30 mmol l^?1) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl^?(15 mmol l^?1)/HCO₃ ^?(27 mmol l^?1) and these activities were inhibited in the presence of picrotoxin (100 μmol l^?1), bumetanide (150 μmol l^?1), and DIDS (1000 μmol l^?1). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~?56 kDa that binds [³H]muscimol and [³H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-³²P and Mg²⁺, as well as Cl^?/HCO₃ ^? sensitive dephosphorylation, is also associated with this 56 kDa peptide. Both preparations also showed one subunit with molecular mass 56 kDa that was immunoreactive with GABA_AR β3 subunit. The use of a fluorescent dye for Cl^? demonstrated that HCO₃ ^?(27 mmol l^?1) causes a twofold increase in Cl^? influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO₃ ^? had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl^? and HCO₃ ^? in the regulation of the Cl^?/HCO₃ ^?-ATPases functioning in neurons with different specializations.     

Purification and characterization of α<Subscript>1</Subscript>-proteinase inhibitor and antithrombin III: major serpins of rainbow trout (<Emphasis Type="Italic">Oncorhynchuss mykiss</Emphasis>) and carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) blood plasma

The main serine proteinase inhibitors of rainbow trout (Oncorhynchuss mykiss) and common carp (Cyprinus carpio) blood plasma were isolated and purified. The investigated inhibitors, α₁-proteinase inhibitor (α₁-PI) and antithrombin III (AT III), act by forming stable complexes with target proteinases. The association rate constants k _on for the interaction of fish plasma inhibitors with several serine proteinases have been determined: k _on for both carp and rainbow trout α₁-PI were >10⁷ M⁻¹ s⁻¹ for human neutrophil elastase, and in the case of bovine trypsin and chymotrypsin k _on values were 2.0–5.2 × 10⁶ M⁻¹ s⁻¹. Association rate constants k _on for the interaction of carp and rainbow trout AT III with bovine trypsin and thrombin were about 1.3 × 10⁴–7.9 × 10⁵ M⁻¹ s⁻¹ without and >10⁷ M⁻¹ s⁻¹ in presence of heparin; so antithrombins require the presence of heparin to become effective proteinase inhibitors. The high degree of homology of the estimated amino acid sequences of fish inhibitors reactive site loops confirms their similarity with other proteinase inhibitors from the serpin family.     

Supercritical CO<Subscript>2</Subscript> extraction and concentration of n−3 polyunsaturated fatty acid ethyl esters from tuna cooking juice

Cooking is an important step in the tuna canning process; it usually produces functional solubilized proteins and lipids. The objective of this study was to recover and increase the concentration of functional n−3 fatty acids from the lipids in cooking juice. The lipids contained 12.98% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) after ethyl esterification and increased to 37.4% with subsequent urea adduct fractionation. Supercritical carbon dioxide extraction was further used to increase the concentration of EPA and DHA ethyl esters. The ratio of EPA+DHA ethyl esters (high-molecular-weight components) to that of C₁₆ and C_18∶1 fatty acid ethyl esters (low-molecular-weight components) was used as a separation index for evaluating the process variables. Experimental results indicated that a high CO₂ density caused a low separation factor. At 1500 psig and 328.2 K, the extraction collected over 600 L CO₂, displaying an accepted concentration factor of EPA+DHA ethyl esters herein. About 80% yield of EPA and DHA was obtained and the ethyl esters increased from 37.4% to 54.3%.     

Cloning,expression, and induction by 17-β estradiol (E<Subscript>2</Subscript>) of a vitellogenin gene in the white cloud mountain minnow <Emphasis Type="Italic">Tanichthys albonubes</Emphasis>

Vitellogenins (Vtgs), the precursors for the yolk proteins, are very important for the embryonic development of teleosts, and have also been studied extensively as biomarkers for environmental estrogenic mimics. The cDNA for a Vtg was isolated from the liver of the female white cloud mountain minnow (Tanichthys albonubes) by 3′- and 5′-RACE methods. It is 4,171 bp in full length, and encodes a putative protein of 1,326 amino acids. This putative Vtg, designated as wcmmVtg, contains complete portions of LVI and PV, but lacks the C-terminal half of LVII and thus belongs to type I vitellogenin. In addition to the liver of the female fish, wcmmVtg was also shown to be expressed in the ovary. During ovarian development, the mRNA expression of wcmmVtg in both the liver and ovary was continuously increased from the previtellogenic to late vitellogenic stages, but then decreased significantly at post-spawning stage. In the male fish, expression of wcmmVtg mRNA was induced in the liver by treatment with E₂ (10 and 100 ng/l) for 14 days. These results suggest that the Vtg originated from the ovary of the white cloud mountain minnow may also contribute to the accumulation of yolk proteins during oocyte growth, and that the male white cloud mountain minnow is sensitive to the estrogenic treatment in terms of Vtg mRNA expression, which could also be applied to monitor the environmental estrogenic mimics.     

