Microsporidian Infection in Mosquitoes (Culicidae) Is Associated with Gut Microbiome Composition and Predicted Gut Microbiome Functional Content

The animal gut microbiota consist of many different microorganisms, mainly bacteria, but archaea, fungi, protozoans, and viruses may also be present. This complex and dynamic community of microorganisms may change during parasitic infection. In the present study, we investigated the effect of the presence of microsporidians on the composition of the mosquito gut microbiota and linked some microbiome taxa and functionalities to infections caused by these parasites. We characterised bacterial communities of 188 mosquito females, of which 108 were positive for microsporidian DNA. To assess how bacterial communities change during microsporidian infection, microbiome structures were identified using 16S rRNA microbial profiling. In total, we identified 46 families and four higher taxa, of which Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae and Pseudomonadaceae were the most abundant mosquito-associated bacterial families. Our data suggest that the mosquito gut microbial composition varies among host species. In addition, we found a correlation between the microbiome composition and the presence of microsporidians. The prediction of metagenome functional content from the 16S rRNA gene sequencing suggests that microsporidian infection is characterised by some bacterial species capable of specific metabolic functions, especially the biosynthesis of ansamycins and vancomycin antibiotics and the pentose phosphate pathway. Moreover, we detected a positive correlation between the presence of microsporidian DNA and bacteria belonging to Spiroplasmataceae and Leuconostocaceae, each represented by a single species, Spiroplasma sp. PL03 and Weissella cf. viridescens, respectively. Additionally, W. cf. viridescens was observed only in microsporidian-infected mosquitoes. More extensive research, including intensive and varied host sampling, as well as determination of metabolic activities based on quantitative methods, should be carried out to confirm our results.Peer reviewe

Microbial identification for an effective biocleaning: the case study of the Santa Lucia alle Malve rupestrian church

Identifying the microbial community that lives on the lithic cultural heritage through phenotypic and genotypic approaches is fundamental for understanding the contribution of colonisers in biodeterioration and selecting the best cleaning solutions with environmentally-friendly products. This was done with the cultivable microbiota present on the internal walls of the Santa Lucia alle Malve (SLM) rock church, recognised by UNESCO as a World Heritage Site. The SLM church is in the Sasso Caveoso area (Matera, South Italy). It is fully excavated in the limestone rock. Numerous frescoes cover the internal surfaces, but the bacterial and fungal colonisation spoils its beauty. The results of this study clearly showed the predominance of bacteria of the phylum Firmicutes and precisely of the genus Bacillus, whose prevalence may be due to the specific environmental conditions, very similar in all sampling sites of the church, and to their ability to produce endospores. These dormant forms allow bacteria to survive in unfavourable conditions. The genotypic approach showed that the various Bacillus species isolated on the surfaces of the experimented church are closely related and similar but show a different phenotypic profile. This diversity of physiological and morphological traits reflects the potential complexity of the metabolomes present in the bacterial communities of the internal walls of the SLM and confirms the need to identify the colonisers correctly and proceed with a non-temporary bio-cleaning. The results of bio-cleaning using Solanum nigrum extracts were effective, but the durability of the treatment has yet to be verified

Microbial identification for an effective biocleaning: the case study of the Santa Lucia alle Malve rupestrian church

Identifying the microbial community that lives on the lithic cultural heritage through phenotypic and genotypic approaches is fundamental for understanding the contribution of colonisers in biodeterioration and selecting the best cleaning solutions with environmentally-friendly products. This was done with the cultivable microbiota present on the internal walls of the Santa Lucia alle Malve (SLM) rock church, recognised by UNESCO as a World Heritage Site. The SLM church is in the Sasso Caveoso area (Matera, South Italy). It is fully excavated in the limestone rock. Numerous frescoes cover the internal surfaces, but the bacterial and fungal colonisation spoils its beauty. The results of this study clearly showed the predominance of bacteria of the phylum Firmicutes and precisely of the genus Bacillus, whose prevalence may be due to the specific environmental conditions, very similar in all sampling sites of the church, and to their ability to produce endospores. These dormant forms allow bacteria to survive in unfavourable conditions. The genotypic approach showed that the various Bacillus species isolated on the surfaces of the experimented church are closely related and similar but show a different phenotypic profile. This diversity of physiological and morphological traits reflects the potential complexity of the metabolomes present in the bacterial communities of the internal walls of the SLM and confirms the need to identify the colonisers correctly and proceed with a non-temporary bio-cleaning. The results of bio-cleaning using Solanum nigrum extracts were effective, but the durability of the treatment has yet to be verified

