Comparative Transcriptomics of Fat Bodies between Symbiotic and Quasi-Aposymbiotic Adult Females of Blattella germanica with Emphasis on the Metabolic Integration with Its Endosymbiont Blattabacterium and Its Immune System.

We explored the metabolic integration of Blattella germanica and its obligate endosymbiont Blattabacterium cuenoti by the transcriptomic analysis of the fat body of quasi-aposymbiotic cockroaches, where the endosymbionts were almost entirely removed with rifampicin. Fat bodies from quasi-aposymbiotic insects displayed large differences in gene expression compared to controls. In quasi-aposymbionts, the metabolism of phenylalanine and tyrosine involved in cuticle sclerotization and pigmentation increased drastically to compensate for the deficiency in the biosynthesis of these amino acids by the endosymbionts. On the other hand, the uricolytic pathway and the biosynthesis of uric acid were severely decreased, probably because the reduced population of endosymbionts was unable to metabolize urea to ammonia. Metabolite transporters that could be involved in the endosymbiosis process were identified. Immune system and antimicrobial peptide (AMP) gene expression was also reduced in quasi-aposymbionts, genes encoding peptidoglycan-recognition proteins, which may provide clues for the maintenance of the symbiotic relationship, as well as three AMP genes whose involvement in the symbiotic relationship will require additional analysis. Finally, a search for AMP-like factors that could be involved in controlling the endosymbiont identified two orphan genes encoding proteins smaller than 200 amino acids underexpressed in quasi-aposymbionts, suggesting a role in the host-endosymbiont relationship.

Gut Microbiota Is Not Essential for Survival and Development in Blattella germanica, but Affects Uric Acid Storage.

Cockroaches harbor two coexisting symbiotic systems: the obligate endosymbiont Blattabacterium cuenotii, and a complex gut microbiota. Blattabacterium is the only bacterium present in the eggs, as the gut microbiota is acquired by horizontal transmission after hatching, mostly through coprophagy. Blattella germanica, a cosmopolitan omnivorous cockroach living in intimate association with humans, is an appropriate model system for studying whether the gut microbiota is essential for the cockroach's survival, development, or welfare. We obtained a germ-free cockroach population (i.e., containing normal amounts of the endosymbiont, but free of microbes on the insects' surface and digestive tract). Non-significant differences with the controls were detected in most fitness parameters analyzed, except for a slight shortening in the hatching time of the second generation and a reduction in female weight at 10 days after adult ecdysis. The latter is accompanied by a decrease in uric acid reserves. This starvation-like phenotype of germ-free B. germanica suggests that the microbiota is not essential in this species for survival and development throughout its complete life cycle, but it could participate in complementation of host nutrition by helping with food digestion and nutrient absorption.

Exploring Gut Microbial Dynamics and Symbiotic Interaction in Blattella germanica Using Rifampicin.

Blattella germanica harbours two cohabiting symbiotic systems: an obligate endosymbiont, Blattabacterium, located inside bacteriocytes and vertically transmitted, which is key in nitrogen metabolism, and abundant and complex gut microbiota acquired horizontally (mainly by coprophagy) that must play an important role in host physiology. In this work, we use rifampicin treatment to deepen the knowledge on the relationship between the host and the two systems. First, we analysed changes in microbiota composition in response to the presence and removal of the antibiotic with and without faeces in one generation. We found that, independently of faeces supply, rifampicin-sensitive bacteria are strongly affected at four days of treatment, and most taxa recover after treatment, although some did not reach control levels. Second, we tried to generate an aposymbiotic population, but individuals that reached the second generation were severely affected and no third generation was possible. Finally, we established a mixed population with quasi-aposymbiotic and control nymphs sharing an environment in a blind experiment. The analysis of the two symbiotic systems in each individual after reaching the adult stage revealed that endosymbiont's load does not affect the composition of the hindgut microbiota, suggesting that there is no interaction between the two symbiotic systems in Blattella germanica.

Human follicular mites: Ectoparasites becoming symbionts.

