16S-23S rRNA Internal Transcribed Spacer Region (ITS) Sequencing: A Potential Molecular Diagnostic Tool for Differentiating Lactococcus garvieae and Lactococcus petauri.

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

NGS analysis revealed that Lactococcus garvieae Lg-Per was Lactococcus petauri in Türkiye.

Lactococcus garvieae Lg-per was originally isolated from rainbow trout cultured in cages located on the Turkish coast of the Black Sea in 2011. A whole genome sequence of Lg-per was performed in the present study. The complete genome of Lg-per mapped to the reference genomes of L. garvieae (GCF_000269925.1) and Lactococcus petauri (GCF_014830225.1) had a total of 1,694,407 and 1,945,297 base pairs, respectively. Lg-per had 1955 protein-coding genes and 4 rRNA, 46 tRNA and 1 tmRNA operons. The orthoANI value was 98.30% between Lg-per and L. petauri (GCF_014830225.1) and 93.1% between Lg-per and L. garvieae (GCF_000269925.1). A phylogenetic tree generated from the whole genome sequences (WGS) of several Lactococcus species found that L. petauri (GCA 002154895) was closely related to the Lg-per strain with 98% similarity. Although L. garvieae Lg-per was confirmed as L. garvieae based on phenotypical, biochemical and 16S rRNA sequence, WGS of the Lg-per strain revealed that Lg-per was L. petauri. Using a 16S rRNA-based PCR detection approach, Lg-per was misdiagnosed as L. garvieae since its 16S rRNA gene was 99.9% similar to that of L. garvieae strains. Consequently, the 16S rRNA-based PCR detection approach may not be adequate for the identification of the Lactococcus genus. This is the first study to document the presence of L. petauri in Türkiye. L. garvieae isolates should be analysed using WGS since the same issue might occur in other countries.

The broad-scale microplastic distribution in surface water and sediments along Northeastern Mediterranean shoreline.

Plastics manufactured to fulfil the unique demands of civilization accumulate in the sea due to their durability. Microplastics (MP) pose a greater threat than macroplastics as they can easily enter the aquatic environment and be hard to detect. MPs potentially impact several components of the marine life and food chain. This study determined MP distribution and characterization by collecting sediment from 47 different stations and surface seawater (SSW) from 29 stations in 2019 along the Turkish coast of the Eastern Mediterranean Sea. Potential MP particles were stained with Nile-Red and verified using ATR-FTIR. While MP abundance in the sediment ranged between 118 ± 97 and 1688 ± 746 MPs kg-1, it varied between 0.18 ± 0.10 MPs m-3 and 2.21 ± 1.75 MPs m-3 in SSW. The MP abundance showed significant spatial variation (p < 0.05). The polymer type in the samples was determined by ATR-FTIR. In both water and sediments, polyethylene was the most common MP type (>59 %), while fragment was the most common MP form (>57.6 %), and >65 % of overall MPs were <1500 µm. The spatial pattern of MPs in the sediments and SSW was affected by the population, the magnitude of the tourism sector, the rim current, and circulation. The monitoring data presented here can provide a remarkable projection of the current trend and form a basis for future MP pollution prevention.

Spatial pattern and characteristics of the benthic marine litter in the southern Black Sea shelf.

The abundance and characteristics of benthic marine litter in the southern Black Sea shelf were demonstrated. During the surveys, 671 litter items weighing 83.93 kg were collected from the stations. The average density by number and weight were 460.70 ± 99.71 n/km2 and 80.68 ± 48.06 kg/km2. The litter density was significantly higher at 10-20 m (646.09 ± 165.63 n/km2) compared to 20-30 m (165.53 ± 81.02 n/km2) and >30 m (413.87 ± 146.59 n/km2) depth classes. Plastic (69.03%) was the most abundant material by number, followed by textile (20.94%) and metal (5.68%). The litter composition differed between the no-trawl zone and trawl ground. Our results indicate that more detailed research is needed to understand the interaction of various factors influencing transportation and accumulation of marine litter in the Black Sea.

Microplastic contamination and characteristics spatially vary in the southern Black Sea beach sediment and sea surface water.

In this study, the abundance, and characteristics of the microplastics on the southern coast of the Black Sea were assessed. More than 70% of the detected microplastics were smaller than 2.5 mm and mostly consisted of fibers and fragments. The average microplastic abundance in the beach sediment and seawater were 64.06 ± 8.95 particles/kg and 18.68 ± 3.01 particles/m3, respectively. The western coast of the study area (Marmara region) was the most polluted area, and a spatially significant difference was determined in terms of abundance. The composition in the beach sediment (particles/kg) was dominated by styrene acrylonitrile copolymer (SAC) (40.53%), polyethylene terephthalate (PET) (38.75%), and polyethylene (PE) (6.91%), whereas the seawater (particles/m3) was dominated by PET (57.26%), PE (13.52%), and polypropylene PP (11.24%). The results of our study can be a baseline for environmental modeling studies and experimental studies on the marine organisms inhabiting the Black Sea.

Characteristics and temporal trends of microplastics in the coastal area in the Southern Black Sea over the past decade.

