Wolbachia Infection through Hybridization to Enhance an Incompatible Insect Technique-Based Suppression of Aedes albopictus in Eastern Spain.

The emergence of insecticide resistance in arbovirus vectors is putting the focus on the development of new strategies for control. In this regard, the exploitation of Wolbachia endosymbionts is receiving increasing attention due to its demonstrated effectiveness in reducing the vectorial capacity of Aedes mosquitoes. Here, we describe the establishment of a naïve Wolbachia infection in a wild Aedes albopictus population of eastern Spain through a hybridization approach to obtain males capable of sterilizing wild females. The obtained lines were compared with the Wolbachia donor, Ae. albopictus ARwP, previously artificially infected with Wolbachia wPip, regarding immature and adult survival, female fecundity, egg fertility, and level of induced sterility. Our results did not show significant differences between lines in any of the biological parameters analyzed, indicating the full suitability of the hybrids to be used as a control tool against Ae. albopictus. In particular, hybrid males induced 99.9% sterility in the eggs of wild females without the need for any preliminary treatment. Being harmless to non-target organisms and the environment, the use of this bacterium for the control of Ae. albopictus deserves further exploration. This is especially relevant in areas such as eastern Spain, where this mosquito species has recently spread and may represent a serious threat due to its competence as a vector for dengue, chikungunya, and Zika viruses.

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.

[Wolbachia pipientis infections in populations of Aedes albopictus in the city of València (Spain): implications for mosquito control.] / Infecciones por Wolbachia pipientis en poblaciones de Aedes albopictus en la ciudad de València (España): implicaciones para el control de mosquitos.

OBJECTIVE: The presence of Aedes albopictus, of high sanitary and social impact, was first reported in Valencia (Eastern Spain) in 2015. Innovative tools for its control include the use of the endosymbiotic bacterium Wolbachia pipientis. The release of mosquito males infected with the wPip strain, has proven very promising for large-scale Incompatible Insect Technique (IIT) applications. Before this strategy can be implemented in Valencia, it is important to know whether the natural local mosquito populations are Wolbachia-infected and, if so, identifying the infecting strains/supergroups, these being the objectives of the present work. METHODS: Eggs were collected from the 19 districts of the València city between May and October 2019. A total of 50 lab-reared adult Ae. albopictus individuals were processed and analyzed for Wolbachia detection and molecular characterization. These actions took place within the framework of a collaboration established with the Department of Health and Consumer Affairs of the city council of Valencia. Fisher's exact test was used to detect the statistical significance of the differences between groups. RESULTS: Our study revealed that 94% of the analyzed samples were naturally infected with Wolbachia. Both wAlbA and wAlbB supergroups were identified, with most samples (72% of the infected ones) carrying co-infections. CONCLUSIONS: These data provide the first characterization of the Wolbachia presence in natural populations of Ae. albopictus in the Mediterranean area of Spain. This information is relevant to evaluate the potential use of Wolbachia strains in order to achieve the suppression of the Asian tiger mosquito populations through massive release of artificially-infected males.

OBJETIVO: La presencia de Aedes albopictus, de alto impacto sanitario y social, se informó por primera vez en Valencia en 2015. Las herramientas innovadoras para su control incluyen el uso de la bacteria endosimbiótica Wolbachia pipientis. La liberación de mosquitos machos infectados con la cepa wPip ha demostrado ser muy prometedora para aplicar la Técnica de Insectos Incompatibles (IIT) a gran escala. Antes de que esta estrategia pueda implementarse, es importante saber si las poblaciones locales de mosquitos silvestres están infectadas por Wolbachia y, de ser así, identificar las cepas/supergrupos infectantes, siendo estos los objetivos del presente trabajo. METODOS: Se recolectaron huevos de los diecinueve distritos de València entre mayo y octubre de 2019, y se mantuvieron en el laboratorio hasta llegar a adultos. Un total de cincuenta individuos adultos de Ae albopictus fueron procesados y analizados para detectar la presencia de Wolbachia y su caracterización molecular. Estas acciones se enmarcaron en la colaboración establecida con la Concejalía de Salud y Consumo del Ayuntamiento de València. La prueba exacta de Fisher fue utilizada para detectar la significación estadística de las diferencias entre grupos. RESULTADOS: El 94% de las muestras analizadas estaban infectadas de forma natural con Wolbachia. Se identificaron los supergrupos wAlbA y wAlbB, y la mayoría de las muestras (72% de las infectadas) presentaban coinfecciones. CONCLUSIONES: Los datos proporcionan la primera caracterización de la presencia de Wolbachia en poblaciones naturales de Ae. albopictus en el área mediterránea de España. Esta información es relevante para evaluar el potencial uso de cepas de Wolbachia de cara a la supresión de poblaciones de mosquito tigre asiático mediante la liberación masiva de machos infectados artificialmente.

