Alejandro Manzano-Marín

Serratia symbiotica from the Aphid Cinara cedri: A Missing Link from Facultative to Obligate Insect Endosymbiont

The genome sequencing of Buchnera aphidicola BCc from the aphid Cinara cedri, which is the smallest known Buchnera genome, revealed that this bacterium had lost its symbiotic role, as it was not able to synthesize tryptophan and riboflavin. Moreover, the biosynthesis of tryptophan is shared with the endosymbiont Serratia symbiotica SCc, which coexists with B. aphidicola in this aphid. The whole-genome sequencing of S. symbiotica SCc reveals an endosymbiont in a stage of genome reduction that is closer to an obligate endosymbiont, such as B. aphidicola from Acyrthosiphon pisum, than to another S. symbiotica, which is a facultative endosymbiont in this aphid, and presents much less gene decay. The comparison between both S. symbiotica enables us to propose an evolutionary scenario of the transition from facultative to obligate endosymbiont. Metabolic inferences of B. aphidicola BCc and S. symbiotica SCc reveal that most of the functions carried out by B. aphidicola in A. pisum are now either conserved in B. aphidicola BCc or taken over by S. symbiotica. In addition, there are several cases of metabolic complementation giving functional stability to the whole consortium and evolutionary preservation of the actors involved.

Settling down: the genome of Serratia symbiotica from the aphid Cinara tujafilina zooms in on the process of accommodation to a cooperative intracellular life.

Particularly interesting cases of mutualistic endosymbioses come from the establishment of co-obligate associations of more than one species of endosymbiotic bacteria. Throughout symbiotic accommodation from a free-living bacterium, passing through a facultative stage and ending as an obligate intracellular one, the symbiont experiences massive genomic losses and phenotypic adjustments. Here, we scrutinized the changes in the coevolution of Serratia symbiotica and Buchnera aphidicola endosymbionts in aphids, paying particular attention to the transformations undergone by S. symbiotica to become an obligate endosymbiont. Although it is already known that S. symbiotica is facultative in Acyrthosiphon pisum, in Cinara cedri it has established a co-obligate endosymbiotic consortium along with B. aphidicola to fulfill the aphid’s nutritional requirements. The state of this association in C. tujafilina, an aphid belonging to the same subfamily (Lachninae) that C. cedri, remained unknown. Here, we report the genome of S. symbiotica strain SCt-VLC from the aphid C. tujafilina. While being phylogenetically and genomically very closely related to the facultative endosymbiont S. symbiotica from the aphid A. pisum, it shows a variety of metabolic, genetic, and architectural features, which point toward this endosymbiont being one step closer to an obligate intracellular one. We also describe in depth the process of genome rearrangements suffered by S. symbiotica and the role mobile elements play in gene inactivations. Finally, we postulate the supply to the host of the essential riboflavin (vitamin B2) as key to the establishment of S. symbiotica as a co-obligate endosymbiont in the aphids belonging to the subfamily Lachninane.

Solving a Bloody Mess: B-Vitamin Independent Metabolic Convergence among Gammaproteobacterial Obligate Endosymbionts from Blood-Feeding Arthropods and the Leech Haementeria officinalis

Endosymbiosis is a common phenomenon in nature, especially between bacteria and insects, whose typically unbalanced diets are usually complemented by their obligate endosymbionts. While much interest and focus has been directed toward phloem-feeders like aphids and mealybugs, blood-feeders such as the Lone star tick (Amblyomma americanum), Glossina flies, and the human body louse (Pediculus humanus corporis) depend on obligate endosymbionts which complement their B-vitamin-deficient diets, and thus are required for growth and survival. Glossiphoniid leeches have also been found to harbor distinct endosymbionts housed in specialized organs. Here, we present the genome of the bacterial endosymbiont from Haementeria officinalis, first of a glossiphoniid leech. This as-yet-unnamed endosymbiont belongs to the Gammaproteobacteria, has a pleomorphic shape and is restricted to bacteriocytes. For this bacterial endosymbiont, we propose the name Candidatus Providencia siddallii. This symbiont possesses a highly reduced genome with high A+T content and a reduced set of metabolic capabilities, all of which are common characteristics of ancient obligate endosymbionts of arthropods. Its genome has retained many pathways related to the biosynthesis of B-vitamins, pointing toward a role in supplementing the blood-restricted diet of its host. Through comparative genomics against the endosymbionts of A. americanum, Glossina flies, and P. humanus corporis, we were able to detect a high degree of metabolic convergence among these four very distantly related endosymbiotic bacteria.