The communities of ectomycorrhizal fungal species associated with Betula pendula ROTH and Pinus sylvestris L. growing in heavy-metal contaminated soils

Aims Pioneer tree species such as Betula pendula and Pinus sylvestris encroach soils contaminated with heavy metals (HMs). This is facilitated by ectomycorrhizal fungi colonizing tree roots. Thus, we evaluated the ectomycorrhizal fungal (EMF) communities of B. pendula and P. sylvestris growing in HMcontaminated soils compared to non-contaminated soils. We also studied the effect of HMs and soil properties on EMF communities and soil fungal biomass. Methods Roots of B. pendula and P. sylvestris were collected from three HM-contaminated sites and from two non-contaminated sites located in Poland. EMF species were identified using DNA barcoding. Soil fungal biomass was determined by soil ergosterol. Results B. pendula and P. sylvestris growing in HMcontaminated soils had similar EMF communities, where Scleroderma, Rhizopogon and Russula as well as ectomycorrhizae of the long-distance exploration type dominated. Among all of the examined soil factors studied, toxicity index (TITotal) was the most significant factor shaping the composition of EMF communities. Despite significant differences in the structure of the EMF communities of trees growing in HMcontaminated sites compared to control sites, no differences in overall diversity were observed. Conclusions Only well-adapted EMF species can survive toxic conditions and form ectomycorrhizal symbiosis with encroaching trees facilitating the forest succession on contaminated soils

Trophic and symbiotic links between obligate-glacier water bears (Tardigrada) and cryoconite microorganisms

Insights into biodiversity and trophic webs are important for understanding ecosystem functions. Although the surfaces of glaciers are one of the most productive and biologically diverse parts of the cryosphere, the links between top consumers, their diet and microbial communities are poorly understood. In this study, for the first time we investigated the relationships between bacteria, fungi and other microeukaryotes as they relate to tardigrades, microscopic metazoans that are top consumers in cryoconite, a biologically rich and productive biogenic sediment found on glacier surfaces. Using metabarcoding (16S rDNA for bacteria, ITS1 for fungi, and 18S rDNA for other microeukaryotes), we analyzed the microbial community structures of cryoconite and compared them with the community found in both fully fed and starved tardigrades. The community structure of each microbial group (bacteria, fungi, microeukaryotes) were similar within each host group (cryoconite, fully fed tardigrades and starved tardigrades), and differed significantly between groups, as indicated by redundancy analyses. The relative number of operational taxonomic units (ZOTUs, OTUs) and the Shannon index differed significantly between cryoconite and tardigrades. Species indicator analysis highlighted a group of microbial taxa typical of both fully fed and starved tardigrades (potential commensals), like the bacteria of the genera Staphylococcus and Stenotrophomonas, as well as a group of taxa typical of both cryoconite and fully fed tardigrades (likely part of the tardigrade diet; bacteria Flavobacterium sp., fungi Preussia sp., algae Trebouxiophyceae sp.). Tardigrades are consumers of bacteria, fungi and other microeukaryotes in cryoconite and, being hosts for diverse microbes, their presence can enrich the microbiome of glaciers

Species diversity of Microsporidia in selected hematophagous arthropods and the impact of the occurrence of microsporidians on the host microbiome

