Microbiome Structure of a Wild Drosophila Community along Tropical Elevational Gradients and Comparison to Laboratory Lines.

Variation along environmental gradients in host-associated microbial communities is not well understood compared to free-living microbial communities. Because elevational gradients may serve as natural proxies for climate change, understanding patterns along these gradients can inform our understanding of the threats hosts and their symbiotic microbes face in a warming world. In this study, we analyzed bacterial microbiomes from pupae and adults of four Drosophila species native to Australian tropical rainforests. We sampled wild individuals at high and low elevations along two mountain gradients to determine natural diversity patterns. Further, we sampled laboratory-reared individuals from isofemale lines established from the same localities to see if any natural patterns are retained in the lab. In both environments, we controlled for diet to help elucidate other deterministic patterns of microbiome composition. We found small but significant differences in Drosophila bacterial community composition across elevation, with some notable taxonomic differences between different Drosophila species and sites. Further, we found that field-collected fly pupae had significantly richer microbiomes than laboratory-reared pupae. We also found similar microbiome composition in both types of provided diet, suggesting that the significant differences found among Drosophila microbiomes are the products of surrounding environments with different bacterial species pools, possibly bound to elevational differences in temperature. Our results suggest that comparative studies between lab and field specimens help reveal the true variability in microbiome communities that can exist within a single species. IMPORTANCE Bacteria form microbial communities inside most higher-level organisms, but we know little about how the microbiome varies along environmental gradients and between natural host populations and laboratory colonies. To explore such effects on insect-associated microbiomes, we studied the gut microbiome in four Drosophila species over two mountain gradients in tropical Australia. We also compared these data to individuals kept in the laboratory to understand how different settings changed microbiome communities. We found that field-sampled individuals had significantly higher microbiome diversity than those from the lab. In wild Drosophila populations, elevation explains a small but significant amount of the variation in their microbial communities. Our study highlights the importance of environmental bacterial sources for Drosophila microbiome composition across elevational gradients and shows how comparative studies help reveal the true flexibility in microbiome communities that can exist within a species.

Microbiome of pear psyllids: A tale about closely related species sharing their endosymbionts.

Psyllids are phloem-feeding insects that can transmit plant pathogens such as phytoplasmas, intracellular bacteria causing numerous plant diseases worldwide. Their microbiomes are essential for insect physiology and may also influence the capacity of vectors to transmit pathogens. Using 16S rRNA gene metabarcoding, we compared the microbiomes of three sympatric psyllid species associated with pear trees in Central Europe. All three species are able to transmit 'Candidatus Phytoplasma pyri', albeit with different efficiencies. Our results revealed potential relationships between insect biology and microbiome composition that varied during psyllid ontogeny and between generations in Cacopsylla pyri and C. pyricola, as well as between localities in C. pyri. In contrast, no variations related to psyllid life cycle and geography were detected in C. pyrisuga. In addition to the primary endosymbiont Carsonella ruddii, we detected another highly abundant endosymbiont (unclassified Enterobacteriaceae). C. pyri and C. pyricola shared the same taxon of Enterobacteriaceae which is related to endosymbionts harboured by other psyllid species from various families. In contrast, C. pyrisuga carried a different Enterobacteriaceae taxon related to the genus Sodalis. Our study provides new insights into host-symbiont interactions in psyllids and highlights the importance of host biology and geography in shaping microbiome structure.

A new symbiotic lineage related to Neisseria and Snodgrassella arises from the dynamic and diverse microbiomes in sucking lice.

The phylogenetic diversity of symbiotic bacteria in sucking lice suggests that lice have a complex history of symbiont acquisition, loss, and replacement throughout their evolution. These processes have resulted in the establishment of different, phylogenetically distant bacteria as obligate mutualists in different louse groups. By combining metagenomics and amplicon screening across several populations of three louse species (members of the genera Polyplax and Hoplopleura) we describe a novel louse symbiont lineage related to Neisseria and Snodgrassella, and show its independent origin in the two louse genera. While the genomes of these symbionts are highly similar, their respective distributions and status within lice microbiomes indicate that they have different functions and history. In Hoplopleura acanthopus, the Neisseriaceae-related bacterium is a dominant obligate symbiont present across several host populations. In contrast, the Polyplax microbiomes are dominated by the obligate symbiont Legionella polyplacis, with the Neisseriaceae-related bacterium co-occurring only in some samples and with much lower abundance. The results thus support the view that compared to other exclusively blood feeding insects, Anoplura possess a unique capacity to acquire symbionts from diverse groups of bacteria.

