Complete genome of Microbacterium plantarum CoE-159-22, isolated from traditionally produced Montenegrin cheese.

Microbacterium plantarum (M. plantarum) was recently described as a new species isolated from copper globemallow (Sphaeralcea angustifolia). Here, we report the complete genome of M. plantarum CoE-159-22, which was obtained from traditionally produced Montenegrin cheese.

Radiation dose fractionation and its potential hormetic effects on male Glossina palpalis gambiensis (Diptera: Glossinidae): a comparative study of reproductive and flight quality parameters.

One of the most critical factors for implementing the sterile insect technique for the management of tsetse is the production of large quantities of highly competitive sterile males in the field. Several factors may influence the biological quality of sterile males, but optimizing the irradiation protocols to limit unwanted somatic cell damage could improve male performance. This study evaluated the effect of fractionation of gamma radiation doses on the fertility and flight quality of male Glossina palpalis gambiensis. Induced sterility was assessed by mating irradiated males with virgin fertile females. Flight quality was assessed using a standard protocol. The male flies were irradiated as pupae on day 23-27 post larviposition with 110 Gy, either in a single dose or in fractionations of 10 + 100 Gy and 50 + 60 Gy separated by 1-, 2- and 3-day intervals or 55 + 55 Gy separated by 4-, 8-, and 24-hour intervals. All treatments induced more than 90% sterility in females mated with irradiated males, as compared with untreated males. No significant differences were found in emergence rate or flight propensity between fractionated and single radiation doses, nor between the types of fractionations. Overall, the 50(D0) + 60(D1) Gy dose showed slightly higher induced sterility, flight propensity, and survival of males under feeding regime. Dose fractionation resulted in only small improvements with respect to flight propensity and survival, and this should be traded off with the required increase in labor that dose fractionation entails, especially in larger control programs.

Title: Fractionnement de la dose de rayonnement et ses effets hormétiques potentiels sur les Glossina palpalis gambiensis mâles (Diptera : Glossinidae) : une étude comparative des paramètres de reproduction et de qualité de vol. Abstract: L'un des facteurs les plus critiques pour la mise en œuvre de la technique de l'insecte stérile pour la gestion des glossines est la production de grandes quantités de mâles stériles hautement compétitifs sur le terrain. Plusieurs facteurs peuvent influencer la qualité biologique des mâles stériles, mais l'optimisation des protocoles d'irradiation pour limiter les dommages indésirables aux cellules somatiques pourrait améliorer les performances des mâles. Cette étude a évalué l'effet du fractionnement de la dose d'irradiation gamma sur la fertilité et la qualité de vol des mâles de Glossina palpalis gambiensis. La stérilité induite a été évaluée en accouplant des mâles irradiés avec des femelles vierges et fertiles. La qualité du vol a été évaluée à l'aide d'un protocole standard. Les mouches mâles ont été irradiées sous forme de pupes agées de 23 à 27 jours après la larviposition avec 110 Gy, soit en dose unique, soit en fractions de 10 + 100 Gy et 50 + 60 Gy séparées par 1, 2 et 3 jours ou 55 + 55 Gy séparés par des intervalles de 4, 8 et 24 heures. Tous les traitements ont induit plus de 90 % de stérilité chez les femelles accouplées avec des mâles irradiés par rapport aux mâles non traités. Aucune différence significative n'a été trouvée dans le taux d'émergence ou la propension au vol entre les doses d'irradiation fractionnées et uniques ni entre les types de fractionnements. Dans l'ensemble, la dose de 50 (J0) + 60 (J1) Gy a montré une stérilité induite, une propension à voler et une survie légèrement plus élevées chez les mâles sous régime alimentaire. Le fractionnement de dose n'a entraîné que de légères améliorations en ce qui concerne la propension à voler et la survie, et cela devrait être compensé par l'augmentation nécessaire du travail qu'implique le fractionnement de dose, en particulier dans les programmes de contrôle de grande envergure.

Enterococcus montenegrensis sp. nov., isolated from artisanal Montenegrin dry sausage.

