Complete mitochondrial DNA sequences of the Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) and Blue tilapia (<Emphasis Type="Italic">Oreochromis aureus</Emphasis>): genome characterization and phylogeny applications

Cichlid fishes have played an important role in evolutionary biology and aquaculture industry. Nile tilapia (Oreochromis niloticus), blue tilapia (Oreochromis aureus) and Mozambique tilapia (Oreochromis mossambicus), the useful models in studying evolutionary biology within Cichlid fishes, are also mainly cultured species in aquaculture with great economic importance. In this paper, the complete nucleotide sequence of the mitochondrial genome for O. niloticus and O. aureus were determined and phylogenetic analyses from mitochondrial protein-coding genes were conducted to explore their phylogenetic relationship within Cichlids. The mitogenome is 16,625 bp for O. niloticus and 16,628 bp for O. aureus, containing the same gene order and an identical number of genes or regions with the other Cichlid fishes, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one putative control region. Phylogenetic analyses using three different computational algorithms (maximum parsimony, maximum likelihood and Bayesian method) show O. niloticus and O. mossambicus are closely related, and O. aureus has remotely phylogenetic relationship from above two fishes.     

Establishment and application of a microsatellite-based parentage identification method for Oreochromis niloticus,O. aureus,and O. niloticus × O. aureus

In genetic fish breeding research, clear pedigree information is of great significance for breeding and parental management. In order to establish a stable, highly accurate, and widely applicable parentage identification method for tilapia, 13 highly polymorphic microsatellite loci within populations of Oreochromis niloticus, O. aureus, O. niloticus × O. aureus, and their mixed population were screened. Four groups of fluorescent-labeled multiple capillary electrophoresis were established for allelic genotyping. The assignment success rate reached 100% when 7, 9, 8 and 12 loci were used in the population of O. niloticus, O. aureus, O. niloticus × O. aureus, and their mixed population, respectively. All 175 progeny individuals of “Yuemin No. 1” tilapia were exclusively assigned to their parental pairs when the 12 loci for the mixed population were used. This study established a fluorescent-labeled microsatellite-based parentage assignment method for O. niloticus, O. aureus, O. niloticus × O. aureus, and their mixed population with high identification accuracy and efficiency, which lays a foundation for pedigree information and population genetic management in tilapia breeding.     

Identification of putative sex chromosomes in the blue tilapia, Oreochromis aureus, through synaptonemal complex and FISH analysis

Sex determination in the blue tilapia, Oreochromis aureus, is primarily a ZW female-ZZ male system. Here, by analysis of the pachytene meiotic chromosomes of O. aureus, we demonstrate the presence of two distinct regions of restricted pairing present only in heterogametic fish. The first, a subterminal region of the largest bivalent is located near to the region of unpairing found in the closely related species O. niloticus, while the second is in a small bivalent, most of which was unpaired. These results suggest that O. aureus has two separate pairs of sex chromosomes.     

Trophic interrelationships between the exotic Nile tilapia, <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> and indigenous tilapiine cichlids in a subtropical African river system (Limpopo River,South Africa)

The stable isotope ratio and seasonal changes in diet of two indigenous (Oreochromis mossambicus, Tilapia rendalli) and one exotic (Oreochromis niloticus) tilapiine cichlids in the subtropical Limpopo River, South Africa were investigated to determine patterns of resource partitioning. Stomach contents of O. niloticus and O. mossambicus indicated high dietary overlap across size class, habitat and season, with both species primarily feeding on vegetative detritus. However, stable isotope analysis revealed that the two Oreochromis species had different stable isotope ratios derived from different food sources. The relatively δ¹³C-depleted O. niloticus indicates a phytoplankton-based diet, while the δ¹³C-enriched O. mossambicus indicates a macrophagous diet dominated by vegetative detritus and periphyton. The high similarity in stomach contents and the interspecific differences in isotopic composition reveal fine-scale patterns of food resource partitioning that could be achieved through selective feeding. Tilapia rendalli was largely macrophagous and fed mainly on aquatic macrophytes and had a low dietary overlap with both O. niloticus and O. mossambicus. In the Limpopo River, detritus and algae are probably the most abundant food resources and the causal factors responsible for the observed patterns of resource partitioning among the tilapiines are usually difficult to ascertain. Fish may be able to perceive food resources in terms of the dynamics that determine their availability. Detailed studies of variation in food resource availability and fish habitat use within the system are needed to evaluate this hypothesis.     

