Isoenzyme electrophoretic patterns in discus fish (Symphysodon aequifasciatus Pellegrin, 1904 and Symphysodon discus Heckel, 1840) from the Central Amazon

The discus is a very popular and expensive aquarium fish belonging to the family Cichlidae, genus Symphysodon, formed by three Amazon basin endemic species: Symphysodon aequifasciatus, S. discus and S. tarzoo. The taxonomic status of these fish is very controversial, with a paucity of molecular research on their population genetic structure and species identification. Information on molecular genetic markers, especially isoenzymes, in search of a better understanding of the population genetic structure and correct identification of fish species, has been receiving more attention when elaborating and implementing commercial fishery management programs. Aiming to contribute to a better understanding of the species taxonomic status, the present study describes the isoenzymatic patterns of 6 enzymes: esterase (Est - EC 3.1.1.1), lactate dehydrogenase (Ldh - EC 1.1.1.27), malate dehydrogenase (Mdh - EC 1.1.1.37), phosphoglucomutase (Pgm - EC 5.4.2.2), phosphoglucose isomerase (Pgi - EC 5.3.1.9), and super oxide dismutase (Sod - EC 1.15.1.1) extracted from skeletal muscle specimens and analyzed by starch gel electrophoresis. Monomorphic patterns, presumably controlled by 11 loci: Est-1, Est-2, Est-3, Ldh-1, Ldh-2, Mdh-1, Mdh-2, Pgi-1, Pgi-2, Pgm-1, and Sod-1 were fixed for the same alleles: Est-1(1), Est-2(1), Est-3(1), Ldh-1(1), Ldh-2(1), Mdh-1(1), Mdh-2(1), Pgi-1(1), Pgi-2(1), Pgm-1(1), and Sod-1(1), respectively, and detected in all 60 specimens examined (27 S. aequifasciatus from Manacapuru and 33 S. discus from Novo Air?o, Central Amazon). The failure in the present study to detect diagnostic loci, which could be very useful for differentiating S. aequifasciatus from S. discus species, and polymorphic loci, which could also be applied for possible identification and delimitation of their stocks, does not rule out the possibility of there existing in other isoenzyme gene loci to be analyzed in the future.     

Ovarian development in the Amazonian red discus, Symphysodon discus Heckel (Osteichthyes: Cichlidae).

The Neotropical red discus fish, Symphysodon discus, originates from the Amazonian basin and has a discoid body. Although this species is popularly used in aquaria and is exported as an ornamental fish, it has not been intensively studied. The purpose of this work was to study the morphological and histological aspects of the ovarian development in the red discus. Forty females of S. discus of varying body sizes and different stages of gonadal development were used in this study. The ovaries were weighed and examined macroscopically in order to observe the maturation stages. Histological staining of hematoxilin-eosin was used for microscopic observations of the ovaries. Macroscopic observations showed immature, maturing, mature and partially spent stages of ovarian development. Microscopic examination of the ovaries revealed five stages and five phases of ovarian development. The ovaries of the mature females showed all the different phases of oocyte development, indicating the multiple spawning habit of this species.     

Patterns of diversification in the discus fishes (Symphysodon spp. Cichlidae) of the Amazon basin

We carried out a phylogeograhic and population genetic analysis of fishes of the taxonomically contentious genus Symphysodon from the Amazon basin in order to test hypotheses of relationships among taxonomic units, and potential processes driving diversification within this genus. We sampled 334 individuals of the genus Symphysodon from 24 localities that span the complete geographic distribution of this genus. The sampling scheme included all known phenotypic groups, species and subspecies. Analyses were based on 474 bp of the mitochondrial control region and 1443 bp of the exon 3 of RAG1 gene. We observed 102 mtDNA haplotypes defined by 89 segregating sites, and 5 nuDNA alleles defined by three segregating sites. Maximum-likelihood, Bayesian-inference and statistical parsimony analyses revealed three well defined monophyletic groups. These clades corresponded to the 'green' and 'blue' groups of Symphysodon aequifasciatus, and to a previously morphologically unrecognized clade from the Xingu River drainage. These three clades were nested within a paraphyletic assemblage consisting of the 'brown' group of S. aequifasciatus and of both described subspecies of S. discus, the 'Heckel' and the 'abacaxi' discus. Nuclear allele sharing was observed among groups, but there were significant differences in frequencies. We inferred several processes including past fragmentation among groups, and restricted gene flow with isolation by distance within the paraphyletic 'brown+Heckel+abacaxi' groups, and suggest that differences among the 'blue', 'Heckel' and 'brown' groups are potentially maintained by differences in water chemistry preferences. We further inferred colonization of the western Amazon basin by an ancestor of the 'green' clade. The 'green' group was the only group with a pattern of haplotype distribution consistent of a demographic expansion, and the divergence of this clade from other groups of discus was consistent with recent geologic evidence on the breach of the Purus Arch which separates western Amazon from eastern Amazon. We further hypothesized that the differentiation of the 'Xingu' clade could be due to vicariance events resulting from Pleistocene sea level, and thus Amazon River level fluctuations. We discuss the bearings of our results on the current taxonomy of this group, and on the biological reality of the different forms, subspecies and species of Symphysodon concluding that we are probably observing a process of diversification, and therefore taxonomy will remain contentious.     