Reinventing the Wheel and Making It Round Again: Evolutionary Convergence in Buchnera-Serratia Symbiotic Consortia between the Distantly Related Lachninae Aphids Tuberolachnus salignus and Cinara cedri.

Virtually all aphids (Aphididae) harbor Buchnera aphidicola as an obligate endosymbiont to compensate nutritional deficiencies arising from their phloem diet. Many species within the Lachninae subfamily seem to be consistently associated also with Serratia symbiotica. We have previously shown that both Cinara (Cinara) cedri and Cinara (Cupressobium) tujafilina (Lachninae: Eulachnini tribe) have indeed established co-obligate associations with both Buchnera and S. symbiotica. However, while Buchnera genomes of both Cinara species are similar, genome degradation differs greatly between the two S. symbiotica strains. To gain insight into the essentiality and degree of integration of S. symbiotica within the Lachninae, we sequenced the genome of both Buchnera and S. symbiotica endosymbionts from the distantly related aphid Tuberolachnus salignus (Lachninae: Tuberolachnini tribe). We found a striking level of similarity between the endosymbiotic system of this aphid and that of C. cedri. In both aphid hosts, S. symbiotica possesses a highly reduced genome and is found exclusively intracellularly inside bacteriocytes. Interestingly, T. salignus’ endosymbionts present the same tryptophan biosynthetic metabolic complementation as C. cedri’s, which is not present in C. tujafilina’s. Moreover, we corroborate the riboflavin-biosynthetic-role take-over/rescue by S. symbiotica in T. salignus, and therefore, provide further evidence for the previously proposed establishment of a secondary co-obligate endosymbiont in the common ancestor of the Lachninae aphids. Finally, we propose that the putative convergent split of the tryptophan biosynthetic role between Buchnera and S. symbiotica could be behind the establishment of S. symbiotica as an obligate intracellular symbiont and the triggering of further genome degradation.

A novel intracellular mutualistic bacterium in the invasive ant Cardiocondyla obscurior

The evolution of eukaryotic organisms is often strongly influenced by microbial symbionts that confer novel traits to their hosts. Here we describe the intracellular Enterobacteriaceae symbiont of the invasive ant Cardiocondyla obscurior, ‘Candidatus Westeberhardia cardiocondylae’. Upon metamorphosis, Westeberhardia is found in gut-associated bacteriomes that deteriorate following eclosion. Only queens maintain Westeberhardia in the ovarian nurse cells from where the symbionts are transmitted to late-stage oocytes during nurse cell depletion. Functional analyses of the streamlined genome of Westeberhardia (533 kb, 23.41% GC content) indicate that neither vitamins nor essential amino acids are provided for the host. However, the genome encodes for an almost complete shikimate pathway leading to 4-hydroxyphenylpyruvate, which could be converted into tyrosine by the host. Taken together with increasing titers of Westeberhardia during pupal stage, this suggests a contribution of Westeberhardia to cuticle formation. Despite a widespread occurrence of Westeberhardia across host populations, one ant lineage was found to be naturally symbiont-free, pointing to the loss of an otherwise prevalent endosymbiont. This study yields insights into a novel intracellular mutualist that could play a role in the invasive success of C. obscurior.