Wydział BiologiiMikrosporydia to wewnątrzkomórkowe, obligatoryjne pasożyty, infekujące szerokie spektrum gospodarzy. Około 15% z nich infekuje komary (Culicidae), będące również gospodarzami wielu patogenów kręgowców. Stawonogi hematofagiczne, takie jak komary i kleszcze (Ixodidae) są najważniejszymi wektorami czynników chorobotwórczych. Stąd, mikrosporydia infekujące te stawonogi stanowią bardzo dobry model do badania oddziaływań ekologicznych między patogenami, jak i składnikami mikrobiomu gospodarza. Celem mojej pracy doktorskiej była próba odpowiedzi na pytania: (1) jaka jest faktyczna różnorodność gatunkowa mikrosporydiów u tych gospodarzy; (2) czy mikrosporydia współwystępujące u tego samego gospodarza oddziałują na siebie; oraz (3) czy infekcja mikrosporydiami wpływa na mikrobiom gospodarza. W celu wykrycia mikrosporydiów opracowałem wysokowydajną metodę, którą wykorzystałem do określenia różnorodności gatunkowej tych pasożytów w badanych modelach. Moje wyniki wskazują, że komary są częstymi gospodarzami mikrosporydiów, spośród których większość jest pasożytami poliksenicznymi. Odnotowałem również relatywnie dużą częstość współwystępowania różnych mikrosporydiów, co skutkowało wzajemnymi oddziaływaniami między koinfekującymi gatunkami. Dodatkowo, stwierdziłem, że infekcja mikrosporydiami prowadzi do zmian w składzie mikrobiomu gospodarza oraz aktywności jego szlaków metabolicznych, głównie w kierunku syntezy antybiotyków i nukleotydów. Wśród kleszczy żerujących zidentyfikowałem tylko jeden gatunek mikrosporydium, który występował ze znikomą prewalencją, co sugeruje, że kleszcze mogły wykształcić mechanizm obronny przeciwko tym patogenom. Wyniki mojej pracy doktorskiej stwarzają nowe możliwości badań w obszarze ekologii pasożytów i mogą mieć zastosowanie w diagnostyce medycznej.Microsporidians are a group of intracellular obligate parasites that infect a wide range of hosts. About 15% of them infect mosquitoes (Culicidae), which are themselves hosts of many pathogens of vertebrates. In fact, the hematophagous arthropods, such as mosquitoes and ticks (Ixodidae) are the most important disease vectors. Therefore, microsporidians infecting these arthropods are a very good model to study ecological interactions between both pathogens and the other components of the host microbiome. The aim of my dissertation was to attempt to answer the questions: (1) what is the actual microsporidian species diversity in these hosts; (2) whether the microsporidians co-occurring in the same host individual interact with each other; and (3) whether the microsporidian infection affects the host's microbiome. To detect microsporidians, I developed a high-throughput method, which I used to determine their species-diversity in the tested models. My results demonstrate that mosquitoes are common hosts of numerous microsporidian species, and most of that species are polyxenic parasites. I also noted a relatively high level of the co occurrences of different microsporidians in mosquitoes, which resulted in interactions between the co-infecting species. Additionally, I found that microsporidian infection leads to changes in the composition of the host microbiome and activities of the microbiome metabolic pathways; especially it concerns the synthesis of antibiotics and nucleotides. Among host-seeking ticks, I identified only one microsporidian species that occurred with negligible prevalence, which indicates that ticks probably have developed a defense mechanism against these pathogens. Results of my dissertation provide new opportunities in such research areas as ecology of parasites and medical diagnostics

Species diversity of Microsporidia in selected hematophagous arthropods and the impact of the occurrence of microsporidians on the host microbiome

Wydział BiologiiMikrosporydia to wewnątrzkomórkowe, obligatoryjne pasożyty, infekujące szerokie spektrum gospodarzy. Około 15% z nich infekuje komary (Culicidae), będące również gospodarzami wielu patogenów kręgowców. Stawonogi hematofagiczne, takie jak komary i kleszcze (Ixodidae) są najważniejszymi wektorami czynników chorobotwórczych. Stąd, mikrosporydia infekujące te stawonogi stanowią bardzo dobry model do badania oddziaływań ekologicznych między patogenami, jak i składnikami mikrobiomu gospodarza. Celem mojej pracy doktorskiej była próba odpowiedzi na pytania: (1) jaka jest faktyczna różnorodność gatunkowa mikrosporydiów u tych gospodarzy; (2) czy mikrosporydia współwystępujące u tego samego gospodarza oddziałują na siebie; oraz (3) czy infekcja mikrosporydiami wpływa na mikrobiom gospodarza. W celu wykrycia mikrosporydiów opracowałem wysokowydajną metodę, którą wykorzystałem do określenia różnorodności gatunkowej tych pasożytów w badanych modelach. Moje wyniki wskazują, że komary są częstymi gospodarzami mikrosporydiów, spośród których większość jest pasożytami poliksenicznymi. Odnotowałem również relatywnie dużą częstość współwystępowania różnych mikrosporydiów, co skutkowało wzajemnymi oddziaływaniami między koinfekującymi gatunkami. Dodatkowo, stwierdziłem, że infekcja mikrosporydiami prowadzi do zmian w składzie mikrobiomu gospodarza oraz aktywności jego szlaków metabolicznych, głównie w kierunku syntezy antybiotyków i nukleotydów. Wśród kleszczy żerujących zidentyfikowałem tylko jeden gatunek mikrosporydium, który występował ze znikomą prewalencją, co sugeruje, że kleszcze mogły wykształcić mechanizm obronny przeciwko tym patogenom. Wyniki mojej pracy doktorskiej stwarzają nowe możliwości badań w obszarze ekologii pasożytów i mogą mieć zastosowanie w diagnostyce medycznej.Microsporidians are a group of intracellular obligate parasites that infect a wide range of hosts. About 15% of them infect mosquitoes (Culicidae), which are themselves hosts of many pathogens of vertebrates. In fact, the hematophagous arthropods, such as mosquitoes and ticks (Ixodidae) are the most important disease vectors. Therefore, microsporidians infecting these arthropods are a very good model to study ecological interactions between both pathogens and the other components of the host microbiome. The aim of my dissertation was to attempt to answer the questions: (1) what is the actual microsporidian species diversity in these hosts; (2) whether the microsporidians co-occurring in the same host individual interact with each other; and (3) whether the microsporidian infection affects the host's microbiome. To detect microsporidians, I developed a high-throughput method, which I used to determine their species-diversity in the tested models. My results demonstrate that mosquitoes are common hosts of numerous microsporidian species, and most of that species are polyxenic parasites. I also noted a relatively high level of the co occurrences of different microsporidians in mosquitoes, which resulted in interactions between the co-infecting species. Additionally, I found that microsporidian infection leads to changes in the composition of the host microbiome and activities of the microbiome metabolic pathways; especially it concerns the synthesis of antibiotics and nucleotides. Among host-seeking ticks, I identified only one microsporidian species that occurred with negligible prevalence, which indicates that ticks probably have developed a defense mechanism against these pathogens. Results of my dissertation provide new opportunities in such research areas as ecology of parasites and medical diagnostics

