A Multistep DNA-Based Methodology for Accurate Authentication of Sturgeon Species.

The sturgeons (order Acipenseriformes) are caviar producers and some of the most valuable fish species worldwide. Due to different reasons, wild populations are now at the brink of extinction. The high demand for caviar has led to the development of aquaculture for restocking and caviar production. Since the caviar from different species has different prices depending on the quality and attempts of commercial fraud based on species substitution have been found, correct species identification is more than necessary. We report a new multistep methodology for an accurate species identification based on both nuclear and mitochondrial markers. Our test integrates data from the analysis of microsatellites (Afu19, Afu34, Afu39, Afu54, Aox27, AoxD234, AnacC11 and AnacE4), nuclear gene markers (RPS7, vimentin and rhodopsin) and mtDNA barcoding to give a reliable molecular diagnostic for five sturgeon species (Huso huso, Acipenser stellatus, Acipenser ruthenus, Acipenser gueldenstaedtii and Acipenser baerii). In addition to species identification, our methodology allows the identification of bester, sterbe and best beluga hybrids, but also the identification of hybrids of unknown origin. This methodology has a good potential to contribute to the conservation of highly threatened sturgeon populations and also to the traceability of their products.

Oxidative Stress and Antioxidant Defense Mechanisms in Response to Starvation and Refeeding in the Intestine of Stellate Sturgeon (Acipenser stellatus) Juveniles from Aquaculture.

Acipenser stellatus is a critically endangered species due to the anthropic influence. It has been intensively captured for decades because of its high economic value, its roe being used in the caviar industry. Therefore, Acipenser stellatus is intensively raised in fish farms for both conservation and economical purposes. Aquaculture aims to optimize the feeding regime of juveniles in order to improve its profitability. The purpose of this study was to investigate if Acipenser stellatus can adapt to a starvation/refeeding regime by assessing the effects of this regime on oxidative stress biomarkers and antioxidant defense mechanisms in juveniles raised under aquaculture conditions. The juveniles were subjected to two regimes: a 7-day starvation period followed by 21 days of refeeding, respectively a 14-day starvation period followed by 21 days of refeeding. The results showed that both starvation/refeeding regimes induced an enhancement of antioxidant enzymes activities in the intestine of the juveniles. The oxidative damage was counteracted at the protein level. However, lipid peroxidation was significantly induced in the intestine of the juveniles subjected to 14/21-day starvation/refeeding regime. The 7/21-day starvation/refeeding regime proved to be more suitable for Acipenser stellatus and therefore, it could be useful to optimize the feeding practice in aquaculture production.

A Broader Perspective on Anti-Ro Antibodies and Their Fetal Consequences-A Case Report and Literature Review.

The presence of maternal Anti-Ro/Anti-La antibodies causes a passively acquired autoimmunity that may be associated with serious fetal complications. The classic example is the autoimmune-mediated congenital heart block (CHB) which is due in most cases to the transplacental passage of Anti-Ro/Anti-La antibodies. The exact mechanisms through which these pathologic events arise are linked to disturbances in calcium channels function, impairment of calcium homeostasis and ultimately apoptosis, inflammation and fibrosis. CHB still represents a challenging diagnosis and a source of debate regarding the best management. As the third-degree block is usually irreversible, the best strategy is risk awareness and prevention. Although CHB is a rare occurrence, it affects one in 20,000 live births, with a high overall mortality rate (up to 20%, with 70% of in utero deaths). There is also concern over the lifelong consequences, as most babies need a pacemaker. This review aims to offer, apart from the data needed for a better understanding of the issue at hand, a broader perspective of the specialists directly involved in managing this pathology: the rheumatologist, the maternal-fetal specialist and the cardiologist. To better illustrate the theoretical facts presented, we also include a representative clinical case.

Assessment of genetic diversity in main local sheep breeds from Romania using microsatellite markers.

The state of the local breeds of farm animals is increasingly precarious worldwide because of the aggressive introduction of breeds with improved economical traits. The preference of the breeders for local breeds is due to their higher adaptability to the particular climate and relief conditions of the mountain areas, to the high rate of assimilation of the feeds from these regions and to their increased resistance to diseases. This study analyzes the genetic variation of the main four local Romanian sheep breeds (Tsurcana, Tsigai, Ratska and Teleorman Blackhead) in terms of stock and economic importance, using 18 microsatellite markers. The mean number of alleles per locus was of 9.764. The values of genetic diversity parameters exhibited a high degree of polymorphism for the analyzed breeds, although inbreeding was highlighted particularly in Tsurcana and Tsigai. These breeds also showed an intense gene flow among them and were less differentiated in comparison with Ratska and Teleorman Blackhead. The results of this study may be useful for breeding programs and conservation plans since the genetic resources of the local breeds must be preserved so as to maintain an adequate level of biodiversity in animal husbandry.