The properties of tilapia sperm and its cryopreservation*

The specific differences between the testis, milt and sperm of six species of tilapia including Oreochromis aureus, O. mossambicus, O. niloticus, Tilapia zillii, O. nilolicus×O. aureus hybrid and red tilapia, Oreochromis sp., were studied. The shape of testis is tubular; the gonadosomatic index varied from 0.07 to 2.71. The pH values of individual milts ranged from 6.2 to 8.2 and the osmolarity from 240 to 380 mOsmol kg^?1. The quantity of milt obtained by stripping averaged only about 0.3 ml, and only in the O. niloticus×O. aureus hybrid did it exceed 3 ml. Sperm motility graded from weak to moderate was determined for the stripped tilapia milt. Sperm concentrations ranged from 7.70 × 10⁸ sperms ml^?1 in T. zillii to 2.74 × 10¹⁰ sperms ml^?1 in O. mossambicus. Tilapia sperm was active in various salinity ranges such as 0–5‰ for O. niloticus, and 0–15‰ for O. mossambicus and T. zillii. Extender containing 15% milk and 5% methanol was used to prepare milt mixture before cooling rapidly to ?35° C and then at 5° C min^?1 to ?75° C for storage in liquid nitrogen (– 196° C). Fertility tests on frozen tilapia milt resulted in a fertilization rate of 72.7% (v. control 85.7%) for the 22-day frozen milt of the O. nilolicus×O. aureus hybrid used to fertilize the eggs of O. honorum, and 93.4% (v. control 90%) for the 304-day frozen sperm of red tilapia used to fertilize eggs of red tilapia.     

Hybridization and phylogeography of the Mozambique tilapia <Emphasis Type="Italic">Oreochromis mossambicus</Emphasis> in southern Africa evidenced by mitochondrial and microsatellite DNA genotyping

Many Oreochromis species utilized in aquaculture were extensively introduced outside their native range in Africa. Given their recent evolutionary radiation, these species hybridize easily, posing a threat to the integrity of local adaptation. The objective of this work was to study the genetic diversity of the Mozambique tilapia (Oreochromis mossambicus) in its native range, southern Africa, and provide a method for identifying hybrids with genetic markers. We genotyped the mitochondrial DNA (mtDNA) control region (385 bp) of wild and farmed O. mossambicus, wild and farmed O. niloticus and morphologic wild hybrids. These data were complemented with published sequences of parapatric and sympatric Oreochromis taxa. Phylogeographic analysis showed the presence of two O. mossambicus lineages, the southernmost representing a recent Holocene radiation. Hybridization of O. mossambicus was indicated by the presence of O. niloticus and O. mortimeri–andersonii mtDNA specimens in the Limpopo basin and of O. karongae mtDNA in specimens from Malawi. We also genotyped seven suspected hybrid individuals from the Limpopo River, and 137 wild and farmed Mozambique and Nile tilapia samples with five microsatellite markers. Factorial Component Analysis, Bayesian clustering and assignment analyses consistently delineated an O. mossambicus and an O. niloticus group, with the putative hybrids positioned in between. Different levels of hybridization were detected by the Bayesian assignment. The complex nature of hybridization and introgression between cichlid species raises major concerns for the long-term integrity of Mozambique tilapia.     

Feeding ecology of Oreochromis niloticus (Linnaeus) & Tilapia Zillii (Gervias) in a Nile canal

Fish stomachs, 1149 for Tilapia zillii and 1698 for Oreochromis niloticus, were collected from April 1984 till April 1985 from a Nile canal in the Egyptian delta. Both species showed a significant overlap of diet (Schoener's index) among individuals ranging from 12 to 17.9 cm standard length. Outside this range differences in the importance of food of animal origin occurred. Macrophytes were the main food of O. niloticus and aquatic insects of the food of T. zillii. This initial diet overlap may have contributed to irregularity in the increase of full stomachs with increasing length, and to a decline of the stomach index (SI = stomach weight × 100/fish weight) with increase in length in O. niloticus. The stomach of either species correlated significantly with water temperature, but with daily photoperiod in O. niloticus only.     

