The circadian and photoperiodic clock of the pea aphid.

The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species that exhibits a remarkable annual life cycle, which is tightly coupled to the seasonal changes in day length. During spring and summer, characterised by longer days, aphid populations consist exclusively of viviparous females that reproduce parthenogenetically. When autumn comes and the days shorten, aphids switch their reproductive mode and generate males and oviparous sexual females, which mate and produce cold-resistant eggs that overwinter and survive the unfavourable season. While the photoperiodic responses have been well described, the nature of the timing mechanisms which underlie day length discrimination are still not completely understood. Experiments from the 1960's suggested that aphids rely on an 'hourglass' clock measuring the elapsed time during the dark night by accumulating a biochemical factor, which reaches a critical threshold at a certain night length and triggers the switch in reproduction mode. However, the photoperiodic responses of aphids can also be attributed to a strongly dampened circadian clock. Recent studies have uncovered the molecular components and the location of the circadian clock in the brain of the pea aphid and revealed that it is well connected to the neurohormonal system controlling aphid reproduction. We provide an overview of the putative mechanisms of photoperiodic control in aphids, from the photoreceptors involved in this process to the circadian clock and the neuroendocrine system.

Pigment-dispersing factor is present in circadian clock neurons of pea aphids and may mediate photoperiodic signalling to insulin-producing cells.

The neuropeptide pigment-dispersing factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far. In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing.

Mapping and quantification of cryptochrome expression in the brain of the pea aphid Acyrthosiphon pisum.

Aphids are paradigmatic photoperiodic animals often used to study the role of the circadian clock in the seasonal response. Previously, we described some elements of the circadian clock core (genes period and timeless) and output (melatonin, AANATs and PTTH) that could have a role in the regulation of the aphid seasonal response. More recently, we identified two opsins (C-ops and SWO4) as candidate input photoperiodic receptors. In the present report, we focus on the study of cryptochromes (cry) as photoreceptors of the circadian clock and discuss their involvement in the seasonal response. We analyse the expression of cry1 and cry2 genes in a circadian and seasonal context, and map their expression sites in the brain. We observe a robust rhythmic expression of cry2 peaking at dusk in phase with core clock genes period and timeless, while cry1 shows a weaker rhythm. Changes in cry1 and cry2 expression correlate with activation of the seasonal response, suggesting a possible link. Finally, we map the expression of cry1 and cry2 genes to clock neurons in the pars lateralis, a region essential for the photoperiodic response. Our results support a role for cry as elements of the aphid circadian clock and suggest a role in photoreception for cry1 and in clock repression for cry2.

Antibodies Against the Clock Proteins Period and Cryptochrome Reveal the Neuronal Organization of the Circadian Clock in the Pea Aphid.

Circadian clocks prepare the organism to cyclic environmental changes in light, temperature, or food availability. Here, we characterized the master clock in the brain of a strongly photoperiodic insect, the aphid Acyrthosiphon pisum, immunohistochemically with antibodies against A. pisum Period (PER), Drosophila melanogaster Cryptochrome (CRY1), and crab Pigment-Dispersing Hormone (PDH). The latter antibody detects all so far known PDHs and PDFs (Pigment-Dispersing Factors), which play a dominant role in the circadian system of many arthropods. We found that, under long days, PER and CRY are expressed in a rhythmic manner in three regions of the brain: the dorsal and lateral protocerebrum and the lamina. No staining was detected with anti-PDH, suggesting that aphids lack PDF. All the CRY1-positive cells co-expressed PER and showed daily PER/CRY1 oscillations of high amplitude, while the PER oscillations of the CRY1-negative PER neurons were of considerable lower amplitude. The CRY1 oscillations were highly synchronous in all neurons, suggesting that aphid CRY1, similarly to Drosophila CRY1, is light sensitive and its oscillations are synchronized by light-dark cycles. Nevertheless, in contrast to Drosophila CRY1, aphid CRY1 was not degraded by light, but steadily increased during the day and decreased during the night. PER was always located in the nuclei of the clock neurons, while CRY was predominantly cytoplasmic and revealed the projections of the PER/CRY1-positive neurons. We traced the PER/CRY1-positive neurons through the aphid protocerebrum discovering striking similarities with the circadian clock of D. melanogaster: The CRY1 fibers innervate the dorsal and lateral protocerebrum and putatively connect the different PER-positive neurons with each other. They also run toward the pars intercerebralis, which controls hormone release via the neurohemal organ, the corpora cardiaca. In contrast to Drosophila, the CRY1-positive fibers additionally travel directly toward the corpora cardiaca and the close-by endocrine gland, corpora allata. This suggests a direct link between the circadian clock and the photoperiodic control of hormone release that can be studied in the future.