Infecciones por Wolbachia pipientis en poblaciones de Aedes albopictus en la ciudad de València (España): implicaciones para el control de mosquitos / Wolbachia pipientis infections in populations of Aedes albopictus in the city of València (Spain): implications for mosquito control

Fundamentos: La presencia deAedes albopictus, de alto impacto sanitario y social, se informó por primera vez en Valenciaen 2015. Las herramientas innovadoras para su control incluyen el uso de la bacteria endosimbióticaWolbachia pipientis. La liberación de mosquitos machos infectados con la cepawPip ha demostrado ser muy prometedora para aplicar la Técnica de InsectosIncompatibles (IIT) a gran escala. Antes de que esta estrategia pueda implementarse, es importante saber si las poblaciones localesde mosquitos silvestres están infectadas porWolbachia y, de ser así, identificar las cepas/supergrupos infectantes, siendo estos losobjetivos del presente trabajo. Métodos: Se recolectaron huevos de los diecinueve distritos de València entre mayo y octubre de 2019, y se mantuvieron en ellaboratorio hasta llegar a adultos. Un total de cincuenta individuos adultos deAe. albopictus fueron procesados y analizados paradetectar la presencia deWolbachia y su caracterización molecular. Estas acciones se enmarcaron en la colaboración establecida conla Concejalía de Salud y Consumo del Ayuntamiento de València. La prueba exacta de Fisher fue utilizada para detectar la significaciónestadística de las diferencias entre grupos. Resultados: El 94% de las muestras analizadas estaban infectadas de forma natural conWolbachia. Se identificaron los supergruposwAlbA ywAlbB, y la mayoría de las muestras (72% de las infectadas) presentaban coinfecciones. Conclusiones: Los datos proporcionan la primera caracterización de la presencia deWolbachia en poblaciones naturales deAe. albopictus en el área mediterránea de España. Esta información es relevante para evaluar el potencial uso de cepas deWolbachiade cara a la supresión de poblaciones de mosquito tigre asiático mediante la liberación masiva de machos infectados artificialmente.(AU)

Background: The presence ofAedes albopictus, of high sanitary and social impact, was first reported in Valencia (Eastern Spain)in 2015. Innovative tools for its control include the use of the endosymbiotic bacteriumWolbachia pipientis. The release of mosquitomales infected with thewPip strain, has proven very promising for large-scale Incompatible Insect Technique (IIT) applications. Beforethis strategy can be implemented in Valencia, it is important to know whether the natural local mosquito populations areWolbachia-infected and, if so, identifying the infecting strains/supergroups, these being the objectives of the present work. Methods: Eggs were collected from the 19 districts of the València city between May and October 2019. A total of 50 lab-reared adultAe. albopictus individuals were processed and analyzed forWolbachia detection and molecular characterization. These actions tookplace within the framework of a collaboration established with the Department of Health and Consumer Affairs of the city council ofValencia. Fisher’s exact test was used to detect the statistical significance of the differences between groups. Results: Our study revealed that 94% of the analyzed samples were naturally infected withWolbachia. BothwAlbA andwAlbBsupergroups were identified, with most samples (72% of the infected ones) carrying co-infections. Conclusions: These data provide the first characterization of theWolbachia presence in natural populations ofAe. albopictusin the Mediterranean area of Spain. This information is relevant to evaluate the potential use ofWolbachia strains in order to achievethe suppression of the Asian tiger mosquito populations through massive release of artificially-infected males.(AU)

The Minimal Translation Machinery: What We Can Learn From Naturally and Experimentally Reduced Genomes.