Nucleotide sequence and mRNA expression analysis of <Emphasis Type="Italic">β</Emphasis>-actin gene in the orange-spotted grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.     

Comparison between α-LNA and DHA in early developmental stages of <Emphasis Type="Italic">Takifugu obscurus</Emphasis> and <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

ABSTRACT:   Rearing experiments were conducted to investigate the essential fatty acid requirements in the early developmental stages of river puffer Takifugu obscurus and tiger puffer T. rubripes using two n-3 series unsaturated fatty acids, α-linolenic acid (18:3n-3, α-LNA) and docosahexaenoic acid (22:6n-3, DHA), under salinity of 30 and 18.5–20.3°C. River and tiger puffer larvae used in this study were 15 and 14 days old after hatching, and their average body weights were 30.1 and 20.8 mg, respectively. The results on fatty acid requirements of these two species were evaluated from fish growth, survival, fatty acid composition of the fish body and activity test results. The DHA groups of both river and tiger puffer exhibited better survival and weight gain. However, there was no difference in the mean final body weights of river puffer between two dietary groups. Also, the DHA group of tiger puffer showed better results in the recovery test from anesthetic condition than that obtained in the LNA group. In an examination of the fatty acid compositions of the whole body, the LNA group containing no dietary DHA resulted in 0.5% DHA in tiger puffer and 1.1% DHA in river puffer . These results suggest that α-LNA from Artemia converted to eicosapentaenoic acid (20:5n-3, EPA) and to DHA successively by their fatty acid metabolism. Symptoms following essential fatty acid deficiency were not observed in any experimental groups. As river puffer did not represent a significant difference in the dietary effects between α-LNA and DHA treatment groups, its essential fatty acid requirement was assumed to be somewhat closer to that of the freshwater fishes in comparison with that of marine fishes, including tiger puffer.     

Effect of nitrite exposure on oxygen-carrying capacity and gene expression of <Emphasis Type="Italic">NF-κB</Emphasis>/<Emphasis Type="Italic">HIF-1α</Emphasis> pathway in gill of bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>)

Nitrite (NO₂^?) contamination of water can severely impact the health of aquatic life and is a major concern for commercial aquaculture. In order to study the effect of nitrite on Aristichthys nobilis, we investigated the oxygen-carrying capacity, NF-κB/HIF-1α pathway, and the gill tissue structure under nitrite stress. In the current study, bighead carp (initial weight 180.05?±?0.092 g) were exposed to nitrite (48.634 mg/L) for 96 h and then for 96 h recovery test. After nitrite exposure for 6 h, hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) mRNA expression increased significantly in the gill of bighead carp (P?<?0.05). After nitrite exposure for 12 h, hemoglobin (Hb) and methemoglobin reductase (MHBR) content in blood decreased significantly (P?<?0.05); TLR4 mRNA expression increased significantly (P?<?0.05). After nitrite exposure for 24 h, methemoglobin (MetHb) content increased significantly (P?<?0.05). After recovery test, all the indicators except TLR4 mRNA expression level recovered to initial level. In conclusion, nitrite exposure can affect hemoglobin dynamics, as oxidization of nitrite by hemoglobin results in the reduction of Hb to MetHb leading to hypoxia and nitrite exposure can also result into gill tissue damage. In the face of nitrite exposure, NF-κB and HIF-1α mRNA expression level increased immediately to protect the body from oxidative damage and eased hypoxic condition caused by nitrite. It was also observed that nitrite damage is recoverable in Aristichthys nobilis, but it may be need more than 96 h.     

Effects of <Emphasis Type="Italic">Euphorbia hirta</Emphasis> plant leaf extract on growth performance,hematological and organosomatic indices of hybrid catfish, <Emphasis Type="Italic">Clarias macrocephalus </Emphasis>× <Emphasis Type="Italic">C. gariepinus</Emphasis>

