Epidemiology of Noble Pen Shell (Pinna nobilis L. 1758) Mass Mortality Events in Adriatic Sea Is Characterised with Rapid Spreading and Acute Disease Progression

From May to October 2019, multiple mass mortality events (MMEs) of Pinna nobilis were observed along Croatian coastline starting from the south-east and rapidly progressing in north-western direction. Time dynamics of the MMEs closely followed general speed and direction patterns of surface sea-currents, advancing approximately 350 km in less than 3 months. Surveillance, clinical evaluation, and sample collection were performed on multiple sites with various degrees of mortality rates. Moribund P. nobilis individuals were collected and subjected to pathological, molecular, and microscopical investigation. Affected animals were positive for Mycobacterium in 70% of the individuals, and Haplosporidium pinnae was present in 58% of the cases. Observed pathological lesions were most severe where concurrent presence of both pathogens was confirmed (in 45.8% of moribund individuals). Moderate to strong lesions were observed in animals positive for Mycobacterium only (25% of cases), and lesions were absent or minor to moderate when only H. pinnae was confirmed (16% of cases). Considering the rapid and severe spread of the MMEs, the areas less exposed to major sea currents appeared to be at lower risk of pathogen transmission. Surveillance activities along the Croatian coastline identified several P. nobilis populations in such “lower risk” areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF THE ESSENTIAL OIL AND SOLVENT EXTRACTS OF MENTHA PULEGIUM L.

We report the total phenolic (TPC; expressed as gallic acid equivalents, GAE, per milligram of dry extract weight) and the total flavonoid contents (TFC; expressed as quercetin equivalents, QE, per milligram of dry extract weight) and antimicrobial and antioxidant activities of the essential oil and hexane, diethyl ether, ethyl acetate and methanol extracts of Mentha pulegium L. (Lamiaceae) collected in Serbia. The total phenolic content was in the range of 129.43-388.29 μg GAE/mg, while TFC ranged from 57.81 to 160.94 QE/mg; the highest TPC and TFC were found in the methanol extract. The antimicrobial activity (against five bacteria and two fungi species) of the essential oil and solvent extracts was assessed using disc-diffusion method. However, the studied samples demonstrated a poor antimicrobial potential. The antioxidant activity was screened using five different tests: 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid radical cation decolorization assay (ABTS), total reducing power (TRP), ferric reducing antioxidant power (FRAP) and cupric reducing antioxidant capacity assay (CUPRAC); the methanol extract showed the strongest antioxidant potential. The results of the different antioxidant assays were correlated mutually and with the total flavonoid and total phenolic contents (regression analysis and agglomerative hierarchical clustering). ANTIOKSIDANTNA I ANTIMIKROBNA AKTIVNOST ETARSKOG ULJA I EKSTRAKATA BILJNE VRSTE MENTHA PULEGIUM L.U ovom radu je određen sadržaj ukupnih fenola (TPC; izražen u ekvivalentima galne kiseline, GAE, po miligramu suvog biljnog ekstrakta) i ukupnih flavonoida (TPC; izražen u ekvivalentima kvarcetina, QE, po miligramu suvog biljnog ekstrakta), kao i antimikrobna i antioksidantna aktivnost etarskog ulja i heksanskog, dietil-etarskog, etil-acetatnog i metanolnog ekstrakta biljne vrste Mentha pulegium L. (Lamiaceae; populacija iz Srbije). Sadržaj ukupnih fenola analiziranih uzoraka se kretao u opsegu od 129,43 do 388,29 μg GAE/mg, dok je TFC bio u interval od 57,81 do 160,94 QE/mg; najviše vrednosti za TPC i TFC nađene su za metanolni ekstrakt. Antimikrobna aktivnost (prema pet bakterijskih i dva soja gljivica) etarskog ulja i ekstrakata je određena disk-difuzionom metodom. Proučavani uzorci su imali slabu antimikrobnu aktivnost. Antioksidantna aktivnost je ispitivana pomoću DPPH (2,2-difenil-1-pikrilhidrazil radikal), ABTS (2,2'-azino-bis(3-etilbenzotijazolin-6-sulfonska kiselina radikal katjon), TRP (ukupna redukciona sposobnost), FRAP (sposobnost redukcije feri-jona) i CUPRAC (sposobnost redukcije Cu(II)-jona) metodama; najviši antioksidantni potencijal je imao metanolni ekstrakt. Rezultati različitih metoda za određivanje antioksidantnog potencijala su korelisani međusobno, kao i sa sadržajem ukupnih fenola i flavonoida (regresiona analiza i aglomerativna hijerarhijska klaster analiza). HIGHLIGHTSThe essential oil and hexane, diethyl ether, ethyl acetate and methanol extracts of Mentha pulegium L. collected in Serbia were studied.Their total phenolic content was in the range of 129.43-388.29 μg GAE/mg, while total flavonoid content ranged from 57.81 to 160.94 QE/mg.Solvent extracts (SE) and essential oil of the studied M. pulegium population were poor antimicrobials.Antioxidant activities of SE were studied using five different methods.Differences between the previous and the present results suggest M. pulegium metabolic profile might be influenced by both genetic and environmental factors

Clastogenic Effect of Atranorin, Evernic acid, and Usnic Acid on Human Lymphocytes

Three lichen secondary metabolites atranorin (1), evernic acid (2), and usnic acid (3), were evaluated for their in vitro clastogenic and antiproliferative effects on human lymphocytes using the cytochalasin-B blocked micronucleus (CBMN) assay at concentrations of 2 mu g/mL, 4 mu g/mL and 6 mu g/mL of final culture solution. The frequency of micronucleus (MN) was scored in binucleated cells, and cytokinesis-block proliferation index (CBPI) was calculated. Among the tested compounds, 3 exhibited the most prominent effect decreasing the frequency of MN in the range of 42.5% - 48.9%, that is about double of the positive control amifostin WR-2721 that reduces MN frequency for 22.0%. The effect of evernic acid was approximately equal to action of amifostin (23.2% -32.9%). Atranorin at concentrations of 2 mu g/mL and 4 mu g/mL decreasing the frequency of MN only for 11.1% and 1.8%, while in concentration of 6 mu g/mL increases the frequency of MN for 9.6 %. The comparable CBPI values of the investigated compounds and control suggested that they did not show a statistically significant inhibitory effect on lymphocyte cell proliferation at applied concentrations

Clastogenic Effect of Atranorin, Evernic acid, and Usnic Acid on Human Lymphocytes

Three lichen secondary metabolites atranorin (1), evernic acid (2), and usnic acid (3), were evaluated for their in vitro clastogenic and antiproliferative effects on human lymphocytes using the cytochalasin-B blocked micronucleus (CBMN) assay at concentrations of 2 mu g/mL, 4 mu g/mL and 6 mu g/mL of final culture solution. The frequency of micronucleus (MN) was scored in binucleated cells, and cytokinesis-block proliferation index (CBPI) was calculated. Among the tested compounds, 3 exhibited the most prominent effect decreasing the frequency of MN in the range of 42.5% - 48.9%, that is about double of the positive control amifostin WR-2721 that reduces MN frequency for 22.0%. The effect of evernic acid was approximately equal to action of amifostin (23.2% -32.9%). Atranorin at concentrations of 2 mu g/mL and 4 mu g/mL decreasing the frequency of MN only for 11.1% and 1.8%, while in concentration of 6 mu g/mL increases the frequency of MN for 9.6 %. The comparable CBPI values of the investigated compounds and control suggested that they did not show a statistically significant inhibitory effect on lymphocyte cell proliferation at applied concentrations