A novel, Gram-positive, facultative anaerobe, coccoid and non-motile bacterium, designated as CoE-012-22T was isolated from dried beef sausage (the original name in Montenegro is Govedji Kulen) manufactured in the municipality of Rozaje (Montenegro) in 2021. Cells of this strain were oxidase- and catalase-negative. Growth occurred at 4-50 °C, at pH 5.0-8.0 and with 0-6.5 % (w/v) NaCl in diverse growth media. MALDI-TOF analysis identified the strain as Enterococcus canintestini (log score 2). Phylogenetic analysis of the 16S rRNA gene and whole genome sequences assigned the strain to the genus Enterococcus. The closest relatives were E. canintestini DSM 21207T and E. dispar ATCC 51266T with 16S rRNA gene sequence pairwise similarities of 99.34 and 98.59 %, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between isolate CoE-012-22T and other enterococci species were below the thresholds for species delineation thresholds (95.0 % ANI; 70.0 % dDDH) with maximum identities of 84.13 % (ANIb), 86.43 % (ANIm) and 28.4 % (dDDH) to E. saigonensis JCM 31193T and 70.97 % (ANIb), 88.99 % (ANIm) and 32.4 % (dDDH) to E. malodoratus ATCC 43197T. Two unknown Enterococcus isolates, Enterococcus sp. MJM12 and Enterococcus SMC-9, showed identities of 99.87 and 99.94 % (16S rRNA), 98.57 and 98.65 % (ANIb), 98.93 and 99.02 % (ANIm), and 89.8 and 90.0 % (dDDH) to strain CoE-012-22T and can therefore be regarded as the same species. Based on the characterization results, strain CoE-012-22T was considered to represent a novel species, for which the name Enterococcus montenegrensis sp. nov. is proposed. The type strain is CoE-012-22T (=DSM 115843T=NCIMB 15468T).

Protein secretion and associated stress in industrially employed filamentous fungi.

Application of filamentous fungi for the production of commercial enzymes such as amylase, cellulase, or xylanase is on the rise due to the increasing demand to degrade several complex carbohydrates as raw material for biotechnological processes. Also, protein production by fungi for food and feed gains importance. In any case, the protein production involves both cellular synthesis and secretion outside of the cell. Unfortunately, the secretion of proteins or enzymes can be hampered due to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) as a result of too high synthesis of enzymes or (heterologous) protein expression. To cope with this ER stress, the cell generates a response known as unfolded protein response (UPR). Even though this mechanism should re-establish the protein homeostasis equivalent to a cell under non-stress conditions, the enzyme expression might still suffer from repression under secretory stress (RESS). Among eukaryotes, Saccharomyces cerevisiae is the only fungus, which is studied quite extensively to unravel the UPR pathway. Several homologs of the proteins involved in this signal transduction cascade are also found in filamentous fungi. Since RESS seems to be absent in S. cerevisiae and was only reported in Trichoderma reesei in the presence of folding and glycosylation inhibitors such as dithiothreitol and tunicamycin, more in-depth study about this mechanism, specifically in filamentous fungi, is the need of the hour. Hence, this review article gives an overview on both, protein secretion and associated stress responses in fungi. KEY POINTS: • Enzymes produced by filamentous fungi are crucial in industrial processes • UPR mechanism is conserved among many fungi, but mediated by different proteins • RESS is not fully understood or studied in industrially relevant filamentous fungi.

Prevalence of Spiroplasma and interaction with wild Glossina tachinoides microbiota.

Tsetse flies (Diptera: Glossinidae) are vectors of the tropical neglected diseases sleeping sickness in humans and nagana in animals. The elimination of these diseases is linked to control of the vector. The sterile insect technique (SIT) is an environment-friendly method that has been shown to be effective when applied in an area-wide integrated pest management approach. However, as irradiated males conserve their vectorial competence, there is the potential risk of trypanosome transmission with their release in the field. Analyzing the interaction between the tsetse fly and its microbiota, and between different microbiota and the trypanosome, might provide important information to enhance the fly's resistance to trypanosome infection. This study on the prevalence of Spiroplasma in wild populations of seven tsetse species from East, West, Central and Southern Africa showed that Spiroplasma is present only in Glossina fuscipes fuscipes and Glossina tachinoides. In G. tachinoides, a significant deviation from independence in co-infection with Spiroplasma and Trypanosoma spp. was observed. Moreover, Spiroplasma infections seem to significantly reduce the density of the trypanosomes, suggesting that Spiroplasma might enhance tsetse fly's refractoriness to the trypanosome infections. This finding might be useful to reduce risks associated with the release of sterile males during SIT implementation in trypanosome endemic areas.

