Widespread infection, diversification and old host associations of Nosema Microsporidia in European freshwater gammarids (Amphipoda).

The microsporidian genus Nosema is primarily known to infect insects of economic importance stimulating high research interest, while other hosts remain understudied. Nosema granulosis is one of the formally described Nosema species infecting amphipod crustaceans, being known to infect only two host species. Our first aim was to characterize Nosema spp. infections in different amphipod species from various European localities using the small subunit ribosomal DNA (SSU) marker. Second, we aimed to assess the phylogenetic diversity, host specificity and to explore the evolutionary history that may explain the diversity of gammarid-infecting Nosema lineages by performing a phylogenetic reconstruction based on RNA polymerase II subunit B1 (RPB1) gene sequences. For the host species Gammarus balcanicus, we also analyzed whether parasites were in excess in females to test for sex ratio distortion in relation with Nosema infection. We identified Nosema spp. in 316 individuals from nine amphipod species being widespread in Europe. The RPB1-based phylogenetic reconstruction using newly reported sequences and available data from other invertebrates identified 39 haplogroups being associated with amphipods. These haplogroups clustered into five clades (A-E) that did not form a single amphipod-infecting monophyletic group. Closely related sister clades C and D correspond to Nosema granulosis. Clades A, B and E might represent unknown Nosema species infecting amphipods. Host specificity seemed to be variable with some clades being restricted to single hosts, and some that could be found in several host species. We show that Nosema parasite richness in gammarid hosts is much higher than expected, illustrating the advantage of the use of RPB1 marker over SSU. Finally, we found no hint of sex ratio distortion in Nosema clade A infecting G. balcanicus. This study shows that Nosema spp. are abundant, widespread and diverse in European gammarids. Thus, Nosema is as diverse in aquatic as in terrestrial hosts.

Microsporidian diversity in the aquatic isopod Asellus aquaticus.

We conducted a molecular survey on microsporidian diversity in different lineages (operational taxonomic units = OTUs) of Asellus aquaticus from 30 sites throughout Europe. Host body length was determined, and DNA was extracted from host tissue excluding the intestine and amplified by microsporidian-specific primers. In total, 247 A. aquaticus specimens were analysed from which 26.7% were PCR-positive for microsporidians, with significantly more infections in larger individuals. Prevalence ranged between 10 and 90%. At 9 sites, no microsporidians were detected. A significant relationship was found between the frequency of infected individuals and habitat type, as well as host OTU. The lowest proportion of infected individuals was detected in spring-habitats (8.7%, n = 46) and the highest in ponds (37.7%, n = 53). Proportion of infected individuals among host OTUs A, D and J was 31.7, 21.7 and 32.1%, respectively. No infections were detected in OTU F. Our results are, however, accompanied by a partially low sample size, as only a minimum of 5 individuals was available at a few locations. Overall, 17 different microsporidian molecular taxonomic units (MICMOTUs) were distinguished with 5 abundant isolates (found in 4­17 host individuals) while the remaining 12 MICMOTUs were "rare" and found only in 1­3 host individuals. No obvious spatio-genetic pattern could be observed. The MICMOTUs predominantly belonged to Nosematida and Enterocytozoonida. The present study shows that microsporidians in A. aquaticus are abundant and diverse but do not show obvious patterns related to host genetic lineages or geography.

First records raise questions: DNA barcoding of Odonata in the middle of the Mediterranean.

We present the results of the first-ever DNA barcoding study of odonates from the Maltese Islands. In total, 10 morphologically identified species were collected during a two-week long expedition in 2018. Eighty cytochrome c oxidase subunit I (COI) barcodes were obtained from the collected specimens. Intra- and interspecific distances ranged from 0.00% to 2.24% and 0.48% to 17.62%, respectively. Successful species identification based on ascribing a single morphological species to a single Barcode Index Number (BIN) was achieved for eight species (80%). In the case of two species, Ischnura genei and Anax parthenope, BINs were shared with other closely related species. The taxonomic status of I. genei is questionable and the phylogenetic relationship between A. imperator/parthenope is not clear. Further studies involving a series of adult specimens collected in a wide spatial range and nuclear markers are necessary to resolve these cases. Therefore, this dataset serves as an initial DNA barcode reference library for Maltese odonates, within a larger project: Aquatic Macroinvertebrates DNA Barcode Library of Malta.

Mitochondrial Genomes, Phylogenetic Associations, and SNP Recovery for the Key Invasive Ponto-Caspian Amphipods in Europe.