Characterization of mitochondrial control region, two intergenic spacers and tRNAs of Zaprionus indianus (Diptera: Drosophilidae)

Total synaptonemal complex (SC) lengths were estimated from Oreochromis aureus Steindachner (which has a WZ/ZZ sex determination system), O. mossambicus Peters and O. niloticus L. (both of which have XX/XY sex determination systems). The total SC length in oocytes was greater than that in spermatocytes in all three species (194 ± 30 μm and 134 ± 13 μm, 187 ± 22 μm and 127 ± 17 μm, 193 ± 37 μm and 144 ± 19 μm, respectively). These sex-specific differences did not appear to be influenced by the type of sex determination system (the female/male total SC length ratio was 1.45 in O. aureus, 1.47 in O. mossambicus and 1.34 in O. niloticus) and do not correlate with the lack of any overall sex-specific length differences in the current Oreochromis linkage map. Although based on data from relatively few species, there appears to be no consistent relationship between sex-specific SC lengths and linkage map lengths in fish. Neomale (hormonally masculinized genetic female) O. aureus and O. mossambicus had total SC lengths of 138 ± 13 μm and 146 ± 13 μm respectively, more similar to normal males than to normal females. These findings agree with data from other vertebrate species that suggest that phenotypic sex, rather than genotype, determines traits such as total SC length, chiasmata position and recombination pattern, at least for the autosomes.     

Utility of DNA barcoding in native Oreochromis species

The importance of Oreochromis in worldwide aquaculture and regional fisheries motivates the study of their genetic diversity in their native range. In this article, all mitochondrial cytochrome c oxidase subunit I gene (COI) sequences of Oreochromis species are retrieved from Barcode of Life Data system to quantify the available DNA barcoding information from wild individuals collected within the native ranges of the respective species. It is found that 70% of the known species in the genus still lack a COI barcode, and only 15% of the available sequences are from within the respective native ranges. Many of the available sequences have been produced from specimens acquired from aquaculture and introduced, naturalized populations, making the assessment of variation within the original native range challenging. Analyses of the wild-collected fraction of available sequences indicated the presence of cryptic lineages within Nile tilapia Oreochromis niloticus and O. schwebischi, the occurrence of potential introgressive hybridization between O. niloticus and blue tilapia O. aureus, and potential ancestral polymorphism between Karonga tilapia O. karongae and black tilapia O. placidus. This article also reports a case of misidentification of O. mweruensis as longfin tilapia O. macrochir. These results stress the importance of improving the knowledge of genetic variation within the native ranges of Oreochromis species for better-informed conservation of these natural resources.     

Genetic structure and gene flow in an endangered native tilapia fish (Oreochromis esculentus) compared to invasive Nile tilapia (Oreochromis niloticus) in Yala swamp, East Africa

The introduction of invasive Nile tilapia (Oreochromis niloticus), and the rapacious predator Nile perch (Lates niloticus), into Lake Victoria resulted in a decline in population sizes, genetic diversity and even extirpation of native species which were previously the mainstay of local fisheries. However, remnant populations of native fish species, including tilapia, still persist in satellite lakes around Lake Victoria where they may coexist with O. niloticus. In this study we assessed population genetic structure, diversity, and integrity of the native critically endangered Singidia tilapia (O. esculentus) in its refugial populations in the Yala swamp, Kenya, and contrasted this diversity with populations of the invasive tilapia O. niloticus in satellite lakes (Kanyaboli, Namboyo and Sare) and Lake Victoria. Based on mtDNA control region sequences and eight nuclear microsatellite loci, we did not detect any mtDNA introgression between the native and the invasive species in Lakes Kanyaboli and Namboyo, but did find low levels of nuclear admixture, primarily from O. niloticus to O. esculentus. Some genetic signal of O. esculentus in O. niloticus was found in Lake Sare, where O. esculentus is not found, suggesting it has recently been extirpated by the O. niloticus invasion. In both species, populations in the satellite lakes are significantly genetically isolated from each other, with private mtDNA haplotypes and microsatellite alleles. For O. niloticus, genetic diversity in satellite lakes was similar to that found in Lake Victoria. Our data imply a low frequency of immigration exchange between the two populations of O. esculentus and we suggest that the populations of this endangered species and important fisheries resource should be conserved separately in Lakes Kanyaboli and Namboyo and with high priority.     

