Sergiu Emil Georgescu

Nuclear Markers of Danube Sturgeons Hybridization

Acipenseriformes are composed of 25 sturgeon species and two paddlefish species distributed exclusively in the northern hemisphere. The Danube River and the Black Sea were originally inhabited by six sturgeon species but two are extinct and only four are still reproducing currently in the Lower Danube: Huso huso, Acipenser stellatus, A. gueldenstaedtii and A. ruthenus. Sturgeon species hybridize more easily than other fish and the determination of pure species or hybrid status is important for conservation and for breeding in fish farms. This survey demonstrated that morphological determination of this status is not reliable and a molecular tool, based on eight microsatellites genotypes is proposed. This method, based on three successive statistical analyses including Factorial Correspondence Analysis (FCA), Structure assignation and NewHybrids status determination, showed a high efficiency in discriminating pure species specimens from F1, F2 and two kinds of backcross individuals involving three of the four reproducing Lower Danube sturgeon species.

Site heteroplasmy in the mitochondrial cytochrome b gene of the sterlet sturgeon Acipenser ruthenus.

Sturgeons are fish species with a complex biology. They are also characterized by complex aspects including polyploidization and easiness of hybridization. As with most of the Ponto-Caspian sturgeons, the populations of Acipenser ruthenus from the Danube have declined drastically during the last decades. This is the first report on mitochondrial point heteroplasmy in the cytochrome b gene of this species. The 1141 bp sequence of the cytb gene in wild sterlet sturgeon individuals from the Lower Danube was determined, and site heteroplasmy evidenced in three of the 30 specimens collected. Two nucleotide sequences were identified in these heteroplasmic individuals. The majority of the heteroplasmic sites are synonymous and do not modify the sequence of amino acids in cytochrome B protein. To date, several cases of point heteroplasmy have been reported in animals, mostly due to paternal leakage of mtDNA. The presence of specific point heteroplasmic sites might be interesting for a possible correlation with genetically distinct groups in the Danube River.

Phylogenetic Relationships of the Mangalitsa Swine Breed Inferred from Mitochondrial DNA Variation

The Mangalitsa pig, a swine breed belonging to the protected gene fund of original and primitive animal breeds of the FAO (Food and Agriculture Organization), has been known to inhabit Romanian territories since the 19th century. The aim of this study was to compare the Mangalitsa breed with several European and Asiatic swine breeds in order to emphasize its uniqueness and to elucidate its origin. For this purpose, we analyzed a 613 bp mitochondrial DNA D-loop fragment and 1140 bp of the cytochrome b gene in a population of Mangalitsa pigs and the polymorphic sites were compared with sequences from GenBank originating from other swine breeds. Taking into account the total of 24 breeds and 5 different Wild Boar populations analyzed, 86 polymorphic sites representing 32 haplotypes were observed, with an average percentage of polymorphic sites of 4.9%. Three Neighbor-Joining phylogenetic trees were constructed based on Kimura 2-parameter distances, using D-loop, cytochrome b and mitochondrial reunited sequences. For the analyzed Mangalitsa population, four distinct haplotypes were identified, including one that was common to other breeds. Our study suggests that the Mangalitsa swine originate from primitive breeds which might be directly derived from the Wild Boar.

Low Danube Sturgeon Identification Using DNA Markers

Sturgeons represent a class of fish with a distinctive importance, both from a scientific and a commercial point of view. This group of fish appeared 200 million years ago (Bemis et al., 1997) and the early diversification of the species took place in a geographical area currently situated in Asia. Since these are some of the oldest fish species in the world, having survived several mass extinction events, authentic “living fossils” with a slow evolution, sturgeons may constitute a study model for the development of all vertebrates. The commercial importance of these species is due to the value of the products obtained from them, especially caviar, which is considered a gourmet delicacy. Unfortunately, due to their economic importance, these species have been overly exploited through overfishing and poaching. As a result, nowadays they are on the brink of extinction.