The Ponto-Caspian region is the main donor of invasive amphipods to freshwater ecosystems, with at least 13 species successfully established in European inland waters. Dikerogammarus spp. and Pontogammarus robustoides are among the most successful, due to their strong invasive impact on local biota. However, genomic knowledge about these invaders is scarce, while phylogeography and population genetics have been based on short fragments of mitochondrial markers or nuclear microsatellites. In this study, we provide: (i) a reconstruction of six mitogenomes for four invasive gammarids (D. villosus, D. haemobaphes, D. bispinosus, and P. robustoides); (ii) a comparison between the structure of the newly obtained mitogenomes and those from the literature; (iii) SNP calling rates for individual D. villosus and D. haemobaphes from different invasion sites across Europe; and (iv) the first time-calibrated full mitogenome phylogeny reconstruction of several Ponto-Caspian taxa. We found that, in comparison to other gammarids, the mitogenomes of Ponto-Caspian species show a translocation between the tRNA-E and tRNA-R positions. Phylogenetic reconstruction using the mitogenomes identified that Ponto-Caspian gammarids form a well-supported group that originated in the Miocene. Our study supports paraphyly in the family Gammaridae. These provided mitogenomes will serve as vital genetic resources for the development of new markers for PCR-based identification methods and demographic studies.

The Use of Yeast Mixed Cultures for Deacidification and Improvement of the Composition of Cold Climate Grape Wines.

Interest in the use of non-Saccharomyces yeast in mixed cultures is increasing due to the perceived improvement in the quality and complexity of the resulting wines. The aim of the study was to determine the ability of monocultures and mixed yeast cultures for deacidification and improvement of the composition of cold climate grape wines. Fermentation of grape musts with increased total acidity was carried out with the use of monocultures of Saccharomyces cerevisiae MH020215 (Sc), Zygosaccharomyces bailii 749 (Zb) and Metschnikowia pulcherrima MG970690 (Mp), and their mixed cultures, inoculated simultaneously and sequentially. Oenological parameters, organic acids and volatile compounds profiles of obtained wines were characterized. The fermentation kinetics and analytical profiles of the obtained wines showed that the use of mixed yeast cultures contributed to the reduction of volatile acidity and acetic acid content in the wines, as well as obtaining a favorable aromatic profile of the wines. The dominant higher alcohols in all wines were 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Significantly higher amounts of the first two compounds were found in wines obtained with M. pulcherrima MG070690, both in monoculture and in mixed cultures. The monocultures of M. pulcherrima MG070690 (Mp) compared with Z. bailli 749 (Zb) synthesized higher levels of esters in wines, including ethyl acetate, ethyl propionate, isobutyl acetate, ethyl pyroracemate and isoamyl acetate.

Dictyocoela microsporidia diversity and co-diversification with their host, a gammarid species complex (Crustacea, Amphipoda) with an old history of divergence and high endemic diversity.

BACKGROUND: Although the processes of co-evolution between parasites and their hosts are well known, evidence of co-speciation remains scarce. Microsporidian intracellular parasites, due to intimate relationships with their hosts and mixed mode of transmission (horizontal but also vertical, from mother to offspring), may represent an interesting biological model for investigating co-speciation. Amphipod crustaceans, especially gammarids, are regular hosts of microsporidian parasites, in particular the Dictyocoela spp., which have so far been found limited to these amphipods and are known to use a vertical mode of transmission. The amphipod genus Gammarus has a diversification history spanning the last 50-60 Mya and an extensive cryptic diversity in most of the nominal species. Here, we investigated the degree of co-diversification between Dictyocoela and Gammarus balcanicus, an amphipod with high degrees of ancient cryptic diversification and lineage endemism, by examining the genetic diversity of these parasites over the entire geographic range of the host. We hypothesised that the strong host diversification and vertical transmission of Dictyocoela would promote co-diversification. RESULTS: Using the parasite SSU rDNA as a molecular marker, analyzing 2225 host specimens from 88 sites covering whole host range, we found 31 haplogroups of Dictyocoela, 30 of which were novel, belonging to four Dictyocoela species already known to infect other Gammarus spp. The relationships between Dictyocoela and gammarids is therefore ancient, with the speciation in parasites preceding those of the hosts. Each novel haplogroup was nevertheless specific to G. balcanicus, leaving the possibility for subsequent co-diversification process during host diversification. A Procrustean Approach to Co-phylogeny (PACo) analysis revealed that diversification of Dictyocoela was not random with respect to that of the host. We found high degrees of congruence between the diversification of G. balcanicus and that of Dictyocoela roeselum and D. muelleri. However, we also found some incongruences between host and Dictyocoela phylogenies, e.g. in D. duebenum, probably due to host shifts between different G. balcanicus cryptic lineages. CONCLUSION: The evolutionary history of Dictyocoela and Gammarus balcanicus represents an example of an overall host-parasite co-diversification, including cases of host shifts.

Environmental factors affecting water mite assemblages along eucrenon-hypocrenon gradients in Mediterranean karstic springs.