The current theoretical proposals of minimal genomes have not attempted to outline the essential machinery for proper translation in cells. Here, we present a proposal of a minimal translation machinery based on (1) a comparative analysis of bacterial genomes of insects' endosymbionts using a machine learning classification algorithm, (2) the empiric genomic information obtained from Mycoplasma mycoides JCVI-syn3.0 the first minimal bacterial genome obtained by design and synthesis, and (3) a detailed functional analysis of the candidate genes based on essentiality according to the DEG database (Escherichia coli and Bacillus subtilis) and the literature. This proposed minimal translational machinery is composed by 142 genes which must be present in any synthetic prokaryotic cell designed for biotechnological purposes, 76.8% of which are shared with JCVI-syn3.0. Eight additional genes were manually included in the proposal for a proper and efficient translation.

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.

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.

DELEAT: gene essentiality prediction and deletion design for bacterial genome reduction.

BACKGROUND: The study of gene essentiality is fundamental to understand the basic principles of life, as well as for applications in many fields. In recent decades, dozens of sets of essential genes have been determined using different experimental and bioinformatics approaches, and this information has been useful for genome reduction of model organisms. Multiple in silico strategies have been developed to predict gene essentiality, but no optimal algorithm or set of gene features has been found yet, especially for non-model organisms with incomplete functional annotation. RESULTS: We have developed DELEAT v0.1 (DELetion design by Essentiality Analysis Tool), an easy-to-use bioinformatic tool which integrates an in silico gene essentiality classifier in a pipeline allowing automatic design of large-scale deletions in any bacterial genome. The essentiality classifier consists of a novel logistic regression model based on only six gene features which are not dependent on experimental data or functional annotation. As a proof of concept, we have applied this pipeline to the determination of dispensable regions in the genome of Bartonella quintana str. Toulouse. In this already reduced genome, 35 possible deletions have been delimited, spanning 29% of the genome. CONCLUSIONS: Built on in silico gene essentiality predictions, we have developed an analysis pipeline which assists researchers throughout multiple stages of bacterial genome reduction projects, and created a novel classifier which is simple, fast, and universally applicable to any bacterial organism with a GenBank annotation file.

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.

The Metabolic Building Blocks of a Minimal Cell.

Defining the essential gene components for a system to be considered alive is a crucial step toward the synthesis of artificial life. Fifteen years ago, Gil and coworkers proposed the core of a putative minimal bacterial genome, which would provide the capability to achieve metabolic homeostasis, reproduce, and evolve to a bacterium in an ideally controlled environment. They also proposed a simplified metabolic chart capable of providing energy and basic components for a minimal living cell. For this work, we have identified the components of the minimal metabolic network based on the aforementioned studies, associated them to the KEGG database and, by applying the MetaDAG methodology, determined its Metabolic Building Blocks (MBB) and reconstructed its metabolic Directed Acyclic Graph (m-DAG). The reaction graph of this metabolic network consists of 80 compounds and 98 reactions, while its m-DAG has 36 MBBs. Additionally, we identified 12 essential reactions in the m-DAG that are critical for maintaining the connectivity of this network. In a similar manner, we reconstructed the m-DAG of JCVI-syn3.0, which is an artificially designed and manufactured viable cell whose genome arose by minimizing the one from Mycoplasma mycoides JCVI-syn1.0, and of "Candidatus Nasuia deltocephalinicola", the bacteria with the smallest natural genome known to date. The comparison of the m-DAGs derived from a theoretical, an artificial, and a natural genome denote slightly different lifestyles, with a consistent core metabolism. The MetaDAG methodology we employ uses homogeneous descriptors and identifiers from the KEGG database, so that comparisons between bacterial strains are not only easy but also suitable for many research fields. The modeling of m-DAGs based on minimal metabolisms can be the first step for the synthesis and manipulation of minimal cells.

Clinical Predictors of Hyperperfusion Syndrome Following Carotid Stenting: Results From a National Prospective Multicenter Study.