Molecular Methods for the Detection of Natural Hybrids in Sturgeon Populations

Abstract Due to construction of the Iron Gates dams, the Lower Danube has suffered a decrease in sturgeon populations. The dams have decreased sturgeon habitat area, which in turn has caused an overlap of reproduction areas for all sturgeon species. The ease with which sturgeon species can create hybrid offsprings gave rise to an increase in the number of hybrid sturgeon species now found in the Lower Danube area. We propose a set of molecular methods for hybrid species using DNA markers represented by microsatellites and mitochondrial DNA. This identification data and methodology is important for use on sturgeon farms due to the need to correctly identify species of sturgeons. Using the proposed methodologies, it is possible to avoid identification errors that might appear when using only morphological criteria to idenfy sturgeons Résumé Méthodes moléculaires pour la détection des hybrides naturelles dans les populations d’esturgeons. En raison de la construction de barrages aux Portes de Fer, le Danube inférieur a subi une diminution des populations d’esturgeon. La diminution de l’aire géographique a induit le chevauchement des sites de reproduction de toutes les espèces d’esturgeons. La facilité d’hybridation caractéristique de ces espèces a conduit à la situation actuelle qui est une augmentation du nombre d’hybrides d’esturgeons. Nous proposons plusieurs méthodes moléculaires pour l’identification des hybrides utilisant deux types de marqueurs d’ADN. Les données sont importantes pour les fermes d’esturgeons de part la nécessité d’une identification correcte des individus. Ainsi, il est possible de pallier les difficultés potentielles liées à une simple identification morphologique Rezumat Metode moleculare de detecṭie a hibrizilor naturali în populaṭii de sturioni. Datorită construcţiei barajelor de la Porţile de Fier, Dunărea Inferioară a suferit o descreştere a populaţiilor de sturioni. Restrângerea arealului a condus la suprapunerea zonelor de reproducere pentru toate speciile de sturioni. Acest fenomen, împreună cu uşurinţa hibridizării în cazul acestor specii, a condus la situaţia actuală, în care există o creştere a numărului hibrizilor naturali de sturioni. Propunem un set de metode moleculare pentru identificarea hibrizilor de sturioni, folosind două tipuri de markeri ADN. Datele obţinute sunt importante pentru fermele piscicole datorită necesităţii identificării corecte a indivizilor. Astfel, se pot evita eventualele dificultăţi apărute în identificarea indivizilor exclusiv pe baza criteriilor morfologice

Molecular analysis of phylogeographic subspecies in three Ponto-Caspian sturgeon species.

Sturgeons (Order Acipenseriformes) represent an extremely valuable natural resource that is now facing depletion. In the current study we evaluate if the traditional classification in subspecies of Acipenser gueldenstaedtii, Acipenser stellatus and Huso huso, endemic to Ponto-Caspian region is sustained by molecular analysis and if these represent Evolutionary Significant Units (ESUs) that should be managed separately in conservation programs. To examine the classification of taxonomic entities we sequenced a fragment of the mitochondrial control region in case of three sturgeon species that inhabit the North-western of Black Sea and migrate for reproduction in the Lower Danube. Beside these sequences, we used previously published sequences from sturgeon individuals sampled in the Black Sea, Azov Sea and Caspian Sea. We determined the genetic diversity and genetic differentiation, conducted a Population Aggregation Analysis (PAA) and inferred an intraspecific molecular phylogeny and haplotype network. The results indicated a low level of genetic differentiation between the geographically designated subspecies and did not support a significant divergence or reciprocal monophyly between them. Our results confirm previous genetic studies with smaller samples sizes, but additional analyses including nuclear markers should be conducted for proper recommendations aiming at the development of conservation programs.