Progress in the characterization of insulin-like peptides in aphids: Immunohistochemical mapping of ILP4.

Aphids were the first animals described as photoperiodic due to their seasonal switch from viviparous parthenogenesis to sexual reproduction (cyclical parthenogenesis) caused by the shortening of the photoperiod in autumn. This switch produces a single sexual generation of oviparous females and males that mate and lay diapausing cold-resistant eggs that can overcome the unfavourable environmental conditions typical of winter in temperate regions. Previous studies have hinted at a possible implication of two insulin-like peptides (ILP1 and ILP4) in the aphid seasonal response, changing their expression levels between different photoperiodic conditions. Moreover, in situ localization of their transcripts in particular neurosecretory cells (NSCs) in the aphid brain supported the idea that these neuropeptides could correspond to the formerly called virginoparin, an uncharacterized factor originally proposed to be transported directly to the aphid embryos to promote their development as parthenogenetic individuals. To further investigate the fate of these ILPs, we raised a specific antiserum against one of them (ILP4) and mapped this neuropeptide by immunohistochemistry (IHC) in Acyrthosiphon pisum and Megoura viciae aphids. Coincident with in situ localization, our results show that ILP4 is synthesized in two groups (one in each brain hemisphere) of four neurosecretory cells in the pars intercerebralis (NSC group I) and then it is transported outside the brain to the corpora cardiaca. From there, three nerves (two laterals and one medial) transport it to the abdomen. Although no precise site of release has been found, the terminations of these nerves near the germaria would be compatible with the proposal of a direct connection between group I of NSCs and the reproductive system by localized release. In addition, we detected some collateral arborizations originating from the eight NSCs going to the pars lateralis, where clock neurons and some photoreceptors have been previously localized, suggesting a possible communication between the circadian and photoperiodic systems.

Correction to: The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

An amendment to this paper has been published and can be accessed via the original article.

The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

BACKGROUND: Although native to North America, the invasion of the aphid-like grape phylloxera Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome of D. vitifoliae as a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture. RESULTS: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world. CONCLUSIONS: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture.

Melatonin in the seasonal response of the aphid Acyrthosiphon pisum.

Aphids display life cycles largely determined by the photoperiod. During the warm long-day seasons, most aphid species reproduce by viviparous parthenogenesis. The shortening of the photoperiod in autumn induces a switch to sexual reproduction. Males and sexual females mate to produce overwintering resistant eggs. In addition to this full life cycle (holocycle), there are anholocyclic lineages that do not respond to changes in photoperiod and reproduce continuously by parthenogenesis. The molecular or hormonal events that trigger the seasonal response (i.e., induction of the sexual phenotypes) are still unknown. Although circadian synthesis of melatonin is known to play a key role in vertebrate photoperiodism, the involvement of the circadian clock and/or of the hormone melatonin in insect seasonal responses is not so well established. Here we show that melatonin levels in the aphid Acyrthosiphon pisum are significantly higher in holocyclic aphids reared under short days than under long days, while no differences were found between anholocyclic aphids under the same conditions. We also found that melatonin is localized in the aphid suboesophageal ganglion (SOG) and in the thoracic ganglionic mass (TGM). In analogy to vertebrates, insect-type arylalkylamine N-acetyltransferases (i-AANATs) are thought to play a key role in melatonin synthesis. We measured the expression of four i-AANAT genes identified in A. pisum and localized two of them in situ in the insect central nervous systems (CNS). Levels of expression of these genes were compatible with the quantities of melatonin observed. Moreover, like melatonin, expression of these genes was found in the SOG and the TGM.