Most humans carry mites in the hair follicles of their skin for their entire lives. Follicular mites are the only metazoans tha continuously live on humans. We propose that Demodex folliculorum (Acari) represents a transitional stage from a host-injuring obligate parasite to an obligate symbiont. Here, we describe the profound impact of this transition on the genome and physiology of the mite. Genome sequencing revealed that the permanent host association of D. folliculorum led to an extensive genome reduction through relaxed selection and genetic drift, resulting in the smallest number of protein-coding genes yet identified among panarthropods. Confocal microscopy revealed that this gene loss coincided with an extreme reduction in the number of cells. Single uninucleate muscle cells are sufficient to operate each of the three segments that form each walking leg. While it has been assumed that the reduction of the cell number in parasites starts early in development, we identified a greater total number of cells in the last developmental stage (nymph) than in the terminal adult stage, suggesting that reduction starts at the adult or ultimate stage of development. This is the first evolutionary step in an arthropod species adopting a reductive, parasitic or endosymbiotic lifestyle. Somatic nuclei show underreplication at the diploid stage. Novel eye structures or photoreceptors as well as a unique human host melatonin-guided day/night rhythm are proposed for the first time. The loss of DNA repair genes coupled with extreme endogamy might have set this mite species on an evolutionary dead-end trajectory.

Of Cockroaches and Symbionts: Recent Advances in the Characterization of the Relationship between Blattella germanica and Its Dual Symbiotic System.

Mutualistic stable symbioses are widespread in all groups of eukaryotes, especially in insects, where symbionts have played an essential role in their evolution. Many insects live in obligate relationship with different ecto- and endosymbiotic bacteria, which are needed to maintain their hosts' fitness in their natural environment, to the point of even relying on them for survival. The case of cockroaches (Blattodea) is paradigmatic, as both symbiotic systems coexist in the same organism in two separated compartments: an intracellular endosymbiont (Blattabacterium) inside bacteriocytes located in the fat body, and a rich and complex microbiota in the hindgut. The German cockroach Blattella germanica is a good model for the study of symbiotic interactions, as it can be maintained in the laboratory in controlled populations, allowing the perturbations of the two symbiotic systems in order to study the communication and integration of the tripartite organization of the host-endosymbiont-microbiota, and to evaluate the role of symbiotic antimicrobial peptides (AMPs) in host control over their symbionts. The importance of cockroaches as reservoirs and transmission vectors of antibiotic resistance sequences, and their putative interest to search for AMPs to deal with the problem, is also discussed.

The transposable element-rich genome of the cereal pest Sitophilus oryzae.

BACKGROUND: The rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions. RESULTS: We sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino acids required for insect development and cuticle biosynthesis. CONCLUSIONS: Here we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes.

Gut Microbiota Cannot Compensate the Impact of (quasi) Aposymbiosis in Blattella germanica.

Blattella germanica presents a very complex symbiotic system, involving the following two kinds of symbionts: the endosymbiont Blattabacterium and the gut microbiota. Although the role of the endosymbiont has been fully elucidated, the function of the gut microbiota remains unclear. The study of the gut microbiota will benefit from the availability of insects deprived of Blattabacterium. Our goal is to determine the effect of the removal (or, at least, the reduction) of the endosymbiont population on the cockroach's fitness, in a normal gut microbiota community. For this purpose, we treated our cockroach population, over several generations, with rifampicin, an antibiotic that only affects the endosymbiont during its extracellular phase, and decreases its amount in the following generation. As rifampicin also affects gut bacteria that are sensitive to this antibiotic, the treatment was performed during the first 12 days of the adult stage, which is the period when the endosymbiont infects the oocytes and lacks bacteriocyte protection. We found that after this antibiotic treatment, the endosymbiont population remained extremely reduced and only the microbiota was able to recover, although it could not compensate for the endosymbiont role, and the host's fitness was drastically affected. This accomplished reduction, however, is not homogenous and requires further study to develop stable quasi-aposymbiotic cockroaches.

Interkingdom Gut Microbiome and Resistome of the Cockroach Blattella germanica.