Identifications of Captive and Wild Tilapia Species Existing in Hawaii by Mitochondrial DNA Control Region Sequence

Background

The tilapia family of the Cichlidae includes many fish species, which live in freshwater and saltwater environments. Several species, such as O. niloticus, O. aureus, and O. mossambicus, are excellent for aquaculture because these fish are easily reproduced and readily adapt to diverse environments. Historically, tilapia species, including O. mossambicus, S. melanotheron, and O. aureus, were introduced to Hawaii many decades ago, and the state of Hawaii uses the import permit policy to prevent O. niloticus from coming into the islands. However, hybrids produced from O. niloticus may already be present in the freshwater and marine environments of the islands. The purpose of this study was to identify tilapia species that exist in Hawaii using mitochondrial DNA analysis.

Methodology/Principal Findings

In this study, we analyzed 382 samples collected from 13 farm (captive) and wild tilapia populations in Oahu and the Hawaii Islands. Comparison of intraspecies variation between the mitochondrial DNA control region (mtDNA CR) and cytochrome c oxidase I (COI) gene from five populations indicated that mtDNA CR had higher nucleotide diversity than COI. A phylogenetic tree of all sampled tilapia was generated using mtDNA CR sequences. The neighbor-joining tree analysis identified seven distinctive tilapia species: O. aureus, O. mossambicus, O. niloticus, S. melanotheron, O. urolepies, T. redalli, and a hybrid of O. massambicus and O. niloticus. Of all the populations examined, 10 populations consisting of O. aureus, O. mossambicus, O. urolepis, and O. niloticus from the farmed sites were relatively pure, whereas three wild populations showed some degree of introgression and hybridization.

Conclusions/Significance

This DNA-based tilapia species identification is the first report that confirmed tilapia species identities in the wild and captive populations in Hawaii. The DNA sequence comparisons of mtDNA CR appear to be a valid method for tilapia species identification. The suspected tilapia hybrids that consist of O. niloticus are present in captive and wild populations in Hawaii.     

Length‐weight relationships of Notopterus notopterus and introduced Oreochromis niloticus from the Indus River,southern Punjab,Pakistan

This study describes the length–weight relationships (LWRs) for Oreochromis niloticus and Notopterus notopterus, collected in 2006 and 2009 respectively from the Indus River, southern Punjab, Pakistan, with W = 0.0393 L^2.72 for O. niloticus and W = 0.0121 L^2.87 for N. notopterus. Negative allometric growth is indicated for both species.     

Relationship between lectin binding properties and the expression of blood group ABH antigens in vascular endothelia and red blood cells from 18 primate species

Summary The reactivity was examined of horseradish peroxidase labelledUlex europaeus agglutinin-I (UEA-I) andGriffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) with red blood cells and vascular endothelium in formalin-fixed, paraffin embedded tissues from 18 primate species. The expression of blood group ABH antigens in these cells as well as secretions from other tissues was also examined by the indirect immunoperoxidase method using monoclonal anti-ABH antibodies as primary antibodies. In Prosimians and New World monkeys which lack ABH antigens on both red blood cells and endothelial cells, but produce these antigens in other tissue secretions, GSAI-B₄ always reacted with both red blood cells and endothelial cells. In Old World monkeys, which express blood group antigens on endothelial cells but not on red blood cells, neither GSAI-B₄ nor UEA-I reactivity were observed, except the endothelial cells from blood group B or O individuals occasionally reacted with GSAI-B₄ or UEA-I, respectively. Although UEA-I reactivity was not observed in the endothelial cells of gibbon, it reacted with these cells from chimpanzees. In these two anthropoid apes, both endothelial cells and red blood cells expressed ABH antigens as in humans. These results suggest the close evolutionary relationship between the expression of blood group ABH antigens and lectin binding properties of red blood cells and endothelial cells in primate species.     