OBJECTIVES: The aim of the HISPANIAS (HyperperfusIon Syndrome Post-carotid ANgIoplasty And Stenting) study was to define CHS rates and develop a clinical predictive model for cerebral hyperperfusion syndrome (CHS) after carotid artery stenting (CAS). BACKGROUND: CHS is a severe complication following CAS. The presence of clinical manifestations is estimated on the basis of retrospective reviews and is still uncertain. METHODS: The HISPANIAS study was a national prospective multicenter study with 14 recruiting hospitals. CHS was classified as mild (headache only) and moderate-severe (seizure, impaired level of consciousness, or development of focal neurological signs). RESULTS: A total of 757 CAS procedures were performed. CHS occurred in 22 (2.9%) patients, in which 16 (2.1%) had moderate-severe CHS and 6 (0.8%) had mild CHS (only headache). The rate of hemorrhages was 0.7% and was associated with high mortality (20%). Pre-operative predictors of moderate-severe CHS in multivariate analysis were female sex (odds ratio [OR]: 3.24; 95% confidence interval [CI]: 1.11 to 9.47; p = 0.03), older patients (OR: 1.09; 95% CI: 1.01 to 1.17; p = 0.02), left carotid artery treated (OR: 4.13; 95% CI: 1.11 to 15.40; p = 0.03), and chronic renal failure (OR: 6.29; 95% CI: 1.75 to 22.57; p = 0.005). The area under the curve of this clinical and radiological model was 0.86 (95% CI: 0.81 to 0.92; p = 0.001). CONCLUSIONS: The rate of CHS in the HISPANIAS study was 2.9%, with moderate-severe CHS of 2.1%. CHS was independently associated with female sex, older age, history of chronic kidney disease, and a treated left carotid artery. Although further investigations are needed, the authors propose a model to identify high-risk patients and develop strategies to decrease CHS morbidity and mortality in the future.

Unity Makes Strength: A Review on Mutualistic Symbiosis in Representative Insect Clades.

Settled on the foundations laid by zoologists and embryologists more than a century ago, the study of symbiosis between prokaryotes and eukaryotes is an expanding field. In this review, we present several models of insect⁻bacteria symbioses that allow for the detangling of most known features of this distinctive way of living, using a combination of very diverse screening approaches, including molecular, microscopic, and genomic techniques. With the increasing the amount of endosymbiotic bacteria genomes available, it has been possible to develop evolutionary models explaining the changes undergone by these bacteria in their adaptation to the intracellular host environment. The establishment of a given symbiotic system can be a root cause of substantial changes in the partners' way of life. Furthermore, symbiont replacement and/or the establishment of bacterial consortia are two ways in which the host can exploit its interaction with environmental bacteria for endosymbiotic reinvigoration. The detailed study of diverse and complex symbiotic systems has revealed a great variety of possible final genomic products, frequently below the limit considered compatible with cellular life, and sometimes with unanticipated genomic and population characteristics, raising new questions that need to be addressed in the near future through a wider exploration of new models and empirical observations.

Natural Occurrence of Secondary Bacterial Symbionts in Aphids from Tunisia, with a Focus on Genus Hyalopterus.

Aphids (Hemiptera: Aphididae) can harbor two types of bacterial symbionts. In addition to the obligate endosymbiont Buchnera aphidicola Munson, Baumann and Kinsey 1991 (Enterobacteriales: Enterobacteriaceae), several facultative symbiotic bacteria, called secondary (S) symbionts, have been identified among many important pest aphid species. To determine interpopulational diversity of S-symbionts, we carried out a survey in a total of 18 populations of six aphid species collected from six localities in Tunisia, by performing a diagnostic polymerase chain reaction analysis of partial 16S-23S rRNA operon sequences. While 61.7% of individuals contained only Buchnera, three S-symbionts were found at different frequencies. Arsenophonus sp. Gherna et al. 1991 (Enterobacteriales: Enterobacteriaceae) was found in all species under study except for Acyrtosiphon pisum (Harris 1776) (Aphidinae: Macrosiphini); Serratia symbiotica Moran et al. 2005 (Enterobacteriales: Enterobacteriaceae) was present in all analyzed individuals of A. pisum but only sporadically in Aphis spiraecola (Patch 1914) (Aphidinae: Aphidini) and Hyalopterus amygdali (Blanchard 1840) (Aphidinae: Aphidini), while Hamiltonella defensa Moran et al. 2005 (Enterobacteriales: Enterobacteriaceae) was found in all analyzed individuals of one population of Aphis gossypii (Glover 1877) (Aphidinae: Aphidini) and sporadically in two populations of Hyalopterus. The lysogenic bacteriophage APSE-1 (A. pisum secondary endosymbiont, type 1) was detected in the three populations infected with H. defensa. This bacteriophage has been associated with moderate protection against braconid parasitoids in pea aphids. The high prevalence of Arsenophonus sp. in our samples is in accordance with previous studies indicating that, among gammaproteobacteria, this genus is one of the most widespread insect facultative symbionts.