Comparative Genomics of Serratia spp.: Two Paths towards Endosymbiotic Life

Symbiosis is a widespread phenomenon in nature, in which insects show a great number of these associations. Buchnera aphidicola, the obligate endosymbiont of aphids, coexists in some species with another intracellular bacterium, Serratia symbiotica. Of particular interest is the case of the cedar aphid Cinara cedri, where B. aphidicola BCc and S. symbiotica SCc need each other to fulfil their symbiotic role with the insect. Moreover, various features seem to indicate that S. symbiotica SCc is closer to an obligate endosymbiont than to other facultative S. symbiotica, such as the one described for the aphid Acirthosyphon pisum (S. symbiotica SAp). This work is based on the comparative genomics of five strains of Serratia, three free-living and two endosymbiotic ones (one facultative and one obligate) which should allow us to dissect the genome reduction taking place in the adaptive process to an intracellular life-style. Using a pan-genome approach, we have identified shared and strain-specific genes from both endosymbiotic strains and gained insight into the different genetic reduction both S. symbiotica have undergone. We have identified both retained and reduced functional categories in S. symbiotica compared to the Free-Living Serratia (FLS) that seem to be related with its endosymbiotic role in their specific host-symbiont systems. By means of a phylogenomic reconstruction we have solved the position of both endosymbionts with confidence, established the probable insect-pathogen origin of the symbiotic clade as well as the high amino-acid substitution rate in S. symbiotica SCc. Finally, we were able to quantify the minimal number of rearrangements suffered in the endosymbiotic lineages and reconstruct a minimal rearrangement phylogeny. All these findings provide important evidence for the existence of at least two distinctive S. symbiotica lineages that are characterized by different rearrangements, gene content, genome size and branch lengths.

Dissecting genome reduction and trait loss in insect endosymbionts

Symbiosis has played a major role in eukaryotic evolution beyond the origin of the eukaryotic cell. Thus, organisms across the tree of life are associated with diverse microbial partners, conferring to the host new adaptive traits that enable it to explore new niches. This is the case for insects thriving on unbalanced diets, which harbor mutualistic intracellular microorganisms, mostly bacteria that supply them with the required nutrients. As a consequence of the lifestyle change, from free‐living to host‐associated mutualist, a bacterium undergoes many structural and metabolic changes, of which genome shrinkage is the most dramatic. The trend toward genome size reduction in endosymbiotic bacteria is associated with large‐scale gene loss, reflecting the lack of an effective selection mechanism to maintain genes that are rendered superfluous by the constant and rich environment provided by the host. This genome‐reduction syndrome is so strong that it has generated the smallest bacterial genomes found to date, whose gene contents are so limited that their status as cellular entities is questionable. The recent availability of data on several endosymbiotic bacteria is enabling us to form a comprehensive picture of the genome‐reduction process and the phenotypic consequences for the dwindling symbiont.

Snapshots of a shrinking partner: Genome reduction in Serratia symbiotica.

Genome reduction is pervasive among maternally-inherited endosymbiotic organisms, from bacteriocyte- to gut-associated ones. This genome erosion is a step-wise process in which once free-living organisms evolve to become obligate associates, thereby losing non-essential or redundant genes/functions. Serratia symbiotica (Gammaproteobacteria), a secondary endosymbiont present in many aphids (Hemiptera: Aphididae), displays various characteristics that make it a good model organism for studying genome reduction. While some strains are of facultative nature, others have established co-obligate associations with their respective aphid host and its primary endosymbiont (Buchnera). Furthermore, the different strains hold genomes of contrasting sizes and features, and have strikingly disparate cell shapes, sizes, and tissue tropism. Finally, genomes from closely related free-living Serratia marcescens are also available. In this study, we describe in detail the genome reduction process (from free-living to reduced obligate endosymbiont) undergone by S. symbiotica, and relate it to the stages of integration to the symbiotic system the different strains find themselves in. We establish that the genome reduction patterns observed in S. symbiotica follow those from other dwindling genomes, thus proving to be a good model for the study of the genome reduction process within a single bacterial taxon evolving in a similar biological niche (aphid-Buchnera).