Transformation of Cells by Photoinactivated Murine Gamma-Herpesvirus 68 during Nonproductive and Quiescent Infection.

Infection of human MRC-5 cells and mouse NIH-3T3 cells with a murine gamma-herpesvirus (MuHV-4 strain 68; MHV-68) photoinactivated by visible light in the presence of methylene blue (MB) resulted in nonproductive infection and the appearance of morphologically transformed cells. Two stably transformed cell lines were derived from both of these cell types and were confirmed to contain both viral DNA and antigen. Next, a quiescent MHV-68 infection in MRC-5 and NIH-3T3 cells was established after cultivation at 41°C in the presence of phosphonoacetic acid. Following the exposure of quiescently infected cells to visible light for 120 s (5 times daily for 6 days) in the presence of MB, both MRC-5 and NIH-3T3 cells were observed to acquire transformed phenotypes. The cytopathic effect was observed in cells after 4-5 passages, after which the cells degenerated. However, when human interferon (IFN)-α and mouse IFN-ß were added to the media of quiescently infected MRC-5 and NIH-3T3 cells during the photoinactivating procedure, 2 stable transformed cell lines containing both viral DNA and the antigen were obtained and resembled those attained following nonproductive infection with photoinactivated virus.

Ticks and bacterial tick-borne pathogens in Piemonte region, Northwest Italy.

A molecular screening for tick-borne pathogens was carried out in engorged and in questing ticks collected in Verbano Cusio Ossola county, Piemonte region, Italy. Engorged ticks were removed from wild and domestic animal hosts. The most abundant and common tick species in the area was Ixodes ricinus (192 adults, 907 nymphs). Few individuals of Ixodes hexagonus (15) and Rhipicephalus sanguineus (7) were found among the ticks removed from domestic animals (46 examined ticks). The presence of Rickettsia spp., Borrelia burgdorferi sensu latu, Francisella tularensis and Coxiella burnetii was evaluated by PCR and sequencing in 392 individuals of I. ricinus (adult and nymphal stages) and 22 individuals of the two other tick species. Five Borrelia species (i.e. B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana and B. lusitaniae), proved or suspected to cause clinical manifestations of Lyme disease in humans, showed 10.5 and 2.2% combined prevalence in questing and engorged I. ricinus, respectively. In addition, two species of rickettsiae (R. helvetica and R. monacensis) were identified and reported with 14.5 and 24.8% overall prevalence in questing and in engorged ticks. The prevalence of F. tularensis in the ticks collected on two wild ungulate species (Capreolus capreolus and Cervus elaphus) was 5.7%. This work provided further data and broadened our knowledge on bacterial pathogens present in ticks in Northwest Italy.

Arsenophonus and Sodalis Symbionts in Louse Flies: an Analogy to the Wigglesworthia and Sodalis System in Tsetse Flies.

Symbiosis between insects and bacteria result in a variety of arrangements, genomic modifications, and metabolic interconnections. Here, we present genomic, phylogenetic, and morphological characteristics of a symbiotic system associated with Melophagus ovinus, a member of the blood-feeding family Hippoboscidae. The system comprises four unrelated bacteria representing different stages in symbiosis evolution, from typical obligate mutualists inhabiting bacteriomes to freely associated commensals and parasites. Interestingly, the whole system provides a remarkable analogy to the association between Glossina and its symbiotic bacteria. In both, the symbiotic systems are composed of an obligate symbiont and two facultative intracellular associates, Sodalis and Wolbachia. In addition, extracellular Bartonella resides in the gut of Melophagus. However, the phylogenetic origins of the two obligate mutualist symbionts differ. In Glossina, the mutualistic Wigglesworthia appears to be a relatively isolated symbiotic lineage, whereas in Melophagus, the obligate symbiont originated within the widely distributed Arsenophonus cluster. Although phylogenetically distant, the two obligate symbionts display several remarkably similar traits (e.g., transmission via the host's "milk glands" or similar pattern of genome reduction). To obtain better insight into the biology and possible role of the M. ovinus obligate symbiont, "Candidatus Arsenophonus melophagi," we performed several comparisons of its gene content based on assignments of the Cluster of Orthologous Genes (COG). Using this criterion, we show that within a set of 44 primary and secondary symbionts, "Ca. Arsenophonus melophagi" is most similar to Wigglesworthia. On the other hand, these two bacteria also display interesting differences, such as absence of flagellar genes in Arsenophonus and their presence in Wigglesworthia. This finding implies that a flagellum is not essential for bacterial transmission via milk glands.

