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.

The rising interest in harmful cyanoprokaryote blooms promotes an increase of phycological and ecological research on potentially toxic species and their hazardous substances. The present study aimed to identify the main microcystin (MC) producers and their contribution to the phytoplankton of shallow waterbodies in Bulgaria, applying different methods. The sampling was performed in August 2019 in nine lakes and reservoirs, two of which (reservoirs Kriva Reka and Izvornik 2) were studied for the first time. The high contribution of cyanoprokaryotes to the total species composition and phytoplankton abundance was proved by light microscopic (LM) observations and HPLC analysis of marker pigments. The LM identification of potential MC-producers was supported by PCR amplification of mcyE and mcyB genes. The MCs amounts, detected by HPLC-DAD, varied by sites with a range from undetectable concentrations to 0.46 µg L⁻¹ with only one recorded variant, namely MC-LR. It was found only in the reservoirs Mandra and Durankulak, while toxigenic MC-strains were obtained by PCR from five more waterbodies. Both LM and PCR demonstrated that the MC-producers were Microcystis aeruginosa and M. wesenbergii, despite their occurrence in low amounts (<0.5–5% of the total biomass) when filamentous cyanoprokaryotes dominated.

Legumes are one of the world's leading sources of nutrition, providing sustainable agriculture. They require minimal amounts of soil improvers (fertilizers) due to the ability to absorb nitrogen through symbiotic interactions with nitrogen-fixing soil microorganisms. The trend for human population growth requires an adequate growth in crop production. This directly depends on the seed size and number as well as on the leaf biomass. In turn, seed size has been the subject of selection programmers for all crops. In this study, we explored the relation between the function of the gene coding Zinc finger CCHC-type protein and flower morphology and seed size in the model legume species Medicago truncatula. M. truncatula lines with modified level of Zinc finger CCHC transcript and transcriptional reporters were developed and analysed by real-time polymerase chain reaction and expression of the GUS (β-glucuronidase) and green fluorescent protein (GFP) reporter genes. A tissue-specific GFP signal was detected in the anthers from overexpessing M. truncatula lines. The M. truncatula lines with knockdown expression showed direct relation between low transcription level of Mt-Zn-CCHC gene and strongly reduced seed size accompanied with short stems length and internodes.

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.

Recently, scientists are focused on the discovery of alternative sources of high-value products such as proteins, lipids and carbohydrates. Microalgal cells have great potential for accumulation of bioactive components important in food, feed, pharmaceutical and cosmetics industry, as sources of biofuel, for waste water remediation, etc. The main advantages of microalgae as producers of bioactive components are fast biomass growth; short reproduction cycle; resistance to harsh conditions and contaminations; smaller area for growing in comparison with agriculture crops, etc. However, the production of high-value components from microalgae biomass is still associated with high costs, so efforts are focused on cost reduction. This study evaluated the possibilities for production of low-cost high-value products from two microalgal strains, Chlorela vulgaris CAUP H1993 and Desmodesmus communis CAUP H522. Microalgal biomass was grown under simple low-cost media supplemented with urea as nitrogen source for extraction of crude protein. To completely utilize the harvested biomass, we applied two consecutive steps of the extraction procedure for protein and lipids accumulated in the exhausted biomass. The media supplemented with urea was promising, especially for growing C. vulgaris H1993 CAUP. The results showed that after two consecutive extraction steps, it is possible to obtain 8–9.0% crude protein and 21–24% lipids from the exhausted dry biomass of D. communis and C. vulgaris, respectively.

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.