Genetic Diversity of Brown Trout Populations Using Mitochondrial Markers In Relatively Similar Geographical and Ecological Conditions – a Carpathian Case Study

ABSTRACT Among the species of salmonids that exist in Romania, the most common is the brown trout (Salmo trutta, morpha fario, Linnaeus, 1758), with a high commercial potential, being used for aquaculture or fishing. Unfortunately, its natural habitat is disrupted by human activities and in order to avoid local extinction of Salmo trutta fario repopulation strategies are being applied. The repopulation activities must be carefully designed and conducted taking into consideration that there is a high risk of hybridization. Our study aims to analyze the genetic diversity of three Romanian brown trout populations found in rivers in the Făgăraş Mountains using mitochondrial markers. Standard DNA extraction protocol with phenol-chloroform was applied to the biological material represented by small fin fragments sampled from 80 individuals of brown trout, followed by PCR amplification of D-loop control region and Sanger sequencing. Alignment and editing of all the sequences obtained were carried out with Bioedit, the phylogenetic tree construction was performed by Neighbour Joining method implemented in MEGA v5, and intra- and interpopulational diversity was evaluated with DNAsp v5 and MEGA v5. For the three brown trout populations, 13 haplotypes were identified for N1, 11 for N2 and 10 for N4, with a haplotype diversity greater than 0.8. The phylogenetic tree topology showed that individuals chosen for this study were placed in the Danubian clade as the other Danubian sequences selected from GenBank. The study proved that the three Salmo trutta fario populations analyzed were genetically distinct and that in Romania there are still are pure Danubian brown trout populations. RÉSUMÉ La diversité génétique des populations de truites brunes à l’aide de marqueurs mitochondriaux dans des conditions géographiques et écologiques relativement similaires - une étude de cas des Carpates. Parmi les espèces de salmonidés qui existent en Roumanie, la plus commune est la truite brune (Salmo trutta fario, Linnaeus, 1758) qui possède un grand potentiel commercial, étant utilisées pour l’aquaculture ou la pêche. Malheureusement, son habitat naturel est perturbé par des activités humaines. Dans le but d’éviter l’extinction de la truite, des stratégies de repeuplement sont appliquées. Les activités de repeuplement doivent être soigneusement conçues et réalisées en tenant compte d’un grand risque d’hybridation. Le but de notre étude est l’analyse la diversité génétique de trois populations de truites brunes roumaines qui se trouvent dans les rivières des Monts Fagărăş à l’aide de la région de contrôle de l’ADN mitochondrial. Le protocole standard d’extraction de l’ADN, au phénol-chloroforme, a été appliqué sur les échantillons biologiques représentés par de petits fragments de nageoire appartenant à 80 individus de truite brune, suivie par l’amplification de la région de contrôle D-loop par PCR ainsi que le séquençage par la méthode Sanger. L’alignement de toutes les séquences obtenues a été réalisé avec Bioedit, la construction de l’arbre phylogénétique a été réalisée par la méthode de Neighbor Joining (logiciel MEGA v5) et la diversité intra- et interpopulationnel a été évaluée avec DNAsp v.5 et MEGAv.5. Pour les trois populations de truites brunes, 13 haplotypes ont été identifiés pour N1, 11 pour N2, et 10 pour N4, avec une diversité d’haplotypes supérieur à 0,8. La topologie de l’arbre phylogénétique a montré que les truites choisies pour cette étude ont été placées dans le clade danubien, comme les autres séquences du Danube choisis dans GenBank. Cette étude a prouvé que les trois populations de Salmo trutta fario analysées sont génétiquement distinctes et qu’en Roumanie, il existe toujours des populations de truite brune pures danubiennes. REZUMAT Analiza diversităţii genetice cu ajutorul markerilor mitocondriali, a unor populaţii de păstrav comun aflate în condiţii geografice şi ecologice relativ similare în Munţii Carpaţi. Păstrăvul comun reprezintă una dintre speciile de Salmonidae din România, cu o importanţă comercială deosebită, utilizat în domeniul acvaculturii şi pentru pescuit sportiv. Din nefericire, influenţa antropică se resimte şi în habitatul lui Salmo trutta fario, iar pentru a contracara influenţele negative, se încearcă aplicarea de strategii de repopulare. Activitățile de repopulare trebuie să fie realizate și desfăşurate cu atenţie, luând în considerare faptul că există un risc ridicat de hibridizare. În cadrul acestui studiu, am avut în vedere analiza diversităţii genetice a trei populaţii de păstrăv comun din regiunea nordică a Munţilor Făgăraş, folosind markeri mitocondriali. Extracţia de ADN a fost realizată din mici fragmente de înotătoare, provenite de la 80 de indivizi, urmând protocolul standard de extracţie cu fenol-cloroform, urmată de amplificare prin PCR a regiunii mitocondriale D-loop şi secvenţiere după metoda Sanger. Alinierea şi editarea secvenţelor obţinute a fost realizată cu programul BioEdit, iar arborele filogenetic a fost construit prin metoda Neighbour Joining din cadrul programului MEGA v.5. Diversitatea inter- şi intrapopulaţională a fost evaluată cu programele DNAsp v.5 şi MEGA v.5. Pentru cele trei populaţii analizate, am observat că există 13 haplotipuri în cadrul populaţiei N1, 11 în populaţia N2 şi 10 în populaţia N4, cu o diversitate haplotipică mai mare de 0,8. Topologia arborelui filogenetic a sugerat că indivizii analizaţi au fost grupaţi în clada danubiană, alături de alte secvenţe aparţinând liniei danubiene utilizate la construcţia dendrogramei. Acest studiu a demonstrat ca cele trei populaṭii de Salmo trutta fario analizate sunt distincte din punct de vedere genetic şi că în România încă există populații pure de păstrăv comun danubian.