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8. We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Happens in the best of subfamilies: Replacement and internalisation of co-obligate Serratia endosymbionts in Lachninae aphids

Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. Most aphids from the Lachninae subfamily have been consistently found to house additional endosymbionts, mainly Serratia symbiotica. This apparent dependence on secondary endosymbionts was proposed to have been triggered by the loss of the riboflavin biosynthetic capability by Buchnera in the Lachninae last common ancestor. However, an integral large-scale analysis of secondary endosymbionts in the Lachninae is still missing, hampering the interpretation of the evolutionary and genomic analyses of these endosymbionts. Here, we analysed the endosymbionts of selected representatives from seven different Lachninae genera and nineteen species, spanning four tribes, both by FISH (exploring the symbionts’ morphology and tissue tropism) and 16S rRNA gene sequencing. We demonstrate that all analysed aphids possess dual symbiotic systems, and while most harbour S. symbiotica, some have undergone symbiont replacement by other phylogenetically-distinct bacterial taxa. We found that these secondary associates display contrasting cell shapes and tissue tropism, and some appear to be lineage-specific. a scenario for symbiont establishment in the Lachninae, followed by changes in the symbiont’s tissue tropism and symbiont replacement events, thereby highlighting the extraordinary versatility of host-symbiont interactions. Originality-Significance Statement A key question in evolutionary biology is that of how mutualism evolves. One way to approach this problem is to investigate recently-established mutualistic associations, particularly by comparing various symbiotic systems in closely related hosts. Here, we present a most comprehensive study to investigate co-obligate symbioses in aphids, focusing in the Lachninae subfamily. While most aphids keep an obligate vertically-transmitted association with intracellular Buchnera bacteria, some, such as members of the Lachninae subfamily, host an additional putative co-obligate symbiont. Thus, the Lachninae dual symbiotic systems offer a unique opportunity to understand the evolutionary dynamics of host-symbiont associations, in particularly how secondary symbionts become obligate and eventually may be replaced. Through genome sequencing of three aphid species belonging to distantly related tribes within the subfamily, we have previously corroborated that they have indeed established co-obligate mutualistic associations with the S. symbiotica secondary endosymbiotic bacterium. This was putatively facilitated by an ancient pseudogenisation of the riboflavin biosynthetic pathway in Buchnera, rendering it unable to provide the essential vitamin to the host. However, not all Lachninae members harbour S. symbiotica, some species being associated to at least four different bacterial taxa. To correctly interpret the genomic data and to understand the evolutionary dynamics of these symbiotic associations, a wide-range analysis of both the phylogenetic relations as well as of the secondary symbionts’ localisation within the bacteriome is needed. To tackle this, we have combined phylogenetic analyses of the symbionts’ 16S rRNA gene sequences and FISH microscopy, to understand the symbiont’s identity as well as the morphological characteristics and tissue tropism. The phylogenetic affinities and patterns of co-divergence of the symbionts, in combination with previously published genomic data, have enabled us to build an evolutionary scenario for the establishment, changes in tissue tropism such as “stable” internalisation into bacteriocytes, and replacements of the putative “ancient” secondary endosymbiont from the Lachninae last common ancestor. Also, we were able to determine through phylogenetic analyses that some putative co-obligate endosymbionts may have evolved from once facultative ones. The evolutionary framework presented here reveals a dynamic pattern for the more recent evolutionary history of these symbioses, including replacement and novel acquisition of phylogenetically different co-obligate symbionts. This study opens new research avenues on this symbiont-diverse subfamily, providing insight into how mutualism in endosymbiotic associations can evolve, and the role these bacteria have played in the species’ adaptation and even in the speciation process.