Title: Prévalence de Spiroplasma et interaction avec le microbiote des Glossina tachinoides sauvages. Abstract: Les mouches tsé-tsé (Diptera : Glossinidae) sont les vecteurs de maladies tropicales négligées, la maladie du sommeil chez l'homme et la nagana chez les animaux. L'élimination de ces maladies est liée à la lutte contre le vecteur. La technique de l'insecte stérile (TIS) est une méthode respectueuse de l'environnement qui s'est révélée efficace lorsqu'elle est appliquée dans le cadre d'une approche de lutte antiparasitaire intégrée à l'échelle d'une zone. Cependant, comme les mâles irradiés conservent leur compétence vectorielle, il existe un risque potentiel de transmission des trypanosomes lors de la libération des mâles sur le terrain. L'analyse de l'interaction entre la mouche tsé-tsé et son microbiote, et entre différents microbiotes et le trypanosome, pourrait fournir des informations importantes pour améliorer la résistance de la mouche à l'infection trypanosomienne. Cette étude sur la prévalence de Spiroplasma dans les populations sauvages de sept espèces de glossines d'Afrique de l'Est, de l'Ouest, centrale et australe a montré que Spiroplasma est présent uniquement chez Glossina fuscipes fuscipes et Glossina tachinoides. Chez G. tachinoides, un écart significatif par rapport à l'indépendance dans la co-infection par Spiroplasma et Trypanosoma spp. a été observé. De plus, les infections à Spiroplasma semblent réduire considérablement la densité des trypanosomes, ce qui suggère que Spiroplasma pourrait renforcer le caractère réfractaire de la mouche tsé-tsé aux infections trypanosomiennes. Cette découverte pourrait être utile pour réduire le risque associé à la libération de mâles stériles lors de la mise en œuvre de la TIS dans les zones d'endémie trypanosomienne.

X-rays are as effective as gamma-rays for the sterilization of Glossina palpalis gambiensis Vanderplank, 1911 (Diptera: Glossinidae) for use in the sterile insect technique.

An area-wide integrated pest management strategy with a sterile insect technique (SIT) component requires a radiation source for the sterilisation of male insects. Self-contained gamma irradiators, which were exclusively used in past SIT programmes, are now facing increasing constraints and challenges due to stringent regulations. As a potential alternative, new generation high output X-ray irradiators have been proposed. The feasibility of using X-ray irradiators was assessed by comparing the effects of both gamma- and X-ray irradiators on biological parameters of Glossina palpalis gambiensis (Vanderplank, 1911), that are important for SIT applications. The gamma irradiator Foss Model 812 and two X-ray irradiators, the Rad Source 2400 and the blood irradiator Raycell Mk2 were used. Glossina palpalis gambiensis males were exposed to radiation as pupae. A radiation dose of 110 Gy or above induced more than 97% sterility in females that mated with the irradiated males for all the irradiators. Adult emergence rate, flight propensity, survival and mating performance did not differ between gamma- and X-rays irradiators. These results suggest that irradiating pupae with a dose of 110 Gy is optimal for both gamma-and X-ray irradiators used in this study, to achieve a sterility of approximately 99%. Similar research on other tsetse species could gradually phase out the use of gamma-ray irradiators in favour of X-rays irradiators, especially for smaller SIT programmes.

Development and characterization of microsatellite markers for the tsetse species Glossina brevipalpis and preliminary population genetics analyses.

Tsetse flies, the vectors of African trypanosomes are of key medical and economic importance and one of the constraints for the development of Africa. Tsetse fly control is one of the most effective and sustainable strategies used for controlling the disease. Knowledge about population structure and level of gene flow between neighbouring populations of the target vector is of high importance to develop appropriate strategies for implementing effective management programmes. Microsatellites are commonly used to identify population structure and assess dispersal of the target populations and have been developed for several tsetse species but were lacking for Glossina brevipalpis. In this study, we screened the genome of G. brevipalpis to search for suitable microsatellite markers and nine were found to be efficient enough to distinguish between different tsetse populations. The availability of these novel microsatellite loci will help to better understand the population biology of G. brevipalpis and to assess the level of gene flow between different populations. Such information will help with the development of appropriate strategies to implement the sterile insect technique (SIT) in the framework of an area-wide integrated pest management (AW-IPM) approach to manage tsetse populations and ultimately address the trypanosomoses problem in these targeted areas.