Morphological,ecological and genetic aspects associated with endemism in the Fly Orchid group

The European genus Ophrys (Orchidaceae) is famous for its insect‐like floral morphology, an adaptation for a pseudocopulatory pollination strategy involving Hymenoptera males. A large number of endemic Ophrys species have recently been described, especially within the Mediterranean Basin, which is one of the major species diversity hotspots. Subtle morphological variation and specific pollinator dependence are the two main perceptible criteria for describing numerous endemic taxa. However, the degree to which endemics differ genetically remains a challenging question. Additionally, knowledge regarding the factors underlying the emergence of such endemic entities is limited. To achieve new insights regarding speciation processes in Ophrys, we have investigated species boundaries in the Fly Orchid group (Ophrys insectifera sensu lato) by examining morphological, ecological and genetic evidence. Classically, authors have recognized one widespread taxon (O. insectifera) and two endemics (O. aymoninii from France and O. subinsectifera from Spain). Our research has identified clear morphological and ecological factors segregating among these taxa; however, genetic differences were more ambiguous. Insights from cpDNA sequencing and amplified fragment length polymorphisms genotyping indicated a recent diversification in the three extant Fly Orchid species, which may have been further obscured by active migration and admixture across the European continent. Our genetic results still indicate weak but noticeable phylogeographic clustering that partially correlates with the described species. Particularly, we report several isolated haplotypes and genetic clusters in central and southeastern Europe. With regard to the morphological, ecological and genetic aspects, we discuss the endemism status within the Fly Orchid group from evolutionary, taxonomical and conservation perspectives.     

ABO blood group polymorphisms and risk for ischemic stroke and peripheral arterial disease

Recent studies have demonstrated association between ABO blood system and thrombosis, indicating that individuals belonging to non-O blood groups (A, B or AB) present an increased risk of venous thrombosis, heart disease, and ischemic stroke (IS) as compared to O blood group carriers. In this study, we investigated the frequency of ABO blood group polymorphisms and its association with IS and peripheral arterial disease. Significant differences were observed for O1 (OR 0.57, 95 % CI 0.35–0.95, p < 0.05) and O2 (OR 3.47, 95 % CI 1.15–10.28, p < 0.05) alleles among IS patients while significant differences were observed for B phenotype (26.3 vs 9.5 %, OR 3.42, 95 % CI 1.32–8.76, p = 0.01, patients vs controls, respectively) and alleles A1 (OR 0.31, 95 % CI 0.11–0.84, p < 0.05), O2 (OR 4.61, 95 % CI 1.59–13.23, p < 0.01) and B (OR 3.42, 95 % CI 1.62–7.13, p < 0.001) alleles for PAD patients. O1 allele was an independent variable (OR 0.27, 95 % CI 0.12–0.57, p < 0.001) for IS patients. These data suggest the relationship of non-O blood groups in pathogenesis of thrombosis events and a possible protective effect of O blood group.     

The use of xenoantigenic antisera for the identification of tilapiine species: comparative laboratory and field studies

Rapid and reliable identification of tilapiine taxa and strains is essential for selective breeding purposes and the conservation of natural genetic resources. There is evidence that antisera‐mediated erythrocyte agglutination assays can fit these requirements. We evaluated the applicability of agglutination tests by studying the capacity of species characteristic antisera to recognize erythrocytes from individuals of 10 natural Ghanaian populations of Oreochromis niloticus and Sarotherodon melanotheron. The vast majority of the 218 tested individuals could be identified based on antisera‐mediated erythrocyte recognition. Controls indicated the specificity of these reactions. Still, erythrocytes from 16% of all tested specimens did not respond to any antiserum (zero responders), indicating the possible existence of blood group properties in tilapias. We discuss the specificity of the antisera, the relevance of zero responses and the applicability of these tests in aquaculture and field studies.     

