Proportions of taxa belonging to the gut core microbiome change throughout the life cycle and season of the bark beetle Ips typographus.

The European spruce bark beetle, Ips typographus, is a serious pest of spruce forests in Europe, and its invasion and development inside spruce tissues are facilitated by microorganisms. We investigated the core gut bacterial and fungal microbiomes of I. typographus throughout its life cycle in spring and summer generations. We used cultivation techniques and molecular identification in combination with DNA and RNA metabarcoding. Our results revealed that communities differ throughout their life cycle and across generations in proportion of dominantly associated microbes, rather than changes in species composition. The bacteriome consisted mostly of the phylum Gammaproteobacteria, with the most common orders and genera being Enterobacteriales (Erwinia and Serratia), Pseudomonadales (Pseudomonas), and Xanthomonadales. The fungal microbiome was dominated by yeasts (Saccharomycetes-Wickerhamomyces, Kuraishia, and Nakazawaea), followed by Sordariomycetes (Ophiostoma bicolor and Endoconidiophora polonica). We did not observe any structure ensuring long-term persistence of microbiota on any part of the gut epithelium, suggesting that microbial cells are more likely to pass through the beetle's gut with chyme. The most abundant taxa in the beetle's gut were also identified as dominant in intact spruce phloem. Therefore, we propose that these taxa are acquired from the environment rather than specifically vectored between generations.

New insight into the bark beetle ips typographus bacteriome reveals unexplored diversity potentially beneficial to the host.

BACKGROUND: Ips typographus (European spruce bark beetle) is the most destructive pest of spruce forests in Europe. As for other animals, it has been proposed that the microbiome plays important roles in the biology of bark beetles. About the bacteriome, there still are many uncertainties regarding the taxonomical composition, insect-bacteriome interactions, and their potential roles in the beetle ecology. Here, we aim to deep into the ecological functions and taxonomical composition of I. typographus associated bacteria. RESULTS: We assessed the metabolic potential of a collection of isolates obtained from different life stages of I. typographus beetles. All strains showed the capacity to hydrolyse one or more complex polysaccharides into simpler molecules, which may provide an additional carbon source to its host. Also, 83.9% of the strains isolated showed antagonistic effect against one or more entomopathogenic fungi, which could assist the beetle in its fight against this pathogenic threat. Using culture-dependent and -independent techniques, we present a taxonomical analysis of the bacteriome associated with the I. typographus beetle during its different life stages. We have observed an evolution of its bacteriome, which is diverse at the larval phase, substantially diminished in pupae, greater in the teneral adult phase, and similar to that of the larval stage in mature adults. Our results suggest that taxa belonging to the Erwiniaceae family, and the Pseudoxanthomonas and Pseudomonas genera, as well as an undescribed genus within the Enterobactereaceae family, are part of the core microbiome and may perform vital roles in maintaining beetle fitness. CONCLUSION: Our results indicate that isolates within the bacteriome of I. typographus beetle have the metabolic potential to increase beetle fitness by proving additional and assimilable carbon sources for the beetle, and by antagonizing fungi entomopathogens. Furthermore, we observed that isolates from adult beetles are more likely to have these capacities but those obtained from larvae showed strongest antifungal activity. Our taxonomical analysis showed that Erwinia typographi, Pseudomonas bohemica, and Pseudomonas typographi species along with Pseudoxanthomonas genus, and putative new taxa belonging to the Erwiniaceae and Enterobacterales group are repeatedly present within the bacteriome of I. typographus beetles, indicating that these species might be part of the core microbiome. In addition to Pseudomonas and Erwinia group, Staphylococcus, Acinetobacter, Curtobacterium, Streptomyces, and Bacillus genera seem to also have interesting metabolic capacities but are present in a lower frequency. Future studies involving bacterial-insect interactions or analysing other potential roles would provide more insights into the bacteriome capacity to be beneficial to the beetle.

Insight into the genomes of dominant yeast symbionts of European spruce bark beetle, Ips typographus.