Title: Développement et caractérisation de marqueurs microsatellites pour l'espèce de mouche tsé-tsé Glossina brevipalpis et analyses génétiques préliminaires des populations. Abstract: Les mouches tsé-tsé, vecteurs des trypanosomes africains, sont d'une importance médicale et économique majeure et l'une des contraintes pour le développement de l'Afrique. La lutte contre la mouche tsé-tsé est l'une des stratégies les plus efficaces et durables utilisées pour contrôler la maladie. La connaissance de la structure de la population et du niveau de flux de gènes entre les populations voisines du vecteur cible est d'une grande importance pour développer des stratégies appropriées pour la mise en œuvre de programmes de gestion efficaces. Les microsatellites sont couramment utilisés pour identifier la structure de la population et évaluer la dispersion des populations cibles et ont été développés pour plusieurs espèces de glossines mais manquaient pour Glossina brevipalpis. Dans cette étude, nous avons criblé le génome de G. brevipalpis pour rechercher des marqueurs microsatellites appropriés et neuf ont été trouvés suffisamment efficaces pour faire la distinction entre différentes populations de glossines. La disponibilité de ces nouveaux locus microsatellites aidera à mieux comprendre la biologie des populations de G. brevipalpis et à évaluer le niveau de flux de gènes entre différentes populations. Ces informations aideront à l'élaboration de stratégies appropriées pour mettre en œuvre la technique de l'insecte stérile dans le cadre d'une approche de lutte antiparasitaire intégrée à l'échelle de la zone pour gérer les populations de glossines et, en fin de compte, résoudre le problème des trypanosomoses dans les zones concernées.

The phenomenon of strain degeneration in biotechnologically relevant fungi.

Fungi are widely exploited for large-scale production in the biotechnological industry to produce a diverse range of substances due to their versatility and relative ease of growing on various substrates. The occurrence of a phenomenon-the so-called fungal strain degeneration-leads to the spontaneous loss or decline of production capacity and results in an economic loss on a tremendous scale. Some of the most commonly applied genera of fungi in the biotechnical industry, such as Aspergillus, Trichoderma, and Penicillium, are threatened by this phenomenon. Although fungal degeneration has been known for almost a century, the phenomenon and its underlying mechanisms still need to be understood. The proposed mechanisms causing fungi to degenerate can be of genetic or epigenetic origin. Other factors, such as culture conditions, stress, or aging, were also reported to have an influence. This mini-review addresses the topic of fungal degeneration by describing examples of productivity losses in biotechnical processes using Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, and Penicillium chrysogenum. Further, potential reasons, circumvention, and prevention methods are discussed. This is the first mini-review which provides a comprehensive overview on this phenomenon in biotechnologically used fungi, and it also includes a collection of strategies that can be useful to minimize economic losses which can arise from strain degeneration. KEY POINTS: • Spontaneous loss of productivity is evident in many fungi used in biotechnology. • The properties and mechanisms underlying this phenomenon are very versatile. • Only studying these underlying mechanisms enables the design of a tailored solution.

Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris).

BACKGROUND: The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. RESULTS: We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. CONCLUSION: The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.

Suitability of Raycell MK2 Blood X-ray Irradiator for the Use in the Sterile Insect Technique: Dose Response in Fruit Flies, Tsetse Flies and Mosquitoes.

The sterile insect technique (SIT) is based on the inundatory field release of a target pest following their reproductive sterilization via exposure to radiation. Until recently, gamma irradiation from isotopic sources has been the most widely used in SIT programs. As isotopic sources are becoming increasingly expensive, especially for small programs, and regulations surrounding their procurement and shipment increasingly strict, irradiation capacity is one of the limiting factors in smaller or newly developing SIT projects. For this reason, the possibility of using X-ray irradiators has been evaluated in the recent decade. The availability of "off-the-shelf" blood X-ray irradiators that meet the technical requirements for insect irradiation can provide irradiation capacity for those SIT projects in which the acquisition of gamma ray irradiators is not feasible. Following the recent technical characterization of a Raycell MK2 X-ray blood irradiator, it was found in this study, that MK2 instruments were suitable for the sterilization of fruit flies, tsetse flies and mosquitoes, inducing comparable, even slightly higher, sterility levels compared to those achieved by gamma ray irradiation. This, together with its estimated processing efficiency, shows that MK2 irradiators are suitable for small- to mid-sized SIT programs.