The A-B-O blood groups of baboons

A new series of 188 baboons, Papio papio, Senegal, have been tested for the human type A-B-O groups with the following results: 2 group O, 27 group A, 93 group B and 66 group AB. This distribution fits the Hardy-Weinberg formula perfectly, using the allele frequencies O = 10.3%, A = 29.0%, and B = 60.8%. Up to date, five series of baboons comprising a total of 684 animals have been tested for their A-B-O groups. On these 684 baboons, from three different species, only three belonged to group O. Nevertheless, there is convincing indirect evidence that in most of the baboon species tested so far the frequency of gene O is about 10%. There are significant differences in the distribution of the blood groups in the various baboon species, comparable to the differences in racial distribution of the A-B-O blood groups in man, e.g., the frequency of gene A ranges from 18.2% in Papio ursinus, South Africa, to 48.3% in Papio cynocephalus. The usefulness of the methods of population genetics, viz, allele frequency analysis, for studies of blood groups in primates is demonstrated. The differences and similarities between the A-B-O blood groups in man and baboons are discussed.     

SNP marker detection and genotyping in tilapia

We have generated a unique resource consisting of nearly 175 000 short contig sequences and 3569 SNP markers from the widely cultured GIFT (Genetically Improved Farmed Tilapia) strain of Nile tilapia (Oreochromis niloticus). In total, 384 SNPs were selected to monitor the wider applicability of the SNPs by genotyping tilapia individuals from different strains and different geographical locations. In all strains and species tested (O. niloticus, O. aureus and O. mossambicus), the genotyping assay was working for a similar number of SNPs (288–305 SNPs). The actual number of polymorphic SNPs was, as expected, highest for individuals from the GIFT population (255 SNPs). In the individuals from an Egyptian strain and in individuals caught in the wild in the basin of the river Volta, 197 and 163 SNPs were polymorphic, respectively. A pairwise calculation of Nei’s genetic distance allowed the discrimination of the individual strains and species based on the genotypes determined with the SNP set. We expect that this set will be widely applicable for use in tilapia aquaculture, e.g. for pedigree reconstruction. In addition, this set is currently used for assaying the genetic diversity of native Nile tilapia in areas where tilapia is, or will be, introduced in aquaculture projects. This allows the tracing of escapees from aquaculture and the monitoring of effects of introgression and hybridization.     

Trophic Niche Segregation in the Nilotic Ichthyofauna of Lake Albert (Uganda, Africa)

Synopsis Nile perch, Lates niloticus, and Nile tilapia, Oreochromis niloticus, were originally transplanted from Lake Albert in western Uganda to the African Great Lakes, Lake Victoria and Lake Kyoga, where they are partially implicated in reduction of the fish species diversity. Lake Albert is facing multiple environmental changes, including declining fish species diversity, hyper-eutrophication, hypoxia, and reduced fish catches. To examine the role of Nile perch and Nile tilapia in the food web in their native Lake Albert, we estimated their diets using stable nitrogen and carbon isotopes. In Lake Albert, the tilapiine congeners (closely related species), Tilapia zillii, Oreochromis leucostictus, and Sarethorodon galilaeus, and the centropomid Nile perch congener, Lates macrophthalmus, have narrower diet breath in the presence of the native O. niloticus and L. niloticus. A computerized parameter search of dietary items for five commercially important fish species (Hydrocynus forskahlii, Bagrus bayad, L. niloticus, Alestes baremose and Brycinus nurse) was completed using a static isotopic mixing model. The outcome of the simulation for most fish species compared favorably to previously published stomach contents data for the Lake Albert fishes dating back to 1928, demonstrating agreement between stable isotope values and analyses of stomach contents. While there were some indications of changes in the diets of L. niloticus and A. baremose diets over the past 20 years in parallel with other changes in the lake, for the most part, food web structure in this lake remained stable since 1928. The Lake Albert fish assemblage provides insight into the invasion success of L. niloticus and O. niloticus.