Insulin-like peptides involved in photoperiodism in the aphid Acyrthosiphon pisum.

Aphids were the first animals reported as photoperiodic as their life cycles are strongly determined by the photoperiod. During the favourable seasons (characterised by long days) aphid populations consist exclusively of viviparous parthenogenetic females (known as virginoparae). Shortening of the photoperiod in autumn is perceived by aphids as the signal that anticipates the harsh season, leading to a switch in the reproductive mode giving place to the sexual morphs (oviparae females and males) that mate and lay winter-resistant (diapause-like) eggs. The molecular and cellular basis governing the switch between the two reproductive modes are far from being understood. Classical experiments identified a group of neurosecretory cells in the pars intercerebralis of the aphid brain (the so called group I of neurosecretory cells) that were essential for the development of embryos as parthenogenetic females and were thus proposed to synthesise a parthenogenesis promoting substance that was termed "virginoparin". Since insulin-like peptides (ILPs) have been implicated in the control of diapause in other insects, we investigated their involvement in aphid photoperiodism. We compared the expression of two ILPs (ILP1 and ILP4) and an Insulin receptor coding genes in A. pisum aphids reared under long- and short-day conditions. The three genes showed higher expression in long-day reared aphids. In addition, we localised the site of expression of the two ILP genes in the aphid brain. Both genes were found to be expressed in the group I of neurosecretory cells. Altogether, our results suggest that ILP1 and ILP4 play an important role in the control of the aphid life-cycle by promoting the parthenogenetic development during long-day seasons while their repression by short days would activate the sexual development. Thus we propose these ILPs correspond to the so called "virginoparin" by early bibliography. A possible connection with the circadian system is also discussed.

Characterisation, analysis of expression and localisation of the opsin gene repertoire from the perspective of photoperiodism in the aphid Acyrthosiphon pisum.

Organisms exhibit a wide range of seasonal responses as adaptions to predictable annual changes in their environment. These changes are originally caused by the effect of the Earth's cycles around the sun and its axial tilt. Examples of seasonal responses include floration, migration, reproduction and diapause. In temperate climate zones, the most robust variable to predict seasons is the length of the day (i.e. the photoperiod). The first step to trigger photoperiodic driven responses involves measuring the duration of the light-dark phases, but the molecular clockwork performing this task is poorly characterized. Photopigments such as opsins are known to participate in light perception, being part of the machinery in charge of providing information about the luminous state of the surroundings. Aphids (Hemiptera: Aphididae) are paradigmatic photoperiodic insects, exhibiting a strong induction to diapause when the light regime mimics autumn conditions. The availability of the pea aphid (Acyrthosiphon pisum) genome has facilitated molecular approaches to understand the effect of light stimulus in the photoperiodic induction process. We have identified, experimentally validated and characterized the expression of the full opsin gene repertoire in the pea aphid. Among identified opsin genes in A. pisum, arthropsin is absent in most insects sequenced to date (except for dragonflies and two other hemipterans) but also present in a crustacean, an onychophoran and chelicerates. We have quantified the expression of these genes in aphids exposed to different photoperiodic conditions and at different times of the day and localized their transcripts in the aphid brain. Clear differences in expression patterns were found, thus relating opsin expression with the photoperiodic response.

Characterisation, analysis of expression and localisation of circadian clock genes from the perspective of photoperiodism in the aphid Acyrthosiphon pisum.