Draft Genome Sequence of Enterococcus dispar CoE-457-22, Isolated from Traditionally Produced Montenegrin Dry Sausage.

Enterococcus dispar was isolated for the first time from synovial fluid and stool cultures and described as a new species in 1991. Here, we report the genome of E. dispar CoE-457-22, which was obtained from traditionally produced Montenegrin dry sausage (sudzuk).

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei.

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

Interactions between Glossina pallidipes salivary gland hypertrophy virus and tsetse endosymbionts in wild tsetse populations.

BACKGROUND: Tsetse control is considered an effective and sustainable tactic for the control of cyclically transmitted trypanosomosis in the absence of effective vaccines and inexpensive, effective drugs. The sterile insect technique (SIT) is currently used to eliminate tsetse fly populations in an area-wide integrated pest management (AW-IPM) context in Senegal. For SIT, tsetse mass rearing is a major milestone that associated microbes can influence. Tsetse flies can be infected with microorganisms, including the primary and obligate Wigglesworthia glossinidia, the commensal Sodalis glossinidius, and Wolbachia pipientis. In addition, tsetse populations often carry a pathogenic DNA virus, the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) that hinders tsetse fertility and fecundity. Interactions between symbionts and pathogens might affect the performance of the insect host. METHODS: In the present study, we assessed associations of GpSGHV and tsetse endosymbionts under field conditions to decipher the possible bidirectional interactions in different Glossina species. We determined the co-infection pattern of GpSGHV and Wolbachia in natural tsetse populations. We further analyzed the interaction of both Wolbachia and GpSGHV infections with Sodalis and Wigglesworthia density using qPCR. RESULTS: The results indicated that the co-infection of GpSGHV and Wolbachia was most prevalent in Glossina austeni and Glossina morsitans morsitans, with an explicit significant negative correlation between GpSGHV and Wigglesworthia density. GpSGHV infection levels > 103.31 seem to be absent when Wolbachia infection is present at high density (> 107.36), suggesting a potential protective role of Wolbachia against GpSGHV. CONCLUSION: The result indicates that Wolbachia infection might interact (with an undefined mechanism) antagonistically with SGHV infection protecting tsetse fly against GpSGHV, and the interactions between the tsetse host and its associated microbes are dynamic and likely species specific; significant differences may exist between laboratory and field conditions.

A bioinformatic-assisted workflow for genome-wide identification of ncRNAs.

With the upcoming of affordable Next-Generation Sequencing technologies, the number of known non-protein coding RNAs increased drastically in recent years. Different types of non-coding RNAs (ncRNAs) emerged as key players in the regulation of gene expression on the RNA-RNA, RNA-DNA as well as RNA-protein level, ranging from involvement in chromatin remodeling and transcription regulation to post-transcriptional modifications. Prediction of ncRNAs involves the use of several bioinformatics tools and can be a daunting task for researchers. This led to the development of analysis pipelines such as UClncR and lncpipe. However, these pipelines are limited to datasets from human, mouse, zebrafish or fruit fly and are not able to analyze RNA sequencing data from other organisms. In this study, we developed the analysis pipeline Pinc (Pipeline for prediction of ncRNA) as an enhanced tool to predict ncRNAs based on sequencing data by removing transcripts that show protein-coding potential. Additionally, a feature for differential expression analysis of annotated genes as well as for identification of novel ncRNAs is implemented. Pinc uses Nextflow as a framework and is built with robust and well-established analysis tools. This will allow researchers to utilize sequencing data from every organism in order to reliably identify ncRNAs.

Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins.

Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.

Prevalence of Trypanosoma and Sodalis in wild populations of tsetse flies and their impact on sterile insect technique programmes for tsetse eradication.

The sterile insect technique (SIT) is an environment friendly and sustainable method to manage insect pests of economic importance through successive releases of sterile irradiated males of the targeted species to a defined area. A mating of a sterile male with a virgin wild female will result in no offspring, and ultimately lead to the suppression or eradication of the targeted population. Tsetse flies, vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and analyse their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and T. brucei sspp were higher in west African countries, whereas tsetse infection with T. congolense and T. simiae, T. simiae (tsavo) and T. godfreyi were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in G. tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (< 0.05) seems to suggest that Sodalis and Trypanosoma infection correlate in G. palpalis gambiensis, G. pallidipes and G. medicorum. Trypanosoma infection seemed significantly associated with an increased density of Sodalis in wild G. m. morsitans and G. pallidipes flies, however, there was no significant impact of Sodalis infection on trypanosome density.