Expression Characterization of Six Genes Possibly Involved in Gonad Development for Stellate Sturgeon Individuals (Acipenser stellatus, Pallas 1771)

Nowadays, in sturgeons aquaculture, there is a necessity for sex identification at early stages in order to increase the efficiency of this commercial activity. The basis for a correct identification is studying the different factors that influence the gonad development. The research has been directed towards molecular methods that have been employed with various degrees of success in identifying genes with different expression patterns between male and female sturgeons during their development stages. For the purpose of understanding the sexual development of 4-year-old stellate sturgeon (Acipenser stellatus) individuals, we have selected six genes (foxl2, cyp17a1, ar, dmrt1, sox9, and star). We analysed the gene expression of the selected genes for gonads, anal fin, liver, body kidney, and white muscle. The cyp17a1, ar, dmrt1, and sox9 genes have a significant higher expression in male gonads than in female gonads, while the data shows no significant differences in the expression of the investigated genes in the other organs. We investigate these genes to shed light on aquaculture sturgeon sexual development.

Genetic profile analysis of aquaculture Russian sturgeon (Acipenser gueldenstaedtii) – implications for restocking measures

The aim of this study is to help the conservation effort of the Acipenser gueldenstaedtii Brandt et Ratzeburg, 1833 population from Lower Danube, ensuring its sustainable development in regards to aquaculture and wild populations. We evaluated the genetic diversity of five aquaculture strains using molecular markers, determining the suitability of employing the individuals in ex situ conservation and in restocking programs. The mitochondrial cytochrome b gene analysis highlighted that some sequences from aquaculture and wild populations are grouped together representing a single haplotype. Results showed that the five aquaculture strains are grouped in 3 haplotypes with a haplotype diversity of 0.55686. The microsatellite analysis highlights that the majority of loci have a tetrasomic profile that makes the inferring of genotypes and the statistical analyses difficult, only two (AciG198 and Spl106) had a disomic profile that could prove useful in future studies. We can conclude that the molecular analysis is important for the genetic characterization of aquaculture strains, shedding light on the genetics of A. gueldenstaedtii.

Analysis of the Microsatellite Variation in the Common Hybrid Between Russian Sturgeon (Acipenser Gueldenstaedtii Brandt and Ratzeburg, 1833) and Siberian Sturgeon (Acipenser Baerii Brandt, 1869) from Aquaculture