Diversification of genes for carotenoid biosynthesis in aphids following an ancient transfer from a fungus.

The pea aphid genome was recently found to harbor genes for carotenoid biosynthesis, reflecting an ancestral transfer from a fungus. To explore the evolution of the carotene desaturase gene family within aphids, sequences were retrieved from a set of 34 aphid species representing numerous deeply diverging lineages of aphids and analyzed together with fungal sequences retrieved from databases. All aphids have at least one copy of this gene and some aphid species have up to seven, whereas fungal genomes consistently have a single copy. The closest relatives of aphids, adelgids, also have carotene desaturase; these sequences are most closely related to those from aphids, supporting a shared origin from a fungal to insect transfer predating the divergence of adelgids and aphids. Likewise, all aphids, and adelgids, have carotenoid profiles that are consistent with their biosynthesis using the acquired genes of fungal origin rather than derivation from food plants. The carotene desaturase was acquired from a fungal species outside of Ascomycota or Basidiomycota and closest to Mucoromycotina among sequences available in databases. In aphids, an ongoing pattern of gene duplication is indicated by the presence of both anciently and recently diverged paralogs within genomes and by the presence of a high frequency of pseudogenes that appear to be recently inactivated. Recombination among paralogs is evident, making analyses of patterns of selection difficult, but tests of selection for a nonrecombining region indicates that duplications tend to be followed by bouts of positive selection. Species of Macrosiphini, which often show color polymorphisms, typically have a larger number of desaturase copies relative to other species sampled in the study. These results indicate that aphid evolution has been accompanied by ongoing evolution of carotenogenic genes, which have undergone duplication, recombination, and occasional positive selection to yield a wide variety of carotenoid profiles in different aphid species.

Reconstructing the phylogeny of aphids (Hemiptera: Aphididae) using DNA of the obligate symbiont Buchnera aphidicola.

Reliable phylogenetic reconstruction, as a framework for evolutionary inference, may be difficult to achieve in some groups of organisms. Particularly for lineages that experienced rapid diversification, lack of sufficient information may lead to inconsistent and unstable results and a low degree of resolution. Coincidentally, such rapidly diversifying taxa are often among the biologically most interesting groups. Aphids provide such an example. Due to rapid adaptive diversification, they feature variability in many interesting biological traits, but consequently they are also a challenging group in which to resolve phylogeny. Particularly within the family Aphididae, many interesting evolutionary questions remain unanswered due to phylogenetic uncertainties. In this study, we show that molecular data derived from the symbiotic bacteria of the genus Buchnera can provide a more powerful tool than the aphid-derived sequences. We analyze 255 Buchnera gene sequences from 70 host aphid species and compare the resulting trees to the phylogenies previously retrieved from aphid sequences, only. We find that the host and symbiont data do not conflict for any major phylogenetic conclusions. Also, we demonstrate that the symbiont-derived phylogenies support some previously questionable relationships and provide new insights into aphid phylogeny and evolution.

Supergroup F Wolbachia with extremely reduced genome: transition to obligate insect symbionts.

BACKGROUND: Wolbachia belong to highly abundant bacteria which are frequently found in invertebrate microbiomes and manifest by a broad spectrum of lifestyles from parasitism to mutualism. Wolbachia supergroup F is a particularly interesting clade as it gave rise to symbionts of both arthropods and nematodes, and some of its members are obligate mutualists. Investigations on evolutionary transitions among the different symbiotic stages have been hampered by a lack of the known diversity and genomic data for the supergroup F members. RESULTS: Based on amplicon screening, short- and long-read WGS approaches, and laser confocal microscopy, we characterize five new supergroup F Wolbachia strains from four chewing lice species. These strains reached different evolutionary stages and represent two remarkably different types of symbiont genomes. Three of the genomes resemble other known members of Wolbachia F supergroup, while the other two show typical signs of ongoing gene inactivation and removal (genome size, coding density, low number of pseudogenes). Particularly, wMeur1, a symbiont fixed in microbiomes of Menacanthus eurysternus across four continents, possesses a highly reduced genome of 733,850 bp. The horizontally acquired capacity for pantothenate synthesis and localization in specialized bacteriocytes suggest its obligate nutritional role. CONCLUSIONS: The genome of wMeur1 strain, from the M. eurysternus microbiome, represents the smallest currently known Wolbachia genome and the first example of Wolbachia which has completed genomic streamlining as known from the typical obligate symbionts. This points out that despite the large amount and great diversity of the known Wolbachia strains, evolutionary potential of these bacteria still remains underexplored. The diversity of the four chewing lice microbiomes indicates that this vast parasitic group may provide suitable models for further investigations. Video Abstract.