FunOrder 2.0 - a method for the fully automated curation of co-evolved genes in fungal biosynthetic gene clusters.

Coevolution is an important biological process that shapes interacting proteins - may it be physically interacting proteins or consecutive enzymes in a metabolic pathway, such as the biosynthetic pathways for secondary metabolites. Previously, we developed FunOrder, a semi-automated method for the detection of co-evolved genes, and demonstrated that FunOrder can be used to identify essential genes in biosynthetic gene clusters from different ascomycetes. A major drawback of this original method was the need for a manual assessment, which may create a user bias and prevents a high-throughput application. Here we present a fully automated version of this method termed FunOrder 2.0. In the improved version, we use several mathematical indices to determine the optimal number of clusters in the FunOrder output, and a subsequent k-means clustering based on the first three principal components of a principal component analysis of the FunOrder output to automatically detect co-evolved genes. Further, we replaced the BLAST tool with the DIAMOND tool as a prerequisite for using larger proteome databases. Potentially, FunOrder 2.0 may be used for the assessment of complete genomes, which has not been attempted yet. However, the introduced changes slightly decreased the sensitivity of this method, which is outweighed by enhanced overall speed and specificity.

Identifying Inorganic Turbidity in Water Samples as Potential Loss Factor During Nucleic Acid Extraction: Implications for Molecular Fecal Pollution Diagnostics and Source Tracking.

Molecular diagnostic methods are increasingly applied for food and environmental analysis. Since several steps are involved in sample processing which can affect the outcome (e.g., adhesion of DNA to the sample matrix, inefficient precipitation of DNA, pipetting errors and (partial) loss of the DNA pellet during DNA isolation), quality control is essential at all processing levels. In soil microbiology, particular attention has been paid to the inorganic component of the sample matrix affecting DNA extractability. In water quality testing, however, this aspect has mostly been neglected so far, although it is conceivable that these mechanisms have a similar impact. The present study was therefore dedicated to investigate possible matrix effects on results of water quality analysis. Field testing in an aquatic environment with pronounced chemo-physical gradients [total suspended solids (TSS), inorganic turbidity, total organic carbon (TOC), and conductivity] indicated a negative association between DNA extractability (using a standard phenol/chloroform extraction procedure) and turbidity (spearman ρ = -0.72, p < 0.001, n = 21). Further detailed laboratory experiments on sediment suspensions confirmed the hypothesis of inorganic turbidity being the main driver for reduced DNA extractability. The observed effects, as known from soil samples, were also indicated to result from competitive effects for free charges on clay minerals, leading to adsorption of DNA to these inorganic particles. A protocol modification by supplementing the extraction buffer with salmon sperm DNA, to coat charged surfaces prior to cell lysis, was then applied on environmental water samples and compared to the standard protocol. At sites characterized by high inorganic turbidity, DNA extractability was significantly improved or made possible in the first place by applying the adapted protocol. This became apparent from intestinal enterococci and microbial source tracking (MST)-marker levels measured by quantitative polymerase chain reaction (qPCR) (100 to 10,000-fold median increase in target concentrations). The present study emphasizes the need to consider inorganic turbidity as a potential loss factor in DNA extraction from water-matrices. Negligence of these effects can lead to a massive bias, by up to several orders of magnitude, in the results of molecular MST and fecal pollution diagnostics.

An overview on current molecular tools for heterologous gene expression in Trichoderma.

Fungi of the genus Trichoderma are routinely used as biocontrol agents and for the production of industrial enzymes. Trichoderma spp. are interesting hosts for heterologous gene expression because their saprotrophic and mycoparasitic lifestyles enable them to thrive on a large number of nutrient sources and some members of this genus are generally recognized as safe (GRAS status). In this review, we summarize and discuss several aspects involved in heterologous gene expression in Trichoderma, including transformation methods, genome editing strategies, native and synthetic expression systems and implications of protein secretion. This review focuses on the industrial workhorse Trichoderma reesei because this fungus is the best-studied member of this genus for protein expression and secretion. However, the discussed strategies and tools can be expected to be transferable to other Trichoderma species.