Cockroaches are intriguing animals with two coexisting symbiotic systems, an endosymbiont in the fat body, involved in nitrogen metabolism, and a gut microbiome whose diversity, complexity, role, and developmental dynamics have not been fully elucidated. In this work, we present a metagenomic approach to study Blattella germanica populations not treated, treated with kanamycin, and recovered after treatment, both naturally and by adding feces to the diet, with the aim of better understanding the structure and function of its gut microbiome along the development as well as the characterization of its resistome.IMPORTANCE For the first time, we analyze the interkingdom hindgut microbiome of this species, including bacteria, fungi, archaea, and viruses. Network analysis reveals putative cooperation between core bacteria that could be key for ecosystem equilibrium. We also show how antibiotic treatments alter microbiota diversity and function, while both features are restored after one untreated generation. Combining data from B. germanica treated with three antibiotics, we have characterized this species' resistome. It includes genes involved in resistance to several broad-spectrum antibiotics frequently used in the clinic. The presence of genetic elements involved in DNA mobilization indicates that they can be transferred among microbiota partners. Therefore, cockroaches can be considered reservoirs of antibiotic resistance genes (ARGs) and potential transmission vectors.

Metagenomic analysis of formalin-fixed paraffin-embedded tumor and normal mucosa reveals differences in the microbiome of colorectal cancer patients.

An increased risk of developing colorectal cancer (CRC) and other types of tumor is associated to Lynch syndrome (LS), an inherited condition caused by germline mutations in mismatch repair genes. We selected a cohort of LS patients that had developed CRC and had undergone surgical resection. Formalin-fixed paraffin embedded (FFPE) tissue blocks from matched colorectal and normal mucosa were used for genomic DNA extraction with a commercial kit and sequenced by high-throughput sequencing. A metagenomic approach enabled the taxonomic and functional identification of the microbial community and associated genes detected in the specimens. Slightly lower taxonomic diversity was observed in the tumor compared to the non-tumor tissue. Furthermore, the most remarkable differences between tumors and healthy tissue was the significant increase in the genus Fusobacterium in the former, in particular the species F. nucleatum, as well as Camplylobacter or Bacteroides fragilis, in accordance with previous studies of CRC. However, unlike prior studies, the present work is not based on directed detection by qPCR but instead uses a metagenomic approach to retrieve the whole bacterial community, and addresses the additional difficulty of using long-term stored FFPE samples.

Insects' potential: Understanding the functional role of their gut microbiome.

The study of insect-associated microbial communities is a field of great importance in agriculture, principally because of the role insects play as pests. In addition, there is a recent focus on the potential of the insect gut microbiome in areas such as biotechnology, given some microorganisms produce molecules with biotechnological and industrial applications, and also in biomedicine, since some bacteria and fungi are a reservoir of antibiotic resistance genes (ARGs). To date, most studies aiming to characterize the role of the gut microbiome of insects have been based on high-throughput sequencing of the 16S rRNA gene and/or metagenomics. However, recently functional approaches such as metatranscriptomics, metaproteomics and metabolomics have also been employed. Besides providing knowledge about the taxonomic distribution of microbial populations, these techniques also reveal their functional and metabolic capabilities. This information is essential to gain a better understanding of the role played by microbes comprising the microbial communities in their hosts, as well as to indicate their possible exploitation. This review provides an overview of how far we have come in characterizing insect gut functionality through omics, as well as the challenges and future perspectives in this field.

Blattella germanica displays a large arsenal of antimicrobial peptide genes.

Defence systems against microbial pathogens are present in most living beings. The German cockroach Blattella germanica requires these systems to adapt to unhealthy environments with abundance of pathogenic microbes, in addition to potentially control its symbiotic systems. To handle this situation, four antimicrobial gene families (defensins, termicins, drosomycins and attacins) were expanded in its genome. Remarkably, a new gene family (blattellicins) emerged recently after duplication and fast evolution of an attacin gene, which is now encoding larger proteins with the presence of a long stretch of glutamines and glutamic acids. Phylogenetic reconstruction, within Blattellinae, suggests that this duplication took place before the divergence of Blattella and Episymploce genera. The latter harbours a long attacin gene (pre-blattellicin), but the absence of the encoded Glx-region suggests that this element evolved recently in the Blattella lineage. A screening of AMP gene expression in available transcriptomic SR projects of B. germanica showed that, while some AMPs are expressed during almost the whole development, others are restricted to shorter periods. Blattellicins are highly expressed only in adult females. None of the available SR tissue projects could be associated with blattellicins' expression, suggesting that it takes place in other tissues, maybe the gut.