Murine herpesvirus-68-related growth factors treatment correlates with decrease of p53 and HIF-1α protein levels.

Murine herpesvirus 68 (MHV-68) belongs to the subfamily Gammaherpesvirinae of the family Herpesviridae. This exceptional murine herpesvirus is an excellent model for the study of human gammaherpesvirus infections. Cells infected with MHV-68 under nonpermissive conditions for viral replication produce substances designated as MHV-68 growth factors (MHGF-68), that can cause transformation of the cells, or on the other side, turn transformed cells into normal. It was already proposed, that the MHGF-68 fractions cause transformation, disruption of the cytoskeleton and slower growth of the tumors in nude mice. Here, we examined newly extracted fractions of MHGF-68 designated as F5 and F8. Both fractions proved to inhibit the growth of the spheroids and also tumours induced in nude mice. What more, the fractions caused the decrease of the protein levels of wt p53 and HIF-1α. Decreased levels of p53 and HIF-1α activity leads to decreased vascularization, slower tumour growth, and lower adaptation to hypoxic conditions. This would propose MHGF-68 fractions, or their human herpesvirus equivalents, as a potential anticancer drugs in combined chemotherapy.

Arsenophonus symbiosis with louse flies: multiple origins, coevolutionary dynamics, and metabolic significance.

IMPORTANCE: Insects that live exclusively on vertebrate blood utilize symbiotic bacteria as a source of essential compounds, e.g., B vitamins. In louse flies, the most frequent symbiont originated in genus Arsenophonus, known from a wide range of insects. Here, we analyze genomic traits, phylogenetic origins, and metabolic capacities of 11 Arsenophonus strains associated with louse flies. We show that in louse flies, Arsenophonus established symbiosis in at least four independent events, reaching different stages of symbiogenesis. This allowed for comparative genomic analysis, including convergence of metabolic capacities. The significance of the results is twofold. First, based on a comparison of independently originated Arsenophonus symbioses, it determines the importance of individual B vitamins for the insect host. This expands our theoretical insight into insect-bacteria symbiosis. The second outcome is of methodological significance. We show that the comparative approach reveals artifacts that would be difficult to identify based on a single-genome analysis.

Division of labor within psyllids: metagenomics reveals an ancient dual endosymbiosis with metabolic complementarity in the genus Cacopsylla.

IMPORTANCE: Heritable beneficial bacterial endosymbionts have been crucial for the evolutionary success of numerous insects by enabling the exploitation of nutritionally limited food sources. Herein, we describe a previously unknown dual endosymbiosis in the psyllid genus Cacopsylla, consisting of the primary endosymbiont "Candidatus Carsonella ruddii" and a co-occurring Enterobacteriaceae bacterium for which we propose the name "Candidatus Psyllophila symbiotica." Its localization within the bacteriome and its small genome size confirm that Psyllophila is a co-primary endosymbiont widespread within the genus Cacopsylla. Despite its highly eroded genome, Psyllophila perfectly complements the tryptophan biosynthesis pathway that is incomplete in the co-occurring Carsonella. Moreover, the genome of Psyllophila is almost as small as Carsonella's, suggesting an ancient dual endosymbiosis that has now reached a precarious stage where any additional gene loss would make the system collapse. Hence, our results shed light on the dynamic interactions of psyllids and their endosymbionts over evolutionary time.

Functionalization of decavanadate anion by coordination to cobalt(II): Binding to proteins, cytotoxicity, and water oxidation catalysis.

