Cuspidothrix Is the First Genetically Proved Anatoxin A Producer in Bulgarian Lakes and Reservoirs.

The paper presents the first proof of planktonic cyanoprokaryote genus Cuspidothrix as an anatoxin A (ATX) producer in Bulgarian wetlands. The results from polymerase chain reaction (PCR) obtained from two summer sampling campaigns in 26 selected lakes and reservoirs demonstrated presence of the anaC gene, responsible for ATX production in 21 strains of the genus. They were found in three waterbodies sampled in 2018 (coastal lake Vaya, coastal reservoir Poroy, inland reservoir Sinyata Reka) and in four waterbodies sampled in 2019 (inland reservoirs Duvanli, Koprinka, Plachidol 2, Sinyata Reka). The detected genetic diversity generally corresponds to the observations conducted by conventional light microscopy, by which we distinguished three species of Cuspidothrix (Cuspidothrix issatschenkoi, Cuspidothrix elenkinii and Cuspidothrix tropicalis, the latter considered alien in the country). Eleven strains showed high similarity to two sequences of C. issatschenkoi available from the National Centre for Biotechnology Information (NCBI). Ten other strains assembled in a group, which-in lack of available from NCBI genetic sequences-were presumed related to C. tropicalis and C. elenkinii after comparison with the results from light microscopy. Cuspidothrix strains found in Bulgarian waterbodies showed high genetic similarity to those isolated and sequenced from Asia (Japan, China) and Northern Europe (Norway, Finland).

First Report on Microcystis as a Potential Microviridin Producer in Bulgarian Waterbodies.

Bulgaria, situated on the Balkan Peninsula, is rich in small and shallow, natural and man-made non-lotic waterbodies, which are threatened by blooms of Cyanoprokaryota/Cyanobacteria. Although cyanotoxins in Bulgarian surface waters are receiving increased attention, there is no information on microviridins and their producers. This paper presents results from a phytoplankton study, conducted in August 2019 in three lakes (Durankulak, Vaya, Uzungeren) and five reservoirs (Duvanli, Mandra, Poroy, Sinyata Reka, Zhrebchevo) in which a molecular-genetic analysis (PCR based on the precursor mdnA gene and subsequent translation to amino acid alignments), combined with conventional light microscopy and an HPLC analysis of marker pigments, were applied for the identification of potential microviridin producers. The results provide evidence that ten strains of the genus Microcystis, and of its most widespread species M. aeruginosa in particular, are potentially toxigenic in respect to microviridins. The mdnA sequences were obtained from all studied waterbodies and their translation to amino-acid alignments revealed the presence of five microviridin variants (types B/C, Izancya, CBJ55500.1 (Microcystis 199), and MC19, as well as a variant, which was very close to type A). This study adds to the general understanding of the microviridin occurrence, producers, and sequence diversity.

Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca).

Hemocyanins are copper-binding proteins that play a crucial role in the physiological processes in crustaceans. In this study, the cDNA encoding hemocyanin subunit 5 from the Black sea crab Eriphia verrucosa (EvHc5) was cloned using EST analysis, RT-PCR and rapid amplification of the cDNA ends (RACE) approach. The full-length cDNA of EvHc5 was 2254 bp, consisting of a 5' and 3' untranslated regions and an open reading frame of 2022 bp, encoding a protein consisting of 674 amino acid residues. The protein has an N-terminal signal peptide of 14 amino acids as is expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph. The 3D model showed the presence of three functional domains and six conserved histidine residues that participate in the formation of the copper active site in Domain 2. The EvHc5 is O-glycosylated and the glycan is exposed on the surface of the subunit similar to Panulirus interruptus. The phylogenetic analysis has shown its close grouping with γ-type of hemocyanins of other crustacean species belonging to order Decapoda, infraorder Brachyura.

Morphological and Molecular Identification of Microcystin-Producing Cyanobacteria in Nine Shallow Bulgarian Water Bodies.