This study was conducted to evaluate the effects of Euphorbia hirta leaf extract on the growth performance, hematological and organosomatic indices of hybrid catfish, Clarias macrocephalus?×?C. gariepinus. The fish were treated with 0 (control), 300, 500 and 800 mg/kg (ppm) for 90 days. The weight gain, average daily growth rate, and specific growth rate were at significantly higher levels in fish from all the treatment groups on days 75 and 90, while the feed efficiency and protein efficiency ratio were consistently higher in fish from all the treatment groups from day 60 up until day 90. The feed conversion rate significantly decreased from day 60 until day 90 in all treatment groups when compared with the control group, and the survival rate was significantly different from day 30 until day 90; a consistently higher rate was observed in fish fed 800 mg/kg. The highest viscerosomatic index and intraperitoneal fat were observed in the group fed 500 mg/kg (p?<?0.05). The hepatosomatic index was significantly increased alongside increasing levels of E. hirta extract. The total white blood cell count in the control group was significantly higher on day 30, but on day 90 all the treatment groups showed higher levels. Hematocrit percentage was significantly different on days 30 and 90. Lymphocyte, eosinophil and thrombocyte levels were shown to be significantly different (p?<?0.05) when different groups of fish were compared. In conclusion, 300 mg/kg of E. hirta leaf extract could improve growth performance, hematological and some organosomatic indices in hybrid catfish.     

Reproductive dynamics and nursery habitat preferences of two commercially important Indo-Pacific red snappers <Emphasis Type="Italic">Lutjanus erythropterus</Emphasis> and <Emphasis Type="Italic">L. malabaricus</Emphasis>

Red snappers were examined for reproductive biology and age-0 habitat preferences. Spawning in red snappers occurred throughout the year in northern Australia and eastern Indonesia; at least 10–30% of females and 40–80% of males were in ripe or spawning condition in most months. Northern Australian populations showed a spawning peak from July to December (L. erythropterus) and September to March (L. malabaricus). Eastern Indonesian L. malabaricus had a less defined pattern with two peaks: January–March and October. Size at first maturity was 240 mm for males and 250–300 mm for females. L ₅₀ estimates were similar between species in northern Australia: 270–280 mm (males) and 350–370 mm (females). Maximum batch fecundity was 676,100 oocytes for L. erythropterus and 997,000 oocytes for L. malabaricus. Higher mean abundances of age-0 L. erythropterus were found in silty and coarse sand/rubble estuarine habitats of northern Australia (456 ± 119 fish/km²) compared with sandy coastal habitats (5 ± 3 fish/km²). Most age-0 snapper caught at Sape (eastern Indonesia) were L. malabaricus (91%) with mean abundances of 312 ± 14 fish/km². The similarities in the reproductive characteristics of red snappers suggest that successful management approaches adopted in northern Australia should be considered in eastern Indonesia.     

Age,growth and mortality estimates for populations of red snappers <Emphasis Type="Italic">Lutjanus erythropterus</Emphasis> and <Emphasis Type="Italic">L. malabaricus</Emphasis> from northern Australia and eastern Indonesia

Lutjanus erythropterus and L. malabaricus were examined for life-history differences among northern Australian and eastern Indonesian populations. Formation of opaque growth increments in otoliths began during April to September for northern Australian fishes and September to April for eastern Indonesian fishes. Maximum observed ages were greater than previously reported: 42 and 48 years for L. erythropterus and L. malabaricus, respectively. Eastern Indonesian red snappers grew faster than northern Australian fish. Growth was similar for northern Australian L. erythropterus populations. However, Kupang and Sape populations of L. erythropterus in eastern Indonesian showed different growth from the Arafura North population, which was similar to northern Australian fish. There were growth differences among northern Australian populations of L. malabaricus. Arafura South and Timor populations were similar, but differed from Groote and Weipa populations. There were no significant differences in growth among populations of L. malabaricus in eastern Indonesia. Total mortality estimates were similar between northern Australian and eastern Indonesian fish: 0.09 and 0.16 year⁻¹ for L. erythropterus and 0.11 and 0.14 year⁻¹ for L. malabaricus, respectively. Life-history characteristics of these red snappers are typical of other tropical snappers: slow-growing and long-lived with low mortality. However, population growth differences suggest that their management should be based on biological information from distinct stocks.     

Effect of trawling with traditional and ‘T90’ trawl codends on fish size and on different quality parameters of cod <Emphasis Type="Italic">Gadus morhua</Emphasis> and haddock <Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>

The effect of trawling on fish size and on different quality parameters of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) was evaluated after conducting 16 valid hauls using two trawls in a double rig fitted with a traditional and a novel ‘T90’ codend, respectively. The total catch volume during the fishing period was 47.6 metric tons, with an average catch per codend of 1.5 (range 0.5–2.9) tons. The mean haul duration was 5 h. The catch was assessed according to fish size, mortality, external damage, initial white muscle pH and development of rigor mortis. Fillet quality (colour, blood spots, gaping) was assessed after 1 week of freeze-storage. Our results showed there was no difference between the two types of nets in terms of catch volume, but significantly slightly bigger fish were caught with T90 than with the traditional trawl net (p < 0.05). Haddock caught with the traditional trawl net had more external injuries related to the trawl gear than haddock caught with the T90 gear (p < 0.05). The gaping frequency for cod caught with the traditional trawl net tended to be higher than cod caught with the T90 gear, but the difference was not significant (p = 0.07). No other differences in fish quality between fish caught in the trawl nets were observed.     