A series of five decavanadates (V10) using a simple, one-pot synthesis, adhering to the model template: transition metal ion - decavanadate - ligands:(Hnicotinamide)2{[Co(H2O)3(nicotinamide)2]2[µ-V10O28]}.6H2O (1), {[Co(H2O)4(isonicotinamide)2]3}V10O28·4H2O (2), {[Co(H2O)4]2[Co(H2O)2(µ-pyrazinamide)2][µ-V10O28]}·4H2O (3) {[Co(H2O)4(µ-pyrazinamide)]3.V10O28}·4H2O (4), and (NH4)2{[Ni(H2O)4(2-hydroxyethylpyridine)]2}V10O28·2H2O (5) was synthesized. X-ray analysis reveals that 1 and 3 are decavanadato complexes, while 2, 4 and 5 are decavanadate complex salts. Moreover, 3 is the first example of a polymeric decavanadato complex, employing direct coordination with the metal center and the organic ligand, in toto. From the solution studies using 51V NMR spectroscopy, it was decoded that 1 and 3 stay stable in the model buffer solution and aqueous media. Binding to model proteins, cytotoxicity and water oxidation catalysis (WOC) was studied primarily for 1 and 3 and concluded that neither 1 nor 3 have an interaction with the model proteins thaumatin, lysozyme and proteinase K, because of the presence of the organic ligands in the Co(II) center, any further interplay with the proteins was blocked. Cytotoxicity studies reveal that 1 is 40% less toxic (0.05 mM) and 26% less toxic (0.1 mM) than the uncoordinated V10 with human cell lines A549 and HeLa respectively. In WOC, 1 performed superior activity, by evolving 143.37 nmol of O2 which is 700% (9-fold) increase than the uncoordinated V10.

Gypsum endolithic phototrophs under moderate climate (Southern Sicily): their diversity and pigment composition.

In this study, we used microscopic, spectroscopic, and molecular analysis to characterize endolithic colonization in gypsum (selenites and white crystalline gypsum) from several sites in Sicily. Our results showed that the dominant microorganisms in these environments are cyanobacteria, including: Chroococcidiopsis sp., Gloeocapsopsis pleurocapsoides, Gloeocapsa compacta, and Nostoc sp., as well as orange pigmented green microalgae from the Stephanospherinia clade. Single cell and filament sequencing coupled with 16S rRNA amplicon metagenomic profiling provided new insights into the phylogenetic and taxonomic diversity of the endolithic cyanobacteria. These organisms form differently pigmented zones within the gypsum. Our metagenomic profiling also showed differences in the taxonomic composition of endoliths in different gypsum varieties. Raman spectroscopy revealed that carotenoids were the most common pigments present in the samples. Other pigments such as gloeocapsin and scytonemin were also detected in the near-surface areas, suggesting that they play a significant role in the biology of endoliths in this environment. These pigments can be used as biomarkers for basic taxonomic identification, especially in case of cyanobacteria. The findings of this study provide new insights into the diversity and distribution of phototrophic microorganisms and their pigments in gypsum in Southern Sicily. Furthemore, this study highlights the complex nature of endolithic ecosystems and the effects of gypsum varieties on these communities, providing additional information on the general bioreceptivity of these environments.

Microbiomes of Blood-Feeding Triatomines in the Context of Their Predatory Relatives and the Environment.

The importance of gut microbiomes has become generally recognized in vector biology. This study addresses microbiome signatures in North American Triatoma species of public health significance (vectors of Trypanosoma cruzi) linked to their blood-feeding strategy and the natural habitat. To place the Triatoma-associated microbiomes within a complex evolutionary and ecological context, we sampled sympatric Triatoma populations, related predatory reduviids, unrelated ticks, and environmental material from vertebrate nests where these arthropods reside. Along with five Triatoma species, we have characterized microbiomes of five reduviids (Stenolemoides arizonensis, Ploiaria hirticornis, Zelus longipes, and two Reduvius species), a single soft tick species, Ornithodoros turicata, and environmental microbiomes from selected sites in Arizona, Texas, Florida, and Georgia. The microbiomes of predatory reduviids lack a shared core microbiota. As in triatomines, microbiome dissimilarities among species correlate with dominance of a single bacterial taxon. These include Rickettsia, Lactobacillus, "Candidatus Midichloria," and Zymobacter, which are often accompanied by known symbiotic genera, i.e., Wolbachia, "Candidatus Lariskella," Asaia, Gilliamella, and Burkholderia. We have further identified a compositional convergence of the analyzed microbiomes in regard to the host phylogenetic distance in both blood-feeding and predatory reduviids. While the microbiomes of the two reduviid species from the Emesinae family reflect their close relationship, the microbiomes of all Triatoma species repeatedly form a distinct monophyletic cluster highlighting their phylosymbiosis. Furthermore, based on environmental microbiome profiles and blood meal analysis, we propose three epidemiologically relevant and mutually interrelated bacterial sources for Triatoma microbiomes, i.e., host abiotic environment, host skin microbiome, and pathogens circulating in host blood. IMPORTANCE This study places microbiomes of blood-feeding North American Triatoma vectors (Reduviidae) into a broader evolutionary and ecological context provided by related predatory assassin bugs (Reduviidae), another unrelated vector species (soft tick Ornithodoros turicata), and the environment these arthropods coinhabit. For both vectors, microbiome analyses suggest three interrelated sources of bacteria, i.e., the microbiome of vertebrate nests as their natural habitat, the vertebrate skin microbiome, and the pathobiome circulating in vertebrate blood. Despite an apparent influx of environment-associated bacteria into the arthropod microbiomes, Triatoma microbiomes retain their specificity, forming a distinct cluster that significantly differs from both predatory relatives and ecologically comparable ticks. Similarly, within the related predatory Reduviidae, we found the host phylogenetic distance to underlie microbiome similarities.