Redescription of Ichthyouris bursata Moravec & Prouza, 1995 (Nematoda: Pharyngodonidae), a parasite of wild and aquarium-reared discus Symphysodon spp. (Osteichthyes)

The oxyuroid nematode Ichthyouris bursata Moravec & Prouza, 1995 (Pharyngodonidae) was recorded from the intestine of discus (Symphysodon spp. and hybrids) cultured in discus farms in Bangkok and Nonthaburi, central Thailand, during 2006 and 2007. This material made it possible to study in detail the morphology of this little known parasite species, using both light and scanning electron microscopy (the latter not previously used for the male). The SEM examination showed taxonomically important morphological features not previously reported or erroneously described, including the presence of three bilobed lips, a pair of sclerotised plate-like structures and a median cone-shaped outgrowth on the posterior cloacal lip, short 'hairs' on the cloacal lips, digital phasmids in the male and, sometimes, up to two filaments on the egg poles. This species is of South American origin, which was confirmed by its recent record from a free-living blue discus S. aequifasciatus Pellegrin in Brazil. The reproduction and transmission of I. bursata in the conditions of aquarium tanks is probably permitted by the direct (homoxenous) life-cycle of this parasite.     

Discus fishes: mitochondrial DNA evidence for a phylogeographic barrier in the Amazonian genus Symphysodon (Teleostei: Cichlidae)

Genetic relationships and variation in meristic counts, body shape and colour were examined in a large sample of Symphysodon collected from several locations in floodplain habitats along the length of the Amazon River. Surprisingly, mitochondrial DNA indicates no difference between the two historically described species, Symphysodon discus and Symphysodon aequifasciatus , but shows that non-clinal variation exists with a distinct lineage found in the western Amazon. This lineage is consistent with a colour form that is distinct from other Symphysodon lineages. This form has a parapatric distribution and is recognized as a distinct species, Symphysodon tarzoo . Adaptation to floodwater habitats supports genetic cohesion across a large range preventing fine scale regional diversification of the genus. Possible explanations for the unusual set of distributions for genetic and colour characters relate to the history of the Amazon basin and the probable division of lowland species when submerged geologic arches influence surface topology.     

Costs and benefits of biparental mucus provisioning in discus fish (<Emphasis Type="Italic">Symphysodon aequifasciatus</Emphasis>)

We tested the manner in which the number of fry influences the costs and benefits of mucus provisioning in discus fish (Symphysodon aequifasciatus) via two experiments. In experiment-1, we found that fry grew more rapidly when they were raised with parents than when raised without parents, suggesting that fry benefits from the mucus provisioning by parents. In experiment-2, a reduction in the body weight of parents correlated with brood size. The growth rate of fry did not linearly correlate with brood size. We discuss the functions, costs, and benefits of mucus provisioning in discus fish.     

Early infection of discus Symphysodon aequifasciatus altricial larvae by Sciadicleithrum variabilum (Monogenea)

The occurrence of Sciadicleithrum variabilum on the gills of discus Symphysodon aequifasciatus larvae 7 days after hatching is reported for the first time. This suggests that altricial larvae are susceptible to Monogenea following gill differentiation and before complete yolk resorption.     

Characterization of 12 single nucleotide polymorphisms (SNPs) in Pacific abalone, Haliotis discus hannai

We report here for the first time 12 polymorphic single nucleotide polymorphisms (SNPs) in a commercially important gastropod, Pacific abalone (Haliotis discus hannai) that were identified by searching expressed sequence tag database. These SNP loci (seven nuclear and five mitochondrial SNPs) were polymorphic among 37 wild abalone individuals, based on a four-primer allele-specific polymerase chain reaction analysis. All loci had two alleles and the minor allele frequency ranged from 0.027 to 0.473. For the seven nuclear SNPs, the expected and observed heterozygosities ranged from 0.053 to 0.499 and from 0.054 to 0.811, respectively.     