Spruce bark beetle Ips typographus can trigger outbreaks on spruce that results in significant losses in the forest industry. It has been suggested that symbiotic microorganisms inhabiting the gut of bark beetles facilitate the colonization of plant tissues as they play a role in the detoxification of plant secondary metabolites, degrade plant cell wall and ameliorate beetle's nutrition. In this study, we sequenced and functionally annotated the genomes of five yeasts Kuraishia molischiana, Cryptococcus sp., Nakazawaea ambrosiae, Ogataea ramenticola, and Wickerhamomyces bisporus isolated from the gut of Ips typographus. Genome analysis identified 5314, 7050, 5722, 5502, and 5784 protein coding genes from K. molischiana, Cryptococcus sp., N. ambrosiae, O. ramenticola, and W. bisporus, respectively. Protein-coding sequences were classified into biological processes, cellular and molecular function based on gene ontology terms enrichment. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation was used to predict gene functions. All analyzed yeast genomes contain full pathways for the synthesis of essential amino acids and vitamin B6, which have nutritional importance to beetle. Furthermore, their genomes contain diverse gene families related to the detoxification processes. The prevalent superfamilies are aldo-keto reductase, ATP-binding cassette and the major facilitator transporters. The phylogenetic relationships of detoxification-related enzymes aldo-keto reductase, and cytochrome P450 monooxygenase, and ATP-binding cassette are presented. Genome annotations also revealed presence of genes active in lignocellulose degradation. In vitro analyses did not confirm enzymatic endolytic degradation of lignocellulose; however, all species can utilize and pectin and produce a large spectrum of exolytic enzymes attacking cellulose, chitin, and lipids.

Comparative eco-physiology revealed extensive enzymatic curtailment, lipases production and strong conidial resilience of the bat pathogenic fungus Pseudogymnoascus destructans.

The genus Pseudogymnoascus encompasses soil psychrophilic fungi living also in caves. Some are opportunistic pathogens; nevertheless, they do not cause outbreaks. Pseudogymnoascus destructans is the causative agent of the white-nose syndrome, which is decimating cave-hibernating bats. We used comparative eco-physiology to contrast the enzymatic potential and conidial resilience of P. destructans with that of phylogenetically diverse cave fungi, including Pseudogymnoascus spp., dermatophytes and outdoor saprotrophs. Enzymatic potential was assessed by Biolog MicroArray and by growth on labelled substrates and conidial viability was detected by flow cytometry. Pseudogymnoascus destructans was specific by extensive losses of metabolic variability and by ability of lipid degradation. We suppose that lipases are important enzymes allowing fungal hyphae to digest and invade the skin. Pseudogymnoascus destructans prefers nitrogenous substrates occurring in bat skin and lipids. Additionally, P. destructans alkalizes growth medium, which points to another possible virulence mechanism. Temperature above 30 °C substantially decreases conidial viability of cave fungi including P. destructans. Nevertheless, survival of P. destructans conidia prolongs by the temperature regime simulating beginning of the flight season, what suggests that conidia could persist on the body surface of bats and contribute to disease spreading during bats active season.

Fungal metabolic profile dataset was not influenced by long-term in vitro preservation of strains.

Comparative ecophysiology is highly valuable approach to reveal adaptive traits linked with specific ecological niches. Although long-term in vitro preserved fungal isolates are often used for analyses, only sparse data is available about the effect of such handling on fungal physiology. The purpose of our data is to show the effect of long-term in vitro preservation of fungal strains on their metabolic profiles. This data is related to research paper "Adaptive traits of bark and ambrosia beetle-associated fungi" (Veselská et al., 2019). Biolog MicroPlates™ for Filamentous fungi were used to compare metabolic profiles between freshly isolated and long-term in vitro preserved strains of two Geosmithia species. Additionally, carbon utilization profiles of 35 Geosmithia species were assessed, including plant pathogen G. morbida and three ambrosia species. Data also shows differences in carbon utilization profiles among diverse ecology types presented in the genus Geosmithia.

Application of flow cytometry for genome size determination in Geosmithia fungi: a comparison of methods.

Genome size has played an important role in the evolution of plants and animals because changes in genome size seem to accompany if not facilitate evolutionary adaptation to environmental conditions. Flow cytometry (FCM) is a widespread method for determining genome size thanks to its high accuracy and speed of measurements. Nevertheless, only a few comparative studies of FCM methods exist in the field of mycology, and reviews are absent. In this study, we compared the suitability of several concentrations and RNAse A incubation times, fixatives and buffers for estimating genome size in fungi. We chose the genus Geosmithia as a model filamentous fungus. We also introduced a new standard, Aspergillus fumigatus CEA10, to determine absolute genome size. We found FCM to be an appropriate method for measuring genome size in fungi, but optimization steps showed that incorrect propidium iodide staining of nuclei can overestimate genome size due to cytoplasmic staining. We identified fixation with methanol:glacial acetic acid (3:1 v/v), 10% DMSO, 0.1% Triton-X 100, and 5 mM EDTA in combination with Tris-MgCl2 buffer as the best treatment.