Lightella neohaematopini: A new lineage of highly reduced endosymbionts coevolving with chipmunk lice of the genus Neohaematopinus.

Sucking lice (Anoplura) are known to have established symbiotic associations multiple times with different groups of bacteria as diverse as Enterobacteriales, Legionellales, and Neisseriales. This diversity, together with absence of a common coevolving symbiont (such as Buchnera, in aphids), indicates that sucking lice underwent a series of symbiont acquisitions, losses, and replacements. To better understand evolution and significance of louse symbionts, genomic and phylogenetic data are needed from a broader taxonomic diversity of lice and their symbiotic bacteria. In this study, we extend the known spectrum of the louse symbionts with a new lineage associated with Neohaematopinus pacificus, a louse species that commonly parasitizes North American chipmunks. The recent coevolutionary analysis showed that rather than a single species, these lice form a cluster of unique phylogenetic lineages specific to separate chipmunk species (or group of closely related species). Using metagenomic assemblies, we show that the lice harbor a bacterium which mirrors their phylogeny and displays traits typical for obligate mutualists. Phylogenetic analyses place this bacterium within Enterobacteriaceae on a long branch related to another louse symbiont, "Candidatus Puchtella pedicinophila." We propose for this symbiotic lineage the name "Candidatus Lightella neohaematopini." Based on the reconstruction of metabolic pathways, we suggest that like other louse symbionts, L. neohaematopini provides its host with at least some B vitamins. In addition, several samples harbored another symbiotic bacterium phylogenetically affiliated with the Neisseriales-related symbionts described previously from the lice Polyplax serrata and Hoplopleura acanthopus. Characterizing these bacteria further extend the known diversity of the symbiotic associations in lice and show unique complexity and dynamics of the system.

Development and validation of an LC-MS/MS method for determination of B vitamins and some its derivatives in whole blood.

Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, suggesting that levels of B vitamins may vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood can provide valuable informative background and understanding of general principles of insect symbiosis. In this work, a chromatographic separation of eight B vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folic acid, and cyanocobalamine), four B vitamin derivatives (niacinamide, pyridoxal-5-phosphate, 4-pyridoxic acid, and tetrahydrofolic acid), and 3 stable isotope labelled internal standards was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Except for vitamin B9 (tetrahydrofolic acid), the instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 µg/L, correlation coefficients from 0.9997 to 1.0000, and QC coefficients from 0.53 to 3.2%. Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. The best results were obtained for zinc sulphate in methanol, but only nine analytes were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120%, precision from 0.5 to 13%, and process efficiency from 65 to 108%. The content of B vitamins in whole blood samples from human and various vertebrates is presented as an application example of this newly developed method.

Fitness of mCherry Reporter Tick-Borne Encephalitis Virus in Tick Experimental Models.