Time-series datasets showing the gradual conformational change in the status and characteristics of microplastics (MP) in marine waters are needed to protect vulnerable marine ecosystems. In this study, the abundance, spatiotemporal distribution, polymer composition, size, and shapes of the MP in the seawater sampled from 0.5 (station-1), 2.5 (station-2), and 5 (station-3) miles off the coast of the southeastern Black Sea between 2009 and 2020 were investigated. The abundance of the MP ranged from 0.181 to 0.944 m-3. The amounts of MP in the stations, from the greatest to the smallest, were 0.65 MP m-3 (2), 0.45 MP m-3 (1), 0.29 MP m-3 (3). Thirteen types of polymers were detected by FTIR, the most abundant types of which were polyethylene (44.9%) and polyethylene terephthalate (25.3%). Sizes of MP ranged from 118 to 4998 µm, 50% being smaller than 2000 µm. The most dominant shape of the MPs was fragments (56.3%). Abundance did not show significant spatiotemporal changes. Periodical data presented herein may represent a remarkable projection and create a baseline for the ecosystem's future functioning and pollution research in the Black Sea.

Comparative trace element trends in Posidonia oceanica and its sediments along the Turkish-Mediterranean Sea coast.

Concentrations of eight trace elements (V, Cr, Ni, Cu, Zn, As, Cd, and Pb) were investigated in Posidonia oceanica leaf blades and sediments sampled from 23 stations along the Turkish-Mediterranean Sea coast. Trace element (TE) concentration in both sediment and P. oceanica showed a statistically significant distribution among sampling stations. Most of the TE concentration in samples varied remarkably among stations without a consistent trend. Concentrations of Zn, Cd, Cu, and Pb in the sediment of all of the sampling stations were determined as non-polluted based on the comparison with the sediment quality guideline index. More than a quarter of the sampling stations were found as moderately or heavily polluted for Cr, Ni, and As. The mean TE concentrations found in the sediment sample in the present study were similar to the concentrations reported from the different parts of the eastern Mediterranean Sea. TE concentrations in P. oceanica were generally lower compared to the concentrations in the sediment except for Cd, Zn, and Cu. While a positive correlation was found for Ni concentrations between sediment and P. oceanica samples, negative correlation was detected for V, Cr, Zn, Cu, As, and Cd concentrations between sediment and P. oceanica. The highest bioaccumulation factor in P. oceanica was calculated for Cd. The study area of the present study, especially the western sites (provinces of Antalya and Mugla), hosts millions of tourists annually and under the influence of intensive human activities in summer. Thus, coastal waters are heavily exposed to TEs and significantly positive correlations were detected between the anthropogenic TE pollutants (As, Cd, Cu, Zn, Pb, and V) and natural sourced TE (Ni and Cr). Based on our data, the Mediterranean Sea coast of Turkey does not present significant levels of Zd, Cd, Cu, and Pb pollution, whereas 65% of the stations were heavily polluted with As. Since As categorized as carcinogenic to humans, seafood sources should be monitored in terms of As concentrations. The current data might be useful in further TE-monitoring studies and TE discharge management strategies.

Population genetic structure of turbot (Scophthalmus maximus L., 1758) in the Black Sea.

Turbot, Scophthalmus maximus, is a commercially important demersal flatfish species distributed throughout the Black Sea. Several studies performed locally with a limited number of specimens using both mitochondrial DNA (mtDNA) and microsatellite markers evidenced notable genetic variation among populations. However, comprehensive population genetic studies are required to help management of the species in the Black Sea. In the present study eight microsatellite loci were used to resolve the population structure of 414 turbot samples collected from 12 sites across the Black Sea. Moreover, two mtDNA genes, COI and Cyt-b, were used for taxonomic identification. Microsatellite markers of Smax-04 and B12-I GT14 were excluded from analysis due to scoring issues. Data analysis was performed with the remaining six loci. Loci were highly polymorphic (average of 17.8 alleles per locus), indicating high genetic variability. Locus 3/20CA17, with high null allele frequency (>30%), significantly deviated from HW equilibrium. Pairwise comparison of the FST index showed significant differences between most of the surveyed sampling sites (P < 0.01). Cluster analysis evidenced the presence of three genetic groups among sampling sites. Significant genetic differentiation between Northern (Sea of Azov and Crimea) and Southern (Turkish Black Sea Coast) Black Sea sampling sites were detected. The Mantel test supported an isolation by distance model of population structure. These findings are vital for long-term sustainable management of the species and development of conservation programs. Moreover, generated mtDNA sequences would be useful for the establishment of a database for S. maximus.

Determination of virulence associated immunogenic proteins in some of Lactococcus garvieae strains.

Lactococcosis disease incident caused by Lactococcus garvieae has been increased with increasing aquaculture productions and outbreaks of the disease have become a threat on farmed species. To prevent lactococcosis, inactivated vaccine has been used, however, it only provides protection when given by injection. Other than inactivated vaccine, various vaccines such as subunit vaccines can be developed. In the present study, total protein profile of 43 strains of L. garvieae isolated from fish, milk and cheese by SDS-PAGE and virulence associated immunogenic proteins of L. garvieae strains using western blot with hyper-immune rabbit sera were determined. After analyzing whole-cell lysate protein of L. garvieae strains with SDS-PAGE, protein bands were ranged between 8.00 and 140.00 kDA. Among strains, variable protein bands were ranged between 17.00 and 48.00 kDa with some variability in the staining intensity of the protein bands and formed in 6 clusters. The immunogenic protein bands were ranged between 25.00 - 75.00 kDa. Only a variable and highly immunogenic protein band was observed between 40.00 and 45.00 kDa. Most of the strain including Lgper had 44.00 kDa immunogenic protein while nonvirulent ATCC strain had 42.50 kDA immunogenic protein. Predominant immuno-reactive proteins encoded by genes can be used as a subunit vaccine.