用微卫星标记分析皱纹盘鲍群体的遗传变异

利用微卫星标记技术, 对皱纹盘鲍山东长岛和辽宁獐子岛的两个野生群体以及山东崆峒岛一个养殖群体的遗传变异进行了分析。对6个微卫星基因座的多态性进行了评估, 各基因座的多态信息含量(PIC)值均大于0.5, 适合对鲍群体遗传结构的分析。结果表明, 这6个基因座在3个皱纹盘鲍群体中共获得57个等位基因, 等位基因数(A)平均为9.50, 有效等位基因数(N_e)平均为5.8572, 平均杂合度观测值(H_o)和期望值(^H_e)分别为0.6925和0.7966; 两个野生群体的杂合度观测值(H_o)和期望值(H_e)均高于养殖群体。上述结果为保护和利用皱纹盘鲍的遗传多样性提供了依据。     

Development of 15 polymorphic genic microsatellite DNA markers of Pacific abalone Haliotis discus hannai

Fifteen polymorphic genic microsatellite DNA markers were developed based on the expressed sequence tags of Pacific abalone Haliotis discus hannai we ourselves generated, which were used to type 32 individuals representing a cultured population. The number of alleles each locus ranged from two to 12. The observed and expected heterozygosities ranged from 0.094 to 0.969 and from 0.091 to 0.878, respectively. Among 15 loci, four were found to deviate significantly from Hardy–Weinberg equilibrium (P_HW < 0.001). No linkage disequilibrium was found between these loci. It is certain that these markers will facilitate the management and exploitation of the genetic resource of Pacific abalone.     

Cytogenetic Analysis and Chromosomal Characteristics of the Polymorphic 18S rDNA of Haliotis discus hannai from Fujian,China

We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82%) and two rare types 2n = 36 = 11M + 7SM (14%) and 2n = 36 = 10M + 7SM + 1ST (4%). The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA)₃ displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.     

Spironucleus vortens, a possible cause of hole-in-the-head disease in cichlids

Hole-in-the-head disease is recorded in 11 discus Symphysodon discus Heckel, 1840 and 1 angelfish Pterophyllum scalare Lichtenstein, 1823 obtained from local aquarists within the Southwest of the UK. Spironucleus vortens Poynton et al. 1995, was isolated from the kidney, liver, spleen and head lesions of discus showing severe signs of the disease and from the intestines of all fish. The hexamitid was also recorded from the head lesions of the angelfish. The identity of these flagellates was confirmed as S. vortens on the basis of topographical features seen with the aid of SEM. A modified in vitro culture method was successfully developed for the detection, isolation and long-term maintenance of S. vortens. The flagellate was sub-cultured at 3 to 5 d intervals, new media being supplemented with fresh liver from Oreochromis niloticus (Linnaeus, 1757) free from infection. The results are discussed in relation to S. vortens as the causative agent for hole-in-the-head disease following systemic infection via the digestive tract.     

Centromere mapping in the Pacific abalone (Haliotis discus hannai) through half-tetrad analysis in gynogenetic diploid families

Centromere mapping is an essential prerequisite for our understanding of the composition and structure of genomes. For centromere mapping, in two meiogynogenetic families of the Pacific abalone (Haliotis discus hannai), we screened 97 microsatellite markers that cover all linkage groups from a currently available abalone linkage map. Microsatellite analysis showed that no unique paternal allele was found in all gynogenetic progeny, which confirmed 100% success of induction of gynogenesis. In the control crosses, all 97 microsatellite loci were compatible with Mendelian inheritance, while in meiogynogenetic progeny, 5.2% of the microsatellite loci showed segregation distortions from an expected 1:1 ratio of two homozygote classes. The second division segregation frequency of the microsatellites ranged from 0.037 to 0.950 with a mean of 0.399, indicating the existence of interference. Heterogeneity among linkage groups in the crossover distribution was observed. Centromere location was mostly in accordance with the abalone karyotype, but differences in marker order between linkage and centromere maps occurred. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in the commercially important abalone species.     