Tremblaya phenacola PPER: an evolutionary beta-gammaproteobacterium collage.

Many insects rely on bacterial endosymbionts to obtain nutrients that are scarce in their highly specialized diets. The most surprising example corresponds to the endosymbiotic system found in mealybugs from subfamily Pseudococcinae in which two bacteria, the betaproteobacterium 'Candidatus Tremblaya princeps' and a gammaproteobacterium, maintain a nested endosymbiotic consortium. In the sister subfamily Phenacoccinae, however, a single beta-endosymbiont, 'Candidatus Tremblaya phenacola', has been described. In a previous study, we detected a trpB gene of gammaproteobacterial origin in 'Ca. Tremblaya phenacola' from two Phenacoccus species, apparently indicating an unusual case of horizontal gene transfer (HGT) in a bacterial endosymbiont. What we found by sequencing the genome of 'Ca. Tremblaya phenacola' PPER, single endosymbiont of Phenacoccus peruvianus, goes beyond a HGT phenomenon. It rather represents a genome fusion between a beta and a gammaproteobacterium, followed by massive rearrangements and loss of redundant genes, leading to an unprecedented evolutionary collage. Mediated by the presence of several repeated sequences, there are many possible genome arrangements, and different subgenomic sequences might coexist within the same population.

Et tu, Brute? Not Even Intracellular Mutualistic Symbionts Escape Horizontal Gene Transfer.

Many insect species maintain mutualistic relationships with endosymbiotic bacteria. In contrast to their free-living relatives, horizontal gene transfer (HGT) has traditionally been considered rare in long-term endosymbionts. Nevertheless, meta-omics exploration of certain symbiotic models has unveiled an increasing number of bacteria-bacteria and bacteria-host genetic transfers. The abundance and function of transferred loci suggest that HGT might play a major role in the evolution of the corresponding consortia, enhancing their adaptive value or buffering detrimental effects derived from the reductive evolution of endosymbionts' genomes. Here, we comprehensively review the HGT cases recorded to date in insect-bacteria mutualistic consortia, and discuss their impact on the evolutionary success of these associations.

Seasonal Changes in the Endosymbiotic Consortia of Aphids from the Genus Cinara.

Buchnera aphidicola is the primary endosymbiont of aphids with which it maintains an obligate mutualistic symbiotic relationship. Insects also maintain facultative symbiotic relationships with secondary symbionts, and Serratia symbiotica is the most common in aphids. The presence of both symbionts in aphids of the subfamily Lachninae has been widely studied by our group. We examined two closely related aphids, Cinara tujafilina and C. cedri in the present study. Even though both B. aphidicola strains have similar genome sizes and gene contents, the genomes of the two S. symbiotica strains were markedly different. The SCc strain has the smallest genome known for this species, while SCt possesses a larger genome in an intermediate stage between the facultative S. symbiotica of Acyrthosiphon pisum (SAp) and the co-obligate S. symbiotica SCc.Aphids are vulnerable to high temperatures. Previous studies indicated that S. symbiotica SAp confers resistance to heat-shock stress. In order to clarify whether S. symbiotica strains from genus Cinara also play a role in heat stress protection, we performed a quantitative determination of the consortium Buchnera/Serratia from two geographically close populations, each of which belonged to the Cinara species examined, over two years in natural environments. We found no variation in the consortium from our C. cedri population, but a positive correlation between both endosymbiont densities and average daily temperatures in the C. tujafilina population. Even though S. symbiotica SCt may retain some protective role against heat stress, this does not appear to be due to the release of protective metabolites by cell lysis.

Bacterial antisense RNAs are mainly the product of transcriptional noise.

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.