Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the β-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.This article belongs to the Special Issue MycovirusesThis research was funded by a grant from the Lithuanian Research Council (No. SIT-7/2015

The bacterial microbiota of edible insects Acheta domesticus and Gryllus assimilis revealed by high content analysis

In the concept of novel food, insects reared under controlled conditions are considered mini livestock. Mass-reared edible insect production is an economically and ecologically beneficial alternative to conventional meat gain. Regarding food safety, insect origin ingredients must comply with food microbial requirements. House crickets (Acheta domesticus) and Jamaican field crickets (Gryllus assimilis) are preferred insect species that are used commercially as food. In this study, we examined cricket-associated bacterial communities using amplicon-based sequencing of the 16S ribosomal RNA gene region (V3–V4). The high taxonomic richness of the bacterial populations inhabiting both tested cricket species was revealed. According to the analysis of alpha and beta diversity, house crickets and Jamaican field crickets displayed significantly different bacterial communities. Investigation of bacterial amplicon sequence variants (ASVs) diversity revealed cricket species as well as surface and entire body-associated bacterial assemblages. The efficiency of crickets processing and microbial safety were evaluated based on viable bacterial counts and identified bacterial species. Among the microorganisms inhabiting both tested cricket species, the potentially pathogenic bacteria are documented. Some bacteria representing identified genera are inhabitants of the gastrointestinal tract of animals and humans, forming a normal intestinal microflora and performing beneficial probiotic functions. The novel information on the edible insect-associated microbiota will contribute to developing strategies for cricket processing to avoid bacteria-caused risks and reap the benefits

Molecular identification of protozoan Sarcocystis in different types of water bodies in Lithuania

Representatives of the genus Sarcocystis are unicellular parasites having a two-host life cycle and infecting mammals, birds, and reptiles. Until now, Sarcocystis spp. have been mainly investigated in definitive and intermediate hosts. Only a few studies have been conducted on the detection of Sarcocystis parasites in water samples. The aim of this research was to examine whether the prevalence of Sarcocystis spp. parasitizing farm animals varies in different types of water bodies. Water samples (n = 150) were collected from the entire territory of Lithuania, dividing water bodies into five groups (lakes, rivers, ponds/canals, swamps, and the inshore zone of the territorial Baltic Sea area). One-liter samples were filtered and subsequently analyzed using nested PCR. At least one of the analyzed Sarcocystis spp. (S. arieticanis, S. bertrami, S. bovifelis, S. capracanis, S. cruzi, S. hirsuta, S. miescheriana, and S. tenella) was determined in all examined samples from water bodies. No significant difference in Sarcocystis spp. prevalence between different types of water sources was detected. Our research proved that selecting appropriate primers is important for the accurate identification of parasites in samples collected from water bodies

Molecular identification of parasitic protozoa Sarcocystis in water samples

Sarcocystis parasites are among the most common parasitic protozoa in farm animals. So far, the diversity of these parasites has been mainly studied in animal carcasses by morphological or molecular methods. Research on parasitic protozoa in environmental samples is scarce due to the lack of an appropriate methodology and low concentrations of parasites. For these reasons, there is a paucity of validated methods for Sarcocystis identification from environmental samples. Therefore, the present study aims to investigate various molecular methods for Sarcocystis parasite identification in water samples. In the present study, the sample volume, sporocysts isolation, and various conventional PCR were evaluated, and species-specific primers for the identification of different Sarcocystis species have been developed. Of the methods studied, based on data the most appropriate method for the identification of analyzed Sarcocystis spp. in water bodies is nested PCR, using species-specific primers targeting the cox1 gene. Sarcocystis DNA was detected in 111 out of 114 (97.4%) samples. This paper represents the first identification of S. bovifelis, S. cruzi, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. bertrami, and S. miescheriana by PCR and sequencing in environmental water samples. Our pilot study is useful in developing techniques for the identification of Sarcocystis species from water samples

Mycobiota in the carposphere of sour and sweet cherries and antagonistic features of potential biocontrol yeasts

Sour cherries (Prunus cerasus L.) and sweet cherries (P. avium L.) are economically important fruits with high potential in the food industry and medicine. In this study, we analyzed fungal communities associated with the carposphere of sour and sweet cherries that were freshly harvested from private plantations and purchased in a food store. Following DNA isolation, a DNA fragment of the ITS2 rRNA gene region of each sample was individually amplified and subjected to high-throughput NGS sequencing. Analysis of 168,933 high-quality reads showed the presence of 690 fungal taxa. Investigation of microbial ASVs diversity revealed plant-dependent and postharvest handling-affected fungal assemblages. Among the microorganisms inhabiting tested berries, potentially beneficial or pathogenic fungi were documented. Numerous cultivable yeasts were isolated from the surface of tested berries and characterized by their antagonistic activity. Some of the isolates, identified as Aureobasidium pullulans, Metschnikowia fructicola, and M. pulcherrima, displayed pronounced activity against potential fungal pathogens and showed attractiveness for disease control

Molecular identification of four Sarcocystis species in cattle from Lithuania, including S. hominis, and development of a rapid molecular detection method

Background: Six Sarcocystis species are known to use cattle (Bos taurus) as the intermediate host, two of which, S. hominis and S. heydorni, are zoonotic. There is a need for a method that will enable rapid identification of the Sarcocystis species in cattle. Methods: The diaphragm muscles of 102 cattle from Lithuania were examined for the presence of Sarcocystis spp., using two different methods for species identification. Individual sarcocysts were isolated from squash preparations of the diaphragm muscle under the light microscope, followed by genetic characterisation of excised cysts using sequence analysis of the 18S rRNA (18S rRNA) and cytochrome c oxidase subunit I (cox1) genes. The same cattle muscle samples were digested and species-specific PCR analyses targeting cox1 were developed to identify the Sarcocystis isolates to the species level. Results: Under the light microscope, sarcocysts were detected in 87.3% of animals, and Sarcocystis infection was verified in all digested samples. Three species, namely S. cruzi (n = 20), S. bovifelis (n = 23) and S. hirsuta (n = 6), were identified by DNA sequence analysis of isolated sarcocysts. Based on sequence analysis of cox1, the level of genetic variability depended on Sarcocystis species and geographical location. Four Sarcocystis species, S. cruzi (96.1%), S. bovifelis (71.6%), S. hirsuta (30.4%) and S. hominis (13.7%), were confirmed in the digested samples. In individual samples, the most common finding was two species of Sarcocystis (44.1%), followed by three species (26.5%), a single species (24.5%) and four species (4.9%). Conclusions: Although examination of tissue preparations under the light microscrope did not detect any sarcocysts belonging to S. hominis, this species was identified in the digested samples subjected to a cox1-specific PCR analysis. These results demonstrate the need for effective molecular diagnosis techniques to detect Sarcocystis spp., which may be present at a lower preva