Driven progressive evolution of genome sequence complexity in Cyanobacteria.

Progressive evolution, or the tendency towards increasing complexity, is a controversial issue in biology, which resolution entails a proper measurement of complexity. Genomes are the best entities to address this challenge, as they encode the historical information of a species' biotic and environmental interactions. As a case study, we have measured genome sequence complexity in the ancient phylum Cyanobacteria. To arrive at an appropriate measure of genome sequence complexity, we have chosen metrics that do not decipher biological functionality but that show strong phylogenetic signal. Using a ridge regression of those metrics against root-to-tip distance, we detected positive trends towards higher complexity in three of them. Lastly, we applied three standard tests to detect if progressive evolution is passive or driven-the minimum, ancestor-descendant, and sub-clade tests. These results provide evidence for driven progressive evolution at the genome-level in the phylum Cyanobacteria.

Unraveling Assemblage, Functions and Stability of the Gut Microbiota of Blattella germanica by Antibiotic Treatment.

Symbiosis between prokaryotes and eukaryotes is a widespread phenomenon that has contributed to the evolution of eukaryotes. In cockroaches, two types of symbionts coexist: an endosymbiont in the fat body (Blattabacterium), and a rich gut microbiota. The transmission mode of Blattabacterium is vertical, while the gut microbiota of a new generation is mainly formed by bacterial species present in feces. We have carried out a metagenomic analysis of Blattella germanica populations, treated and non-treated with two antibiotics (vancomycin and ampicillin) over two generations to (1) determine the core of bacterial communities and potential functions of the gut microbiota and (2) to gain insights into the mechanisms of resistance and resilience of the gut microbiota. Our results indicate that the composition and functions of the bacteria were affected by treatment, more severely in the case of vancomycin. Further results demonstrated that in an untreated second-generation population that comes from antibiotic-treated first-generation, the microbiota is not yet stabilized at nymphal stages but can fully recover in adults when feces of a control population were added to the diet. This signifies the existence of a stable core in either composition and functions in lab-reared populations. The high microbiota diversity as well as the observed functional redundancy point toward the microbiota of cockroach hindguts as a robust ecosystem that can recover from perturbations, with recovery being faster when feces are added to the diet.

The Bacterial Microbiome of Meloidogyne-Based Disease Complex in Coffee and Tomato.

The Meloidogyne-based disease complexes (MDCs) are caused by the interaction of different root-knot nematode species and phytopathogenic fungi. These complexes are devastating several important crops worldwide including tomato and coffee. Despite their relevance, little is known about the role of the bacterial communities in the MDCs. In this study 16s rDNA gene sequencing was used to analyze the bacterial microbiome associated with healthy and infested roots, as well with females and eggs of Meloidogyne enterolobii and M. paranaensis, the causal agents of MDC in tomato and coffee, respectively. Each MDC pathosystems displayed a specific taxonomic diversity and relative abundances constituting a very complex system. The main bacterial drivers of the MDC infection process were identified for both crops at order level. While corky-root coffee samples presented an enrichment of Bacillales and Burkholderiales, the corcky-root tomato samples presented an enrichment on Saprospirales, Chthoniobacterales, Alteromonadales, and Xanthomonadales. At genus level, Nocardia was common to both systems, and it could be related to the development of tumor symptoms by altering both nematode and plant systems. Furthermore, we predicted the healthy metabolic profile of the roots microbiome and a shift that may result in an increment of activity of central metabolism and the presence of pathogenic genes in both crops.

An update on the Symbiotic Genomes Database (SymGenDB): a collection of metadata, genomic, genetic and protein sequences, orthologs and metabolic networks of symbiotic organisms.

