Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48-72 hours

Fish photobacteriosis—The importance of rapid and accurate identification of Photobacterium damselae subsp. piscicida

MALDI-TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on-target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24-hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48-hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI-TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate-dependent

Biological, Biochemical and Elemental Traits of Clavelina oblonga, an Invasive Tunicate in the Adriatic Sea

Clavelina oblonga is an invasive tropical tunicate recently introduced into the Adriatic Sea as a consequence of globalization and climate change. Mussel aquaculture sites provide an ideal environment for this colonial ascidian, where it has recently become the dominant fouling species. This study represents the first investigation of its biological and physical characteristics, as well as its proximal, fatty acid, macroelement, trace element, and toxic metal composition. The entire-tissue chemical composition of C. oblonga resulted in 95.44% moisture. Its composite structure revealed several strong peaks, attributed to O-H, C-H, C-N, and C=O stretching, along with cellulose components overlapping with proteins and carbohydrates. The major fatty acids were palmitic, stearic, and docosahexaenoic acid, followed by docosanoic, elaidic, linoleic, and myristic acid. The saturated fatty acids, polyunsaturated fatty acids, and monounsaturated fatty acids comprised 51.37, 26.96, and 15.41% of the total fatty acids, respectively. Among the analysed trace and macroelements, aluminium and sodium were predominant. C. oblonga exhibited different concentrations of toxic metals, such as arsenic and lead, compared to fouled mussels in the Istria region. It appears that the tunicate has adapted to the environmental conditions of the Adriatic, reaching its maximum spread and biomass in mid-autumn. There is a strong possibility that C. oblonga could colonize and establish itself permanently in the Adriatic. This would have a strong negative impact on shellfish farming, the structure of the ecosystem, plankton biomass, and the distribution of other marine species. However, it also represents a biomass resource with high potential of utilization in different industries

Baseline levels of environmental genotoxicity and potential confounding factors using common sole ( Solea solea ) as sentinel organism

Micronuclei (MN) and nuclear buds (NB) frequency was assessed monthly over a one-year period in erythrocytes of common soles (Solea solea) from the Basque continental shelf, a minor disturbed area, in order to determine baseline levels and potential biotic and abiotic confounding factors within biomonitoring purposes. Both genotoxic parameters presented seasonal variations, although only NB frequency was positively correlated with environmental variables (i.e. water temperature and salinity). On the contrary, MN and NB were not affected by age and gender. Therefore, samples of combined age and gender can be used for biomonitoring genotoxic effects, although sampling period together with water temperature and salinity should be thoroughly considered for NB. Overall, these findings are the first attempt to establish baseline MN (0.10-0.78 parts per thousand) and NB (0.13-0.82 parts per thousand) frequencies in common sole from Basque marine waters, which are useful for upcoming data comparisons and integration within genotoxicity evaluation procedures of similar marine environments under biomonitoring scopes

Evaluation of micronucleus and erythrocytic nuclear abnormalities in Balkan whip snake Hierophis gemonensis

The aim of the study was to evaluate the frequency of micronuclei (MN), nuclear abnormalities (NA) including vacuolated nuclei (VN) and cytoplasmic vacuoles (CV) in erythrocytes of Balkan whip snake Hierophis gemonensis and establish the level of spontaneous appearance during the annual cycle. Average frequency of NA was 10.89 ± 4.72 % while the MN (0.03 ± 0.03 %) and VN(0.04 ± 0.08 %) were seldom detected. NA significantly positively correlated with MN (r = 0.319 ; P < 0.05) and VN (r = 0.363 ; P < 0.05). Appearance of CV did not correlate with other measured parameters and average frequency was 11.06 ± 8.33 %. Significant seasonal variation was found in NA appearance with the lowest value in spring and the highest in winter. VN increase was observed in autumn. MN and CV levels varied between seasons but not significantly. Considering the biological cycle, frequency of NA, VN, MN and CV recorded in pre-hibernation/hibernation increased compared to the active phase, but only NA elevation was significant. Although the obtained results showed differences according to sex, statistical analysis of measured parameters showed the same pattern of seasonal variation in both sexes