Cyclodepsipeptide Biosynthesis in Hypocreales Fungi and Sequence Divergence of The Non-Ribosomal Peptide Synthase Genes

Fungi from the Hypocreales order synthesize a range of toxic non-ribosomal cyclic peptides with antimicrobial, insecticidal and cytotoxic activities. Entomopathogenic Beauveria, Isaria and Cordyceps as well as phytopathogenic Fusarium spp. are known producers of beauvericins (BEAs), beauvenniatins (BEAEs) or enniatins (ENNs). The compounds are synthesized by beauvericin/enniatin synthase (BEAS/ESYN1), which shows significant sequence divergence among Hypocreales members. We investigated ENN, BEA and BEAE production among entomopathogenic (Beauveria, Cordyceps, Isaria) and phytopathogenic (Fusarium) fungi; BEA and ENNs were quantified using an LC-MS/MS method. Phylogenetic analysis of partial sequences of putative BEAS/ESYN1 amplicons was also made. Nineteen fungal strains were identified based on sequence analysis of amplified ITS and tef-1α regions. BEA was produced by all investigated fungi, with F. proliferatum and F. concentricum being the most efficient producers. ENNs were synthesized mostly by F. acuminatum, F. avenaceum and C. confragosa. The phylogeny reconstruction suggests that ancestral BEA biosynthesis independently diverged into biosynthesis of other compounds. The divergent positioning of three Fusarium isolates raises the possibility of parallel acquisition of cyclic depsipeptide synthases in ancient complexes within Fusarium genus. Different fungi have independently evolved NRPS genes involved in depsipeptide biosynthesis, with functional adaptation towards biosynthesis of overlapping yet diversified metabolite profiles

Challenges of Comparing Marine Microbiome Community Composition Data Provided by Different Commercial Laboratories and Classification Databases

In the high-throughput sequencing (HTS) era, a metabarcoding technique based on the bacterial V3–V4 hypervariable region of 16S rRNA analysis requires sophisticated bioinformatics pipelines and validated methods that allow researchers to compare their data with confidence. Many commercial laboratories conduct extensive HTS analyses; however, there is no available information on whether the results generated by these vendors are consistent. In our study, we compared the sequencing data obtained for the same marine microbiome community sample generated by three commercial laboratories. Additionally, as a sequencing control to determine differences between commercial laboratories and two 16S rRNA databases, we also performed a “mock community” analysis of a defined number of microbial species. We also assessed the impact of the choice of two commonly used 16S rRNA databases, i.e., Greengenes and SILVA, on downstream data analysis, including taxonomic classification assignment. We demonstrated that the final results depend on the choice of the laboratory conducting the HTS and the reference database of ribosomal sequences. Our findings showed that the number of produced ASVs (amplicon sequence variants) ranged from 137 to 564. Different putative bacterial endosymbionts could be identified, and these differences correspond to the applied 16S rRNA database. The results presented might be of particular interest to researchers who plan to perform microbiome community analysis using the 16S rRNA marker gene, including the identification of putative bacterial endosymbionts, and serve as a guide for choosing the optimum pipeline to obtain the most accurate and reproducible data