Aphids are typical photoperiodic insects that switch from viviparous parthenogenetic reproduction typical of long day seasons to oviparous sexual reproduction triggered by the shortening of photoperiod in autumn yielding an overwintering egg in which an embryonic diapause takes place. While the involvement of the circadian clock genes in photoperiodism in mammals is well established, there is still some controversy on their participation in insects. The availability of the genome of the pea aphid Acyrthosiphon pisum places this species as an excellent model to investigate the involvement of the circadian system in the aphid seasonal response. In the present report, we have advanced in the characterisation of the circadian clock genes and showed that these genes display extensive alternative splicing. Moreover, the expression of circadian clock genes, analysed at different moments of the day, showed a robust cycling of central clock genes period and timeless. Furthermore, the rhythmic expression of these genes was shown to be rapidly dampened under DD (continuous darkness conditions), thus supporting the model of a seasonal response based on a heavily dampened circadian oscillator. Additionally, increased expression of some of the circadian clock genes under short-day conditions suggest their involvement in the induction of the aphid seasonal response. Finally, in situ localisation of transcripts of genes period and timeless in the aphid brain revealed the site of clock neurons for the first time in aphids. Two groups of clock cells were identified: the Dorsal Neurons (DN) and the Lateral Neurons (LN), both in the protocerebrum.

Determination of melatonin in Acyrthosiphon pisum aphids by liquid chromatography-tandem mass spectrometry.

Melatonin is a hormone mainly involved in the regulation of circadian and seasonal rhythms in both invertebrates and vertebrates. Despite the identification of melatonin in many insects, its involvement in the insect seasonal response remains unclear. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for melatonin analysis in aphids (Acyrthosiphon pisum) for the first time. After comparing two different procedures and five extraction solvents, a sample preparation procedure with a mixture of methanol/water (50:50) was selected for melatonin extraction. The method was validated by analyzing melatonin recovery at three spiked concentrations (5, 50 and 100 pg/mg) and showed satisfactory recoveries (75-110%), and good repeatability, expressed as relative standard deviation (<10%). Limits of detection (LOD) and quantitation (LOQ) were 1 pg/mg and 5 pg/mg, respectively. Eight concentration levels were used for constructing the calibration curves which showed good linearity between LOQ and 200 times LOQ. The validated method was successfully applied to 26 aphid samples demonstrating its usefulness for melatonin determination in insects. This is -to our knowledge- the first identification of melatonin in aphids by LC-MS/MS.

Aggressive mimicry coexists with mutualism in an aphid.

Understanding the evolutionary transition from interspecific exploitation to cooperation is a major challenge in evolutionary biology. Ant-aphid relationships represent an ideal system to this end because they encompass a coevolutionary continuum of interactions ranging from mutualism to antagonism. In this study, we report an unprecedented interaction along this continuum: aggressive mimicry in aphids. We show that two morphs clonally produced by the aphid Paracletus cimiciformis during its root-dwelling phase establish relationships with ants at opposite sides of the mutualism-antagonism continuum. Although one of these morphs exhibits the conventional trophobiotic (mutualistic) relationship with ants of the genus Tetramorium, aphids of the alternative morph are transported by the ants to their brood chamber and cared for as if they were true ant larvae. Gas chromatography-mass spectrometry analyses reveal that the innate cuticular hydrocarbon profile of the mimic morph resembles the profile of ant larvae more than that of the alternative, genetically identical nonmimic morph. Furthermore, we show that, once in the brood chamber, mimic aphids suck on ant larva hemolymph. These results not only add aphids to the limited list of arthropods known to biosynthesize the cuticular chemicals of their deceived hosts to exploit their resources but describe a remarkable case of plastic aggressive mimicry. The present work adds a previously unidentified dimension to the classical textbook paradigm of aphid-ant relationships by showcasing a complex system at the evolutionary interface between cooperation and exploitation.

Two mitochondrial haplotypes in Pterochloroides persicae (Hemiptera: Aphididae: Lachninae) associated with different feeding sites.