The changes in the biochemical compositions and enzymatic activities of rotifer (<Emphasis Type="Italic">Brachionus plicatilis</Emphasis>, Müller) and <Emphasis Type="Italic">Artemia</Emphasis> during the enrichment and starvation periods

The changes in the biochemical compositions and enzymatic activities of rotifer (Brachionus plicatilis) and Artemia, enriched and stored at 4°C temperature, were determined. The total starvation period was 16 h and samples were taken at the end of the 8th and 16th hours. In present study, the rotifer and nauplii catabolized a large proportion of the protein during the enrichment period. Lipid contents of both live preys increased during the enrichment period and decreased in nauplii and metanauplii throughout the starvation period but lipid content of the rotifer remained relatively constant during the starvation period. The changes observed in the amino acid compositions of Artemia and the rotifer were statistically significant (P < 0.05). The conspicuous decline the essential amino acid (EAA) and nonessential amino acid (NEAA) content of the rotifer was observed during the enrichment period. However, the essential amino acid (EAA) and nonessential amino acid (NEAA) contents of Artemia nauplii increased during the enrichment period. The unenriched and enriched rotifers contained more monounsaturated fatty acid (MUFAs) than polyunsaturated fatty acid (PUFAs) and saturated fatty acids (SFA). However, Artemia contained more PUFAs than MUFAs and SFA during the experimental period. A sharp increase in the amounts of docosahexaenoic acid (DHA) during the enrichment of the rotifer and Artemia nauplii was observed. However, the amount of DHA throughout the starvation period decreased in Artemia metanauplii but not in Artemia nauplii. Significant differences in tryptic, leucine aminopeptidase N (LAP), and alkaline phosphatase (AP) enzyme activities of Artemia and rotifer were observed during the enrichment and starvation period (P < 0.05). The digestive enzymes derived from live food to fish larvae provided the highest contribution at the end of the enrichment period. In conclusion, the results of the study provide important contributions to determine the most suitable live food offering time for marine fish larvae. Rotifer should be offered to fish larvae at the end of the enrichment period, Artemia nauplii just after hatching and before being stored at 4°C, and Artemia metanauplii at the end of the enrichment and throughout the starvation period.     

The effects of hairtail protein hydrolysate–Fe<Superscript>2+</Superscript> complexes on growth and non-specific immune response of red swamp crayfish (<Emphasis Type="Italic">Procambarus clarkii</Emphasis>)

The experiment was conducted to investigate the effects of prepared hairtail protein hydrolysate–Fe²⁺ (PH–Fe²⁺) complexes on growth and non-specific immune responses of crayfish (Procambarus clarkii). The diets with five different levels of PH–Fe²⁺ (200, 400, 600, 800, and 1000 mg/kg) were fed to crayfish for 60 days. The results indicated that the survival rate, weight gain percentage, specific growth rate, and muscle index of crayfish with 400–1000 mg/kg of PH–Fe²⁺ feeding were significantly higher, 9.94–10.10, 8.55–8.72, 0.24–0.29 %/day, and 2.39–3.05 %, respectively, than the control values after 60 days of feeding. Additionally, after 30 days of feeding, 200–400 mg/kg of PH–Fe²⁺ showed no significant (P > 0.05) improvement effect on activities of superoxide dismutase (SOD), phenoloxidase (PO), lysozyme (LSZ), and acid phosphatase (ACP) in serum or hepatopancreas of crayfish. However, significant improvement effects were observed in 800–1000 mg/kg groups. After 60 days of feeding, 400–600 mg/kg of PH–Fe²⁺ significantly (P < 0.05) improved the activity of SOD and LSZ, but did not affect the activity of PO and ACP significantly (P > 0.05). Moreover, the activities of SOD, PO, LSZ, and ACP in serum and hepatopancreas were all significantly enhanced upon treatment with 800–1000 mg/kg of PH–Fe²⁺. For these reasons, PH–Fe²⁺ can be recommended as a supplement in crayfish feed to increase the growth and immunity.     