The Symbiotic Genomes Database (SymGenDB; http://symbiogenomesdb.uv.es/) is a public resource of manually curated associations between organisms involved in symbiotic relationships, maintaining a catalog of completely sequenced/finished bacterial genomes exclusively. It originally consisted of three modules where users could search for the bacteria involved in a specific symbiotic relationship, their genomes and their genes (including their orthologs). In this update, we present an additional module that includes a representation of the metabolic network of each organism included in the database, as Directed Acyclic Graphs (MetaDAGs). This module provides unique opportunities to explore the metabolism of each individual organism and/or to evaluate the shared and joint metabolic capabilities of the organisms of the same genera included in our listing, allowing users to construct predictive analyses of metabolic associations and complementation between systems. We also report a ~25% increase in manually curated content in the database, i.e. bacterial genomes and their associations, with a final count of 2328 bacterial genomes associated to 498 hosts. We describe new querying possibilities for all the modules, as well as new display features for the MetaDAGs module, providing a relevant range of content and utility. This update continues to improve SymGenDB and can help elucidate the mechanisms by which organisms depend on each other.

gNOMO: a multi-omics pipeline for integrated host and microbiome analysis of non-model organisms.

The study of bacterial symbioses has grown exponentially in the recent past. However, existing bioinformatic workflows of microbiome data analysis do commonly not integrate multiple meta-omics levels and are mainly geared toward human microbiomes. Microbiota are better understood when analyzed in their biological context; that is together with their host or environment. Nevertheless, this is a limitation when studying non-model organisms mainly due to the lack of well-annotated sequence references. Here, we present gNOMO, a bioinformatic pipeline that is specifically designed to process and analyze non-model organism samples of up to three meta-omics levels: metagenomics, metatranscriptomics and metaproteomics in an integrative manner. The pipeline has been developed using the workflow management framework Snakemake in order to obtain an automated and reproducible pipeline. Using experimental datasets of the German cockroach Blattella germanica, a non-model organism with very complex gut microbiome, we show the capabilities of gNOMO with regard to meta-omics data integration, expression ratio comparison, taxonomic and functional analysis as well as intuitive output visualization. In conclusion, gNOMO is a bioinformatic pipeline that can easily be configured, for integrating and analyzing multiple meta-omics data types and for producing output visualizations, specifically designed for integrating paired-end sequencing data with mass spectrometry from non-model organisms.

Erratum: gNOMO: a multi-omics pipeline for integrated host and microbiome analysis of non-model organisms.

[This corrects the article DOI: 10.1093/nargab/lqaa058.].

Inferring Horizontal Gene Transfer with DarkHorse, Phylomizer, and ETE Toolkits.

In this chapter, we describe how to use DarkHorse2.0 to search for xenologs in the genome of the cyanobacterium Synechococcus elongatus PCC 7942. DarkHorse is an implicit phylogenetic method that uses BLAST searches to identify proteins having close homologs of unexpected taxonomic affiliation. Once a set of putative xenologs are identified, Phylomizer is used to reconstruct phylogenetic trees. Phylomizer reproduces all the necessary steps to perform a basic phylogenetic analysis. The combined use of DarkHorse and Phylomizer allows the identification of genes incorporated into a given genome by HGT.

The Gut Microbiota Composition of the Moth Brithys crini Reflects Insect Metamorphosis.

Lepidoptera is a highly diverse insect order with major importance in agriculture as many species are considered pests. The role of the gut microbiota in insect physiology is still poorly understood, despite the research undertaken in recent years. Furthermore, Lepidoptera are holometabolous insects and few studies have addressed the influence of the changes taking place on the gut microbiome composition and diversity during metamorphosis, especially in monophagous species. The V3-V4 region of the 16S rRNA gene was sequenced to investigate the microbiota composition and diversity of the monophagous moth Brithys crini during three different life stages: egg, larvae (midgut and hindgut), and adult (gut). Our results showed that the microbiota composition of B. crini was stage specific, indicating that the developmental stage is a main factor affecting the gut microbiome in composition and potential functions. Moreover, the diversity of the gut microbiome reflected the developmental process, since a drop in diversity occurred between the larval and the adult phase, when the intestine is completely renewed. In spite of the changes in the gut microbiota during metamorphosis, 29 genera were conserved throughout the three developmental stages, mainly belonging to the Proteobacteria phylum, which define the core microbiome of B. crini. These genera seem to contribute to host physiology by participating in food digestion, nutrition, and detoxification mechanisms.