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.     

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.     

Effect of starvation on activities and mRNA expression of lipoprotein lipase and hormone-sensitive lipase in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis> × <Emphasis Type="Italic">O. areus</Emphasis>)

A 4-week study was conducted to determine the effect of starvation on activities and mRNA expression of lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) in hybrid tilapia (Oreochromis niloticus × O. areus). The tissue samples were sampled once a week. Results showed that body weight (BW) and hepatosomatic index (HSI) were decreased significantly (P < 0.05) during starvation. The percentages of crude fat and crude protein in the whole body and the fat content in muscle decreased significantly (P < 0.05), while the rate of moisture and crude ash increased significantly (P < 0.05). The response of LPL, HSL activities and mRNA expression in tissues was tissue dependent. The activities of LPL and HSL in muscle at day 7 were elevated by 2.5 times (P < 0.05) and 11.8 times (P < 0.05) of the value at day 0, respectively, and both then decreased to pre-starvation levels at day 14 and finally stabilized at a certain level afterward. LPL and HSL mRNA abundance in muscle remained relatively stable between 0 and 14 day; then, a significant increase was seen after 14 days. In the liver, LPL activity maintained a significantly increasing trend during starvation, while HSL activity rose dramatically at day 7 of starvation by 2.35 times (P < 0.05) and finally stabilized at a certain level. The mRNA abundance of liver LPL increased significantly during the whole process of starvation (P < 0.05), whereas the mRNA abundance of liver HSL decreased significantly at day 7 of starvation, elevating significantly afterward (P < 0.05).     

Talking to the dead: using <Emphasis Type="Italic">Post</Emphasis>-<Emphasis Type="Italic">mortem</Emphasis> data in the assessment of stress in tiger sharks (<Emphasis Type="Italic">Galeocerdo cuvier</Emphasis>) (Péron and Lesueur, 1822)

Sharks are very sensitive to stress and prone to a high mortality rate after capture. Since approximately 50 million of sharks are caught as bycatch every year, and current recommendations to reduce the impact of commercial fishing strongly support immediate release, it is imperative to better understand post-release mortality caused by the stress of capture and handling. Blood samples allow the assessment of stress levels which are valuable tools to reduce mortality in commercial, recreational and scientific fishing, being essential for the improvement in those conservation measures. Biochemical analyses are widely used for sharks as stress indicators, with secondary plasma parameters (lactate, glucose and ions) being the most often employed assays. However, it is virtually impossible to determine baseline plasma parameters in free-ranging sharks, since blood withdrawal involves animal capture and restrain, which are stressful procedures. This study aims at analyzing secondary parameters of five healthy tiger sharks captured with circular hooks and handlines in Fernando de Noronha (Northeastern Brazil) and comparing them with secondary parameters of three dead tiger sharks caught off Recife (also Northeastern Brazil). The results showed that the analysis of some plasma constituents in dead animals may be an efficient tool to assess stress and lethality. However, traditional parameters such as glucose and calcium, need to be used with caution. The results also demonstrated the extreme importance of urea and phosphorus for assessing stress response and mortality in tiger sharks, both parameters frequently neglected and of utmost importance for shark’s homeostasis.     

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.     

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.     

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.     

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.     

Optimum time for weaning South African <Emphasis Type="Italic">Scylla serrata</Emphasis> (Forskål) larvae from rotifers to <Emphasis Type="Italic">Artemia</Emphasis>

To determine the optimum time at which to wean Scylla serrata larvae from rotifers onto Artemia two experiments were conducted, approximately 1 month apart, using larvae from two different female crabs. In the first experiment, the larvae in three treatment groups, with nine replicates each, were fed rotifers for the first 8 days after hatching. Artemia were introduced on days after hatch (DAH) 0 – during the first zoeal instar (treatment R + A); on DAH 4 – during the second zoeal instar (treatment R4A); on DAH 8 – during the third zoeal instar (treatment R8A). In a control (ROT) larvae were fed with rotifers exclusively for 18 days until the completion of metamorphosis to megalopa. In the second experiment, the same four feeding schedules as in experiment 1 were used with an additional group of larvae (treatment AC) that were fed only on Artemia throughout the rearing period. Similar results were recorded in the two experiments. Larvae in treatments R + A and R4A performed significantly better than those in treatments R8A, ROT and AC. This was particularly evident when examining the proportion of zoeae which successfully completed metamorphosis to megalopa. Poor performance of larvae in treatments AC and ROT implied that rotifers are needed as a first food, but that rotifers alone do not fill the nutritional requirements of S. serrata larvae. Poor performance of larvae in treatment R8A suggested that the diet should be supplemented with Artemia before the end of the zoea 3 stage.     

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.     

Protective effect of squilla chitosan–silver nanoparticles for <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> larvae infected with <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

Antimicrobial nanoparticle therapy was proposed as an alternative strategy to reduce the use of antibiotics in larval-rearing systems. Antibacterial potential of the prepared squilla chitosan–silver nanoparticles and its protective effect on Dicentrarchus labrax (sea bass) larvae in the early stages were studied against Vibrio angularium. Different concentrations of squilla chitosan (Csq) and squilla chitosan–silver nanoparticles (Csq–AgNps) (1, 2, 5, 10, and 20 %) were, in vitro, tested against V.anguillarum and expressed as a role of Log₁₀ mean. Sea bass larvae were treated using: 10 % Csq and 5 % Csq–AgNps as effective inhibitory concentrations against the pathogen either encapsulated during the feeding regime or added directly to the model system via the water from the onset of 4 weeks. The long-term administration of Csq–AgNps through enriched food for both non-infected and infected systems had survival % of 74.5 ± 1.5 and 72.5 ± 2.5, respectively. Larval clinical observations using Csq–AgNps were studied compared with the two controls. The current study found that 5 % encapsulated Csq–AgNps was enough to suppress infection and considered as an alternative to antibiotics in controlling virulent fish pathogens.     