Pterochloroides persicae (Cholodkovsky) is an aphid species belonging to the subfamily Lachninae that uses different members of Rosaceae (specially Prunus spp.) as hosts. Partial sequences from the mitochondrial cytochrome c oxidase 1 (COI) and the nuclear long-wave opsin genes were obtained for approximately 100 P. persicae aphid individuals sampled from 34 colonies collected mainly in Tunisia and other Mediterranean locations. The variability found at the mitochondrial locus revealed the presence of two maternal haplotypes in the studied area that differed in a single nucleotide. The nuclear gene analyzed, however, failed to reveal any variability in this species. The variability found at the COI locus was related to the season of aphid sampling and with the site of feeding, with haplotype I mostly detected in samples collected in spring and summer on trunks and branches and haplotype II only detected in aphids collected in autumn on roots. The observed pattern of molecular variation suggests the presence of two clonal races of P. persicae coexisting in the studied area differentially adapted to conditions prevalent in the alternative seasons and/or to different feeding sites.

A new species of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomentosa (Poaceae, Bambusoideae) from Costa Rica.

The new species Rhopalosiphum chusqueae Pérez Hidalgo & Villalobos Muller, is described from apterous viviparous females caught on Chusquea tomentosa in Cerro de la Muerte (Costa Rica). The identity of the species is supported both by the morphological features and by a molecular phylogenetic analysis based on a fragment of the mitochondrial DNA containing the 5' region of the cytochrome c oxidase 1 (COI) and on the nuclear gene coding for the Elongation factor-1 alpha (EF1α). The taxonomic position of the new species is discussed. An identification key to the Aphidinae species living on plants of Bambusoideae (Poaceae) is presented.

Combination of molecular data support the existence of three main lineages in the phylogeny of aphids (Hemiptera: Aphididae) and the basal position of the subfamily Lachninae.

The first molecular studies on the phylogeny of aphids (Hemiptera: Aphididae) bumped into a striking lack of phylogenetic structure for taxa levels higher than tribe, probably as a consequence of the rapid adaptive radiation that this group of insects went through during the Late Cretaceous. Here we present a new attempt to infer the relationships between major aphid taxa by the separate and combined analysis of two nuclear sequences (the long-wavelength opsin gene and the elongation factor 1 alpha gene) and two mitochondrial sequences (the genes encoding the subunit 6 of the F-ATPase and the subunit II of the cytochrome oxidase). Our results confirm previous results with the grouping of the subfamilies analysed in three main lineages, that are named A+D (subfamilies Aphidinae, Calaphidinae, Chaitophorinae, Drepanosiphinae and Pterocommatinae), E+T (subfamilies Anoeciinae, Eriosomatinae, Hormaphidinae, Mindarinae and Thelaxinae) and L (subfamily Lachninae). Furthermore, phylogenetic reconstructions generally support the early branching of the subfamily Lachninae in the phylogeny of aphids. Although some relationships among subfamilies inside lineages are not highly supported, our results are compatible with a scenario for the evolution of aphid life cycles with only four transitions of feeding from gymnosperms to angiosperms and two origins of host alternation.

Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera).

Aphids are the leading pests in agricultural crops. A large-scale sequencing of 40,904 ESTs from the pea aphid Acyrthosiphon pisum was carried out to define a catalog of 12,082 unique transcripts. A strong AT bias was found, indicating a compositional shift between Drosophila melanogaster and A. pisum. An in silico profiling analysis characterized 135 transcripts specific to pea-aphid tissues (relating to bacteriocytes and parthenogenetic embryos). This project is the first to address the genetics of the Hemiptera and of a hemimetabolous insect.

Molecular systematics of aphids (Homoptera: Aphididae): new insights from the long-wavelength opsin gene.