ABSTRACT Sturgeons such as Acipenser baerii and Acipenser gueldenstaedtii are the most common species farm raised worldwide in aquaculture, because of the dwindling natural sources of caviar and meat. Also, these species can easily participate in the formation of an intraspecific hybrid with a great potential for growth in aquaculture. Microsatellites are nuclear markers consisting of short repetitive sequence, dispersed across the entire genome with characteristics such as relatively small size and high level of polymorphism. The aims of the present study were to optimize a protocol for microsatellite multiplexing and analysis of genetic diversity in hybrid sturgeons farmed in Romania. Genomic DNA was isolated from fins, and four pairs of primers were designed to amplify microsatellite loci: LS 19, LS 68, Aox 9, and Aox 45. Amplification of the microsatellite loci was carried out in one 3-Plex reaction for LS 19, LS 68, and Aox 9, and monoplex reaction for Aox 45. For an individual locus we obtained four alleles for Aox 45, eleven alleles for Aox 9, six alleles for LS 68, and eight alleles for LS 19. The results will be applied to test the broodstocks at Romanian hatcheries and to increase the efficiency of breeding. RÉSUMÉ Analyse de la variation des microsatellites de l’hybride commun d’aquaculture de l’esturgeon russe (Acipenser gueldenstaedtii) et de l’esturgeon sibérien (Acipenser baerii). Les espèces d’esturgeons Acipenser baerii et Acipenser gueldenstaedtii sont parmi les plus communes dans l’aquaculture de monde entier suite de la diminution des ressources naturelles de caviar et de viande. De surcroît, ces espèces peuvent facilement participer à la formation d’un hybride intraspecifique possèdant un fort potentiel d’élevage. Les microsatellites sont des marqueurs nucléaires représentés par des séquences répétitives courtes dispersées dans le génome entier et définit par une taille relativement petite ainsi qu’un haut niveau de polymorphisme. Le but de la présente étude est l’optimisation d’un protocole d’amplification de loci microsatellites par PCR multiplex et l’analyse de la diversité génétique des hybrides d’esturgeons d’aquaculture en Roumanie. L’ADN génomique a été isolée à nageoire. Ainsi, quatre paires d’amorces on été utilisées pour amplifier les microsatellites LS 19, LS 68, Aox 9 et Aox 45. Une réaction d’amplification 3-Plex a été réalisée pour LS 19, LS 68 et Aox 9 ainsi qu’une monoplex pour Aox 45. Pour les loci individuels, nous avons obtenu quatre allèles pour Aox 45, onze allèles pour Aox 9, six allèles pour LS 68 et huit allèles pour LS 19. Le protocole sera appliqué afin de tester la pureté des stocks d’aquaculture des fermes piscicoles de la Roumanie et également pour augmenter l’efficacité de la reproduction. REZUMAT Analiza variaţiei microsateliţilor la hibridul comun dintre nisetru (Acipenser gueldenstaedtii) şi sturionul siberian (Acipenser baerii) din acvacultură. Sturionii precum Acipenser baerii şi Acipenser gueldenstaedtii sunt printre cele mai comune specii crescute în acvacultură, în întrega lumea datorită diminuării resurselor naturale de caviar şi carne. Totodată, aceste specii au o capacitate ridicată pentru a participa la formarea unui hibrid interspecific, cu un înalt potenţial pentru creşterea în acvacultură. Microsateliţii sunt markeri nucleari reprezentaţi de secvenţe repetitive scurte, dispersate în întreg genomul, având caracteristici precum dimensiunile reduse şi un înalt nivel de polimorfism. Scopul prezentului studiu a fost optimizarea unui protocol de PCR multiplex pentru amplificarea de microsateliţi şi analiza diversităţii genetice a hibrizilor de sturioni crescuţi în condiţii de acvacultură în România. ADN genomic a fost izolat din înotătoare şi patru perechi de primeri au fost desemnaţi pentru amplificarea microsateliţilor LS 19, LS 68, Aox 9 şi Aox 45. Amplificarea a fost realizată într-o reacţie 3-Plex pentru locii LS 19, LS 68 şi Aox 9 şi o reacţie monoplex pentru Aox 45. La nivelul locilor individuali, s-au obţinut four alele pentru Aox 45, unsprezece alele pentru Aox 9, şase alele pentru LS 68 şi opt alele pentru LS 19. Rezultatele vor fi utilizate pentru testarea purităţii stocurilor aparţinând crescătoriilor din România şi pentru creşterea eficienţei reproducerii.