The paper presents results from the first application of polyphasic approach in studies of field samples from Bulgaria. This approach, which combined the conventional light microscopy (LM) and molecular-genetic methods (based on PCR amplified fragments of microcystin synthetase gene mcyE), revealed that almost all microcystin-producers in the studied eutrophic waterbodies belong to the genus Microcystis. During the molecular identification of toxin-producing strains by use of HEPF × HEPR pair of primers, we obtained 57 sequences, 56 of which formed 28 strains of Microcystis, spread in six clusters of the phylogenetic tree. By LM, seven Microcystis morphospecies were identified (M. aeruginosa, M. botrys, M. flos-aquae, M. natans, M. novacekii, M. smithii, and M. wesenbergii). They showed significant morphological variability and contributed from <1% to 98% to the total biomass. All data support the earlier opinions that taxonomic revision of Microcystis is needed, proved the presence of toxigenic strains in M. aeruginosa and M. wesenbergii, and suppose their existence in M. natans. Our results demonstrated also that genetic sequencing, and the use of HEPF × HEPR pair in particular, can efficiently serve in water quality monitoring for identifying the potential risk from microcystins, even in cases of low amounts of Microcystis in the water.

Development- and cold-regulated accumulation of cold shock domain proteins in wheat.

Cold shock domain (CSD) proteins, or Y-box proteins, are nucleic acid-binding proteins that are widely distributed from bacteria to higher plants and animals. Bacterial CSD proteins play an essential role in cold adaptation by destabilizing RNA secondary structures. WHEAT COLD SHOCK DOMAIN PROTEIN 1 (WCSP1) shares biochemical functions with bacterial CSD proteins and is possibly involved in cold adaptation. In this study, the temporal and spatial distribution of the wheat cold shock domain protein family (WCSPs) was serologically characterized with regard to plant development and cold adaptation. Four WCSP genes were identified through database analysis and were classified into three classes based on their molecular masses and protein domain structures. Class I (20 kD) and class II (23 kD) WCSPs demonstrated a clear pattern of accumulation in root and shoot meristematic tissues during vegetative growth. In response to cold, marked increases in WCSP levels were observed but the pattern of accumulation differed by tissue. Accumulation of WCSPs in crown tissue during cold acclimation was observed in the winter cultivar 'Chihokukomugi' but not in the spring cultivar 'Haruyutaka', suggesting a possible function for WCSPs in cold acclimation. During flower and seed development, protein levels of class I and class II WCSPs remained high. The class III WCSP (27 kD) was detected only during seed development. The highest level of class III WCSP accumulation was observed at the milky seed stage. Together, the results of this study provide a view of CSD protein accumulation throughout the life cycle of wheat and suggest that WCSPs function differentially in plant development and cold adaptation.

Assessment of genetic variation in Bulgarian tomato (Solanum lycopersicum L.) genotypes, using fluorescent SSR genotyping platform.

Genetic variability in modern crops is limited due to domestication and selection processes. Genetic variation in eight Bulgarian tomato varieties and breeding lines (variety Plovdivska karotina, variety IZK Alya, L21ß, L53ß, L1140, L1116, L975, L984) differing in their morphological and biochemical composition was assessed using a highly efficient and low-cost fluorescent simple sequence repeat (SSR) genotyping platform. Genotyping was conducted with 165 publicly available microsatellite markers developed from different research groups under a number of projects in tomato (SOL Genomics SSRs, Kazusa TGS and TES, SLM, TMS and LEMDDNa) among which only five (3.03%) failed to amplify the expected PCR fragments. Of the remaining markers, 81 (50.62%) were polymorphic in the whole collection of eight genotypes. Among the marker groups used, SLM markers were most polymorphic, followed by TMS and SOL Genomics SSR markers. The total number of amplified alleles was 299, with a mean of 1.869; and the average polymorphic information content (PIC) was 0.196. The genetic diversity within the collection was relatively low (0.2222). Nei's genetic distance varied from 0.0953 to 0.3992. Cluster analysis using the un-weighted pair group method with arithmetic mean (UPGMA) method indicated that the studied tomato genotypes are grouped in four main clusters, which is to some extent consistent with the morpho- and hemo-types of the studied tomatoes. Variety IZK Alya (cherry type) and two of the breeding lines (L1140, L1116) formed three separate and more distant clusters. The fourth cluster includes the other five genotypes. The observed grouping of these genotypes in two sub-clusters reflects their similar morphological and biochemical composition. The genetic distance information from this study might be useful for further implementation of breeding strategies and crosses among these inbred lines.