Species differences and effects of soft coral extracts from <Emphasis Type="Italic">Sinnularia maximus</Emphasis> on the expression of cytochrome P4501A and 2N in butterflyfishes (<Emphasis Type="Italic">Chaetodon</Emphasis> spp.)

Cytochrome P450 (CYP) has been shown to confer resistance in numerous terrestrial insects that consume potentially toxic secondary metabolites in plants, but fewer studies have examined the role of critical biotransformation enzymes in allowing marine organisms to consume chemically defended foods. This study examined the expression of CYP1A and CYP2N mRNAs in several butterflyfish species, which can feed on numerous chemically defended soft and hard corals. In addition, the effect of an extract from a soft coral (Sinnularia maxima) on expression of hepatic CYP1A and CYP2 mRNAs was also examined. Fish were fed extracts on days 1, 3 and 5, and expression was examined on day 5. Phylogenetic analyses of the CYP1A cDNA from 12 species of butterflyfish (DNA, amino acid) indicate well-separated groupings according to their feeding strategies. The non-coralline feeding Chaetodon xanthurus exhibited a 7-fold higher basal expression of CYP2N8 relative to the other species studied. Although induction of CYP2N7 expression was observed in C. punctatofasciatus, CYP1A and CYP2N was largely unaffected or diminished by extract treatment in the other species of butterflyfish. These results indicated groupings of feeding strategy with CYP1A phylogeny in Chaetodon, but generally unaltered expression of CYP1A and CYP2N following dietary treatment with an extract from a chemically defended soft coral suggesting an inconclusive role of these isoforms in the detoxification of chemicals in these extracts.     

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.     

Acute fluoxetine treatment increases aggressiveness in juvenile matrinxã (<Emphasis Type="Italic">Brycon amazonicus)</Emphasis>

Fluoxetine (FLX) is a selective serotonin (5-HT) reuptake inhibitor known for its effects modifying aggressiveness, personality traits, and anxiety-like behaviors. The aim of the present study was to evaluate the influence of the acute treatment, by immersion, with FLX on aggressive behavior of resident Brycon amazonicus fish. Fish pretreated with FLX presented an increase in aggressiveness, evidenced by the increase on the number of bites and chases against the intruder and a decrease in latency for the first attack, when compared to control fish. Together with previous studies, these results show the complexity of the neural modulation of the aggressive behavior in fish by 5-HTergic system.     

Molecular characterization and expression analysis of AMPK α subunit isoform genes from <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> responding to salinity stress

AMP-activated protein kinase (AMPK) is a highly conserved and multi-functional protein kinase that plays important roles in both intracellular energy balance and cellular stress response. In the present study, molecular characterization, tissue distribution and gene expression levels of the AMPK α1 and α2 genes from turbot (Scophthalmus maximus) under salinity stress are described. The complete coding regions of the AMPK α1 and α2 genes were isolated from turbot through degenerate primers in combination with RACE using muscle cDNA. The complete coding regions of AMPK α1 (1722 bp) and α2 (1674 bp) encoded 573 and 557 amino acids peptides, respectively. Multiple alignments, structural analysis and phylogenetic tree construction indicated that S. maximus AMPK α1 and α2 shared a high amino acid identity with other species, especially fish. AMPK α1 and α2 genes could be detected in all tested tissues, indicating that they are constitutively expressed. Salinity challenges significantly altered the gene expression levels of AMPK α1 and α2 mRNA in a salinity- and time-dependent manners in S. maximus gill tissues, suggesting that AMPK α1 and α2 played important roles in mediating the salinity stress in S. maximus. The expression levels of AMPK α1 and α2 mRNA were a positive correlation with gill Na⁺, K⁺-ATPase activities. These findings will aid our understanding of the molecular mechanism of juvenile turbot in response to environmental salinity changes.     

Comparative analysis of mitochondrial control region in polyploid hybrids of red crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>) × blunt snout bream (<Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>)

The entire sequences of the mitochondrial (mt)DNA control region (CR) and portions of its flanking genes in the red crucian carp (RC) and blunt snout bream (BSB) as well as their polyploid hybrids (3nRB, 4nRB and 5nRB) were determined and subjected to a comparative analysis. The mtDNA-CRs of these five fish species ranged from 923 to 937 bp in length, they had the same flanking gene arrangement as other vertebrates and the pattern of nucleotide substitution bias was also similar to that in other vertebrates. Our data are consistent with the viewpoint of three domains [extended terminal associated sequence (ETAS domain), central conserved sequence block domain and conserved sequence block (CSB) domain] within the mtDNA-CR of mammals. On the basis our comparative analysis of the mtDNA-CRs of these five fish species, we were able to identify the consensus sequences of functional conserved units, including the ETAS, CSB-F, CSB-D, CSB-E, CSB1, CSB2 and CSB3 and putative promoter. The percentage of variable nucleotide positions (41.98%) in the central domain was lower than those in the ETAS and conserved domain (71.70 and 47.12%, respectively), suggesting that the central domain was the most conserved part of the mtDNA-CR. These results provide useful and important information for the further study of mtDNA-CR structure in fish. The sequence similarities of mtDNA-CR among the 3nRB, 4nRB, 5nRB hybrids and their respective female parents were higher than those among the 3nRB, 4nRB, 5nRB hybrids and their respective male parents, providing the direct evidence of stringent maternal inheritance of mtDNA-CR in the 3nRB, 4nRB and 5nRB hybrids.