Dietary ascorbic acid influences the intestinal morphology and hematology of hybrid sorubim catfish (<Emphasis Type="Italic">Pseudoplatystoma reticulatum</Emphasis> × <Emphasis Type="Italic">P. corruscans</Emphasis>)

This study evaluated the effect of graded levels of dietary ascorbic acid (AA) (12.47, 20.27, 115.44, 475.50, 737.72, and 850.70 mg kg^?1) on growth, hematology, intestinal morphometry, and phagocyte activity of hybrid sorubim Pseudoplatystoma reticulatum × Pseudoplatystoma corruscans. Fish (n = 420, 14.57 ± 2.71 g, 15.11 ± 0.90 cm) were distributed in 30 polyethylene tanks (80 l) (5 replicates per treatment with 14 fish per tank) and fed for 45 days. Dietary treatment did not have a significant effect on growth metrics (P > 0.05). Fish fed 737.72 mg AA kg^?1 had a higher villi height (289.80 ± 19.96 μm) (P < 0.05) than fish fed 850.70 mg AA kg^?1 (245.4 ± 18.25 μm). Hemoglobin in fish fed 850.70 mg AA kg^?1 (5.34 ± 0.96 g dl^?1) was higher (P < 0.05) than fish fed 12.47 mg AA kg^?1 (3.42 ± 0.55 g dl^?1) and 20.27 mg AA kg^?1 (3.06 ± 1.26 g dl^?1). The erythrocyte number of hybrid sorubim fed 115.40 mg AA kg^?1 (1.73 ± 0.27 × 10⁶ μl^?1) and 475.50 mg AA kg^?1 (1.70 ± 0.28 × 10⁶ μl^?1) were higher (P < 0.05) than in those fed diets containing 20.27 mg AA kg^?1 (1.11 ± 0.34 × 10⁶ μl^?1). There was no significant effect (P > 0.05) of dietary AA on leukocyte and thrombocyte and on phagocyte activity and phagocyte index. Inclusion of AA in feed seems to increase the integrity of the intestinal mucosa and stimulate erythropoiesis in hybrid sorubim catfish.     

Experimental infection of <Emphasis Type="Italic">Betanodavirus</Emphasis> in freshwater fish <Emphasis Type="Italic">Gambusia affinis</Emphasis> (Baird and Girard, 1853)—a potential infection model for viral encephalopathy and retinopathy

Betanodaviruses are the causative agents of the disease known as viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in a variety of marine and freshwater fish species. The aim of this study was to demonstrate experimental infection of an isolate of betanodavirus (RGNNV genotype) in freshwater fish, Gambusia affinis, for elucidation of transmission mechanism and potential use as a laboratory model. Morbidity and mortality rate was significantly higher by injection route of infection as compared to immersion by bath and resembled the natural infection of juvenile marine fish. The fish in disease affected group showed severe neurological disorders accompanied by extensive vacuolar degeneration and mild to moderate neuronal necrosis of the brain in comparison to control. Amplification of ~?427 bp product in the variable region of the coat protein gene of betanodavirus was achieved by RT-PCR with 100% sequence homology to RGNNV genotype.     

Genetic and morphological evidence of hybridization between <Emphasis Type="Italic">Nematalosa japonica</Emphasis> and <Emphasis Type="Italic">N. come</Emphasis> (Clupeiformes: Clupeidae) off Okinawa Island,Ryukyu Archipelago,Japan

Two morphologically similar species of gizzard shad, Nematalosa japonica Regan and N. come (Richardson), sympatrically distributed off Okinawa Island, Japan, were examined using an allozyme locus (SOD*) and two nuclear polymerase chain reaction (PCR)-based DNA markers (ITS-1 and CaM), which provided diagnostic identification of each species. In addition, a multiplex PCR-based mitochondrial DNA (mtDNA) marker (16S) was used to characterize the distribution of mtDNA haplotypes among specimens. The species composition of sympatric and allopatric population samples from Tungkang, southern Taiwan, to Okinawa and the Shikoku Islands, Japan, were also examined. Gizzard shad with hybrid genotypes were detected in three populations from Okinawa Island, with hybrid frequency ranging from 1 to 67%. A backcross level of 2% was detected in the dominant hybrid frequencies of one population sample only. Morphological examination of hybrids showed intermediate forms, with hybrid indices of three meristic characters falling between those of the parental species (range 39–53; mean 45). Although principal component analysis showed differences between N. japonica and N. come based on the first principal component scores, hybrids were difficult to identify. Accordingly, satisfactory identification of species and hybrids could be achieved only using genetic tools. We also discuss the cause of hybridization and its relationship with recently conducted reclamation on Okinawa Island.