Springs are often recognized as biodiversity hotspots on the regional scale but at the same time they are among the most endangered freshwater habitats. Water mites are among the aquatic animal groups with highest share of crenobiotic (= spring-dwelling) species and, therefore, are possibly the best indicators of the ecological status of spring habitats. We studied water mites and environmental factors correlated with their distribution pattern along a eucrenon-hypocrenon gradient. The sampling was conducted in 14 karstic springs located in the Mediterranean part of Montenegro. We collected 17 water mite species of which four species were crenobiotic. We did not find significant differences between the water mite assemblages from the source and springbrook. Similarly, there were no significant differences in number of species and abundance between the studied spring sections, neither for crenobiotic taxa nor for non-crenobiotic taxa. We found that the number of non-crenobiotic taxa was predicted mainly by water depth, whereas the abundance of crenobionts was most strongly associated with temperature. No significant predictors for the number of crenobiotic species in spring habitats were found. Our results revealed also that distance from the nearby water body was the main driver of the crenobiotic species abundance in eucrenon suggesting the large effects of the local flooding events on crenobiotic species. Water mites may help in assessing the response of crenobiotic assemblages in those spring habitats which likely to be flooded in future as the results of ongoing climatic changes.

Evidence for interactions between homocysteine and genistein: insights into stroke risk and potential treatment.

Elevated plasma homocysteine (2-amino-4-sulfanylbutanoic acid) level is a risk factor for stroke. Moreover, it has been suggested that high levels of homocysteine in the acute phase of an ischemic stroke can predict mortality, especially in stroke patients with the large-vessel atherosclerosis subtype. In clinical studies, supplementation with genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) decreased plasma homocysteine levels considerably. Therefore, genistein could be considered as a potential drug for prevention and/or treatment of stroke. However, the mechanism of the effect of genistein on homocysteine level remains to be elucidated. In this report, direct functional interactions between homocysteine and genistein are demonstrated in in vitro experimental systems for determination of methylenetetrahydrofolate reductase (MetF) and glutathione peroxidase (GPx) activities, reconstructed with purified compounds, and in a simple in vivo system, based on measurement of growth rate of Vibrio harveyi and Bacillus subtilis cultures. Results of molecular modelling indicated that homocysteine can directly interact with genistein. Therefore, genistein-mediated decrease in plasma levels of homocysteine, and alleviation of biochemical and physiological effects of one of these compounds by another, might be ascribed to formation of homocysteine-genistein complexes in which biological activities of these molecules are abolished or alleviated.

The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression.

The formation of continental Europe in the Neogene was due to the regression of the Tethys Ocean and of the Paratethys Sea. The dynamic geology of the area and repetitious transitions between marine and freshwater conditions presented opportunities for the colonization of newly emerging hydrological networks and diversification of aquatic biota. Implementing mitochondrial and nuclear markers in conjunction with a large-scale sampling strategy, we investigated the impact of this spatiotemporal framework on the evolutionary history of a freshwater crustacean morphospecies. The Gammarus balcanicus species complex is widely distributed in the area previously occupied by the Paratethys Sea. Our results revealed its high diversification and polyphyly in relation to a number of other morphospecies. The distribution of the studied amphipod is generally characterized by very high local endemism and divergence. The Bayesian time-calibrated reconstruction of phylogeny and geographical distribution of ancestral nodes indicates that this species complex started to diversify in the Early Miocene in the central Balkans, partially in the shallow epicontinental sea. It is possible that there were several episodes of inland water colonization by local brackish water lineages. Subsequent diversification within clades and spread to new areas could have been induced by Alpine orogeny in the Miocene/Pliocene and, finally, by Pleistocene glaciations. The present distribution of clades, in many cases, still reflects Miocene palaeogeography of the area. Our results point out that investigations of the historical aspect of cryptic diversity in other taxa may help in a general understanding of the origins of freshwater invertebrate fauna of Europe.

The model homologue of the partially defective human 5,10-methylenetetrahydrofolate reductase, considered as a risk factor for stroke due to increased homocysteine level, can be protected and reactivated by heat shock proteins.

The A222 V substitution in the human MTHFR gene product (5,10-methylenetetrahydrofolate reductase) is responsible for a decreased activity of this enzyme. This may cause an increased homocysteine level, considered as a risk factor for arteriosclerosis and stroke. The bacterial homologue of the human enzyme, MetF, has been found to be a useful model in genetic and biochemical studies. The similarity of Escherichia coli MetF and human MTHFR proteins is so high that particular mutations in the corresponding human gene can be reflected by the bacterial mutants. For example, the A222 V substitution in MTHFR (caused by the C667T substitution in the MTHFR gene) can be ascribed to the A117 V substitution in MetF. Here, it is reported that a temperature-sensitive MetF117 (A117 V) protein can be partially protected from a thermal inactivation by the heat shock proteins from the Hsp70/100 systems. Moreover, activity of the thermally denatured enzyme can be partially restored by the same heat shock proteins. High temperature protein G (HtpG) had no effect on MetF117 activity in both experimental systems. The presented results indicate that functions of heat shock proteins may be required for maintenance of the MetF117 function. This may have implications for the mechanisms of arteriosclerosis and stroke, especially in the light of previous findings that the A222 V MTHFR polymorphism may be a risk factor for stroke, as well as recently published results which demonstrated the increased levels of antibodies against heat shock proteins in stroke patients.