Effects of starvation and refeeding on growth performance and stress defense mechanisms of stellate sturgeon Acipenser stellatus juveniles from aquaculture.

Acipenser stellatus represents a species of great economical interest due to its roe used for caviar production. Therefore, it has been intensively captured for decades and nowadays, this species is on the verge of extinction. As a consequence, Acipenser stellatus is intensively raised in fish farms. Aquaculture is focused on optimizing the feeding regime of juveniles. The aim of this study was to investigate if Acipenser stellatus can adapt to a starvation/refeeding regime by assessing the effects of this regime on growth performance, oxidative stress biomarkers and heat shock protein (hsp) gene expression in juveniles raised under aquaculture conditions. The juveniles were subjected to two starvation/refeeding regimes: a 7-day starvation period followed by 21 days of refeeding, and a14-day starvation period followed by 21 days of refeeding. The results had shown that the juveniles subjected to 7/21-day starvation/refeeding regime presented a complete compensatory growth, they were able to counteract the oxidative stress by enhancing activities of the antioxidant enzymes and they presented no significant changes in hsp gene expression. In contrast, 14/21-day starvation/refeeding regime negatively influenced growth performance, it induced a high level of oxidative stress that was impossible to counteract and it determined major changes in the hsp gene expression level in the liver of Acipenser stellatus. Thus, Acipenser stellatus seems to be able to adapt only to the 7/21-day starvation/refeeding regime that does not threaten the growth performance and the welfare of juveniles. Therefore, it could be useful to optimize the feeding practice in aquaculture production.

Gene expression involved in the sexual development of Best Beluga hybrid sturgeons.

The scope of this study is to investigate the expression of dmrt1, foxl2, ar, star and sox9 genes in the context of the gonad development stage of 21 month-old Best Beluga individuals. No significant difference was observed between males and females but the cyp17a1 gene showed higher expression in male than in female gonads. The results suggest that during sampling the females were in perinucleolar stage and the males in early spermatogenesis stage which led to specific patterns of expression for the investigated genes.

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

Nowadays, in sturgeon's 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.

Occurrence and first molecular characterization of Sarcocystis spp. in wild boars (Sus scrofa) and domestic pigs (Sus scrofa domesticus) in Romania: Public health significance of the isolates.

Domestic and wild pigs, as intermediate hosts, can harbor tissue cysts of three Sarcocystis species namely S. miescheriana, S. suihominis and S. porcifelis. Out of them, S. suihominis is zoonotic. Romania is a country with high consumption of raw and/or undercooked traditional pork products. This fact may greatly favor the acquiring of the zoonotic Sarcocystis infections by humans, as definitive host. Based on this consideration and in order to investigate the occurrence and public health significance of Sarcocystis spp. in two western counties (Caras-Severin and Timis) of Romania, a total of 165 heart samples from hunted wild boars (Sus scrofa, n=101) and home slaughtered domestic pigs (Sus scrofa domesticus, n=64) were screened using microscopic fresh examination and molecular methods. Microscopic examination revealed the presence of sarcocysts in 60.4% of wild boars, and 23.4% of domestic pigs. Genetic characterization of isolates through the PCR-RFLP procedure, targeting the 18S rRNA gene, was successfully achieved for all microscopically positive samples, indicating the presence of a single species, S. miescheriana, in both hosts. The identity of 13 selected S. miescheriana isolates was also confirmed through sequencing. The tested hosts older than 27 months were found to be significantly higher infected (p<0.05) with Sarcocystis than the 6 to ≤27months age group. Although the human infective S. suihominis has not been registered, for a more reliable epidemiological picture, further molecular studies enrolling a larger number of animals and diagnosis on human intestinal Sarcocystis infections are still necessary.

Molecular Survey of Hepatozoon canis in Red Foxes (Vulpes vulpes) from Romania.

Blood samples of 119 red foxes, originating from 44 hunting grounds of 3 western counties (Arad, Hunedoara, and Timis) of Romania, have been examined for the presence of Hepatozoon canis infection using the conventional polymerase chain reaction (PCR) of the fragment of 18S rRNA gene. Overall, 15 (12.6%) samples were found to be PCR-positive. Of the sampled hunting grounds, 29.5% (13/44) were found positive. Positive samples were recorded in all screened counties with the prevalence of 14.8% (9/61) in Arad, 9.8% (5/51) in Timis, and 14.3% (1/7) in Hunedoara, respectively. No correlation was found (P > 0.05) between H. canis positivity and gender or territorial distribution of the infection. To confirm PCR results, 9 randomly selected amplicons were sequenced. The obtained sequences were identical to each other, confirmed the results of the conventional PCR, and showed 98-100% homology to other H. canis sequences. The results of the current survey support the role of red foxes as sylvatic reservoirs of H. canis in Romania.

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.

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.

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.