The tick-borne encephalitis virus (TBEV) causes a most important viral life-threatening illness transmitted by ticks. The interactions between the virus and ticks are largely unexplored, indicating a lack of experimental tools and systematic studies. One such tool is recombinant reporter TBEV, offering antibody-free visualization to facilitate studies of transmission and interactions between a tick vector and a virus. In this paper, we utilized a recently developed recombinant TBEV expressing the reporter gene mCherry to study its fitness in various tick-derived in vitro cell cultures and live unfed nymphal Ixodes ricinus ticks. The reporter virus was successfully replicated in tick cell lines and live ticks as confirmed by the plaque assay and the mCherry-specific polymerase chain reaction (PCR). Although a strong mCherry signal determined by fluorescence microscopy was detected in several tick cell lines, the fluorescence of the reporter was not observed in the live ticks, corroborated also by immunoblotting. Our data indicate that the mCherry reporter TBEV might be an excellent tool for studying TBEV-tick interactions using a tick in vitro model. However, physiological attributes of a live tick, likely contributing to the inactivity of the reporter, warrant further development of reporter-tagged viruses to study TBEV in ticks in vivo.

Methodological Insight Into Mosquito Microbiome Studies.

Symbiotic bacteria affect competence for pathogen transmission in insect vectors, including mosquitoes. However, knowledge on mosquito-microbiome-pathogen interactions remains limited, largely due to methodological reasons. The current, cost-effective practice of sample pooling used in mosquito surveillance and epidemiology prevents correlation of individual traits (i.e., microbiome profile) and infection status. Moreover, many mosquito studies employ laboratory-reared colonies that do not necessarily reflect the natural microbiome composition and variation in wild populations. As a consequence, epidemiological and microbiome studies in mosquitoes are to some extent uncoupled, and the interactions among pathogens, microbiomes, and natural mosquito populations remain poorly understood. This study focuses on the effect the pooling practice poses on mosquito microbiome profiles, and tests different approaches to find an optimized low-cost methodology for extensive sampling while allowing for accurate, individual-level microbiome studies. We tested the effect of pooling by comparing wild-caught, individually processed mosquitoes with pooled samples. With individual mosquitoes, we also tested two methodological aspects that directly affect the cost and feasibility of broad-scale molecular studies: sample preservation and tissue dissection. Pooling affected both alpha- and beta-diversity measures of the microbiome, highlighting the importance of using individual samples when possible. Both RNA and DNA yields were higher when using inexpensive reagents such as NAP (nucleic acid preservation) buffer or absolute ethanol, without freezing for short-term storage. Microbiome alpha- and beta-diversity did not show overall significant differences between the tested treatments compared to the controls (freshly extracted samples or dissected guts). However, the use of standardized protocols is highly recommended to avoid methodological bias in the data.

Ontogeny, species identity, and environment dominate microbiome dynamics in wild populations of kissing bugs (Triatominae).

BACKGROUND: Kissing bugs (Triatominae) are blood-feeding insects best known as the vectors of Trypanosoma cruzi, the causative agent of Chagas' disease. Considering the high epidemiological relevance of these vectors, their biology and bacterial symbiosis remains surprisingly understudied. While previous investigations revealed generally low individual complexity but high among-individual variability of the triatomine microbiomes, any consistent microbiome determinants have not yet been identified across multiple Triatominae species. METHODS: To obtain a more comprehensive view of triatomine microbiomes, we investigated the host-microbiome relationship of five Triatoma species sampled from white-throated woodrat (Neotoma albigula) nests in multiple locations across the USA. We applied optimised 16S rRNA gene metabarcoding with a novel 18S rRNA gene blocking primer to a set of 170 T. cruzi-negative individuals across all six instars. RESULTS: Triatomine gut microbiome composition is strongly influenced by three principal factors: ontogeny, species identity, and the environment. The microbiomes are characterised by significant loss in bacterial diversity throughout ontogenetic development. First instars possess the highest bacterial diversity while adult microbiomes are routinely dominated by a single taxon. Primarily, the bacterial genus Dietzia dominates late-stage nymphs and adults of T. rubida, T. protracta, and T. lecticularia but is not present in the phylogenetically more distant T. gerstaeckeri and T. sanguisuga. Species-specific microbiome composition, particularly pronounced in early instars, is further modulated by locality-specific effects. In addition, pathogenic bacteria of the genus Bartonella, acquired from the vertebrate hosts, are an abundant component of Triatoma microbiomes. CONCLUSION: Our study is the first to demonstrate deterministic patterns in microbiome composition among all life stages and multiple Triatoma species. We hypothesise that triatomine microbiome assemblages are produced by species- and life stage-dependent uptake of environmental bacteria and multiple indirect transmission strategies that promote bacterial transfer between individuals. Altogether, our study highlights the complexity of Triatominae symbiosis with bacteria and warrant further investigation to understand microbiome function in these important vectors. Video abstract.