Settlement, growth and survival of abalone, Haliotis discus hannai, in response to eight monospecific benthic diatoms

The settlement, early growth and survival of the larval abalone Haliotis discus hannai in response to eight monospecific benthic diatoms were examined in the laboratory. Postlarvae showed active settling and feeding behaviour in all diatom species and in naturally occurring diatoms. Larval settlement rates differed significantly between experimental substrata after 24 h and 48 h. Nitzschia sp. (96.67 %), Hantzschia amphioxys var. leptocephala (95.00 %) and Navicula seminulum (90.00 %) strongly induced larval settlement of H. discus hannai. Postlarvae could feed on benthic diatoms (< 36 μm in shell length) with both weak and strong adhesion on the 4th day after settlement. Greatest growth (shell length) occurred on Nitzschia sp. (786.84 μm ± 2.50 SE and 773.09 μm ± 2.09 SE). Survival of postlarvae was also greatest on Nitzschia sp. (95.33 % ± 1.45 SE). These results indicate the effectiveness of Nitzschia sp., H. amphioxys var. leptocephala, N. seminulum, Rhaphoneis surirella and Navicula corymbosa as single species over natural diatoms in larval settlement and postlarvae growth of H. discus hannai. Thus, Nitzschia sp. has the best potential diet for larval settlement and postlarvae growth of H. discus hannai. H. amphioxys var. leptocephala and N. seminulum can be used as cues to induce larvae settlement, and R. surirella and N. corymbosa can be used as food for growing postlarval.     

Vibrio neonatus sp. nov. and Vibrio ezurae sp. nov. isolated from the gut of Japanese abalones

Five alginolytic, facultative anaerobic, non-motile bacteria were isolated from the gut of Japanese abalones (Haliotis discus discus, H. diversicolor diversicolor and H. diversicolor aquatilis). Phylogenetic analyses based on 16S rRNA gene and gap gene sequences indicated that these strains are closely related to V. halioticoli. DNA-DNA hybridizations, FAFLP fingerprintings, and phylogenies of gap and 16S rRNA gene sequences showed that the five strains represent two species different from all currently described vibrios. The names Vibrio neonatus sp. nov. (IAM 15060T = LMG 19973T = HDD3-1T; mol% G+C of DNA is 42.1-43.9), and Vibrio ezurae sp. nov. (IAM 15061T = LMG 19970T = HDS1-1T; mol% G+C of DNA is 43.6-44.8) are proposed to encompass these new taxa. The two new species can be differentiated from V. halioticoli on the basis of several features, including beta-galactosidase activity, assimilation of glycerol, D-mannose and D-gluconate.     

Variability of 18S rDNA locus among Symphysodon fishes: chromosomal rearrangements

Three species of cichlids belonging to the genus Symphysodon have demonstrated interspecific and intraspecific variation in nucleolus organizer regions (NOR) detected with silver nitrate. In order to understand the evolution of this marker in the genus, the structural variability of these sequences in mitotic chromosomes from Symphysodon aequifasciatus, Symphysodon discus and Symphysodon haraldi was investigated using both silver nitrate impregnation and hybridization of the 18S rRNA gene probe. For the three species, the two markers were intraspecifically and interspecifically variable both in the number and in the size of the sites. This polymorphism may stem from duplications and translocations, which suggests that structural chromosome rearrangements effectively act in the karyoevolution of wild Symphysodon species and may have favoured the adaptability of these fishes to diverse aquatic environments in the Amazon.     

Segregation of Microsatellite Alleles in Gynogenetic Diploid Pacific Abalone (Haliotis discus hannai)

Inheritance of 9 microsatellite loci was examined in 3 families of gynogenetic Pacific abalone Haliotis discus hannai produced by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division. The proportion of heterozygous progeny was used to estimate marker-centromere (M-C) distances. All loci conformed to Mendelian segregation in the control crosses when null alleles were accounted for. The absence of paternal alleles confirmed the gynogenetic origin of the offspring and indicated 100% success for 3 families. Estimated recombinant frequencies ranged from 0.10 to 0.60, which is lower than those observed in other gynogenetic diploid animals. The mean recombination frequency was 0.22, corresponding to a fixation index of 0.78 in one generation. This is 3.12 times the increase in homozygosity expected after one generation of sib mating (0.25), suggesting meiotic gynogenesis may be an effective means of rapid inbreeding in the abalone. M-C map distances for the 9 loci varied between 5 and 30 cM under the assumption of complete interference. The information about M-C distances will be useful for future gene mapping in H. discus hannai.     

Isolation and characterization of microsatellite DNA markers in the Pacific abalone,Haliotis discus hannai

We developed 17 new microsatellite markers in Haliotis discus hannai. All loci were found to be polymorphic with an average of 13.1 alleles per locus (range 3–28). The mean observed and expected heterozygosities were 0.77 (range 0.17–1.00) and 0.79 (range 0.42–0.96), respectively. Six loci deviated significantly from Hardy–Weinberg proportions, and thus should be used with caution. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for studies of trait mapping, kinship and population genetics.     

Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai)

Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker–centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72‐h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second‐division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y‐values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y‐values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species.