Viviparous aphids (Aphididae) constitute a monophyletic group within the Homoptera with more than 4000 extant species worldwide but higher diversity in temperate regions. Several aspects of their biology account for attention paid to this group of insects. Their plant-sap-sucking way of feeding with many species transmitting viruses to crop plants has important implications on crop management strategies. Cyclical parthenogenesis associated in many groups to host alternation and elaborate polyphenisms is of special interests for evolutionists. Finally, the ancient association of most aphid species with intracellular endosymbiotic bacteria (Buchnera sp.) has also received much attention from evolutionists interested in mechanisms involved in the symbiotic process. Knowing the phylogenetic relationships among major aphid taxa is of special interest to evolutionists interested in the above issues. However, until recently, molecular approaches to aphid phylogeny were absent and discussions on the evolution of aphid life-cycles and on evolutionary aspects of their symbiotic association with Buchnera were framed by morphology-based phylogenies. Recently, two reports using molecular approaches attempted to address the yet unresolved phylogeny of Aphididae with limited although somehow different conclusions. In the present report we study the utility of the long-wave opsin gene in resolving phylogenetic relationships among seven subfamilies within the Aphididae. Our results corroborate some previously proposed relationships and suggest a revision of some others. In particular, our data support grouping the analysed aphid species into three main clades, being the subfamily Lachninae one of them, which contradicts its generally accepted sistership relationship with the subfamily Aphidinae. Moreover, our data also suggest a basal position of Lachninae which has implications on current discussions about the ancestrality of conifer-feeding in modern aphids.

Identification of a gene overexpressed in aphids reared under short photoperiod.

Most aphids develop a cyclic parthenogenesis life-cycle. After several generations of viviparously produced parthenogenetic females, follows a single annual generation of sexual individuals, usually in autumn, that mate and lay the sexual eggs. Shortening of photoperiod at the end of the summer (together with temperature) is a key factor inducing the sexual response. Currently no genes involved in the cascade of events that lead to the appearance of sexual forms have been reported. After a Differential Display RT-PCR survey performed on Acyrthosiphon pisum aphids, we identified a gene that is overexpressed in aphids reared under short photoperiod conditions that induce sexuality in this species. This cDNA (called ApSDI-1) shows similarities with a protein involved in amino acid transport in GABAergic neurons. Since several studies implicate GABAergic transmission in the generation and modulation of circadian rhythmicity, we propose that ApSDI-1 could be involved in the transduction of the photoperiodic message and therefore be a candidate to participate at some point in processes that trigger the sexual response in aphids. This is the first gene identified in aphids whose expression is governed by the photoperiod.

Molecular evolution of aphids and their primary (buchnera sp) and secondary endosymbionts: implications for the role of symbiosis in insect evolution

Los pulgones mantienen una asociación endosimbiótica obligada con Buchnera sp., una bacteria estrechamente relacionada con Escherichia coli. La bacteria se encuentra alojada en estructuras celulares llamadas bacteriomas en el hemocele de los pulgones, y son maternalmente transmitidas. Los estudios filogenéticos indican que la asociación tuvo un único origen hace aproximadamente 200-250 millones de años, y desde entonces hospedero y endosimbionte han evolucionado en paralelo. Sin embargo, el patrón de ramificación en la familia de los áfidos no ha sido resuelto, lo cual impide la evaluación de, por ejemplo, el papel de la transferencia horizontal de genes en la evolución temprana de Buchnera. El papel principal de Buchnera en esta asociación es la biosíntesis y aprovisionamiento de aminoácidos esenciales a su áfido hospedero. Esta afirmación está apoyada por estudios fisiológicos, metabólicos y recientemente por estudios genéticos. Las transformaciones genéticas experimentadas por Buchnera en su adaptación a la vida endosimbiótica son variadas. Entre ellas, la reducción de su genoma comparando con bacterias de vida libre, el incremento en A+T, tasas de evolución diferentes, incremento relativo de las tasas de substitución no sinónimas y la amplificación génica mediada por plásmidos. Pero la endosimbiosis es un proceso activo en los insectos, como se puede comprobar por el estado intermedio de los endosimbiontes secundarios, los cuales presentan valores intermedios de las características anteriormente citadas en comparación con las bacterias de vida libre y Buchnera