Endosymbionts (Buchnera) of the Aphid Uroleucon sonchi Contain Plasmids with trpEG and Remnants of trpE Pseudogenes

The aphid Uroleucon sonchi contains a prokaryotic endosymbiont (Buchnera) with plasmids having trpEG as well as remnants of trpE pseudogenes. In this respect it resembles Buchnera from the aphid Diuraphis noxia. Phylogenetic trees based on trpE (plasmid gene) and trpB (chromosomal genes) from eight species of aphids are congruent, indicating a lack of exchange of plasmids among endosymbionts from different aphid species. Received: 16 December 1996 / Accepted: 26 December 1996     

Endosymbionts (Buchnera) from the Aphids Schizaphis graminum and Diuraphis noxia Have Different Copy Numbers of the Plasmid Containing the Leucine Biosynthetic Genes

The prokaryotic endosymbiont (Buchnera) of the aphid Schizaphis graminum contains 24 copies of a plasmid that has genes encoding enzymes of the leucine biosynthetic pathway while the endosymbiont of the related aphid Diuraphis noxia has only one copy of this plasmid. These results, in conjunction with similar results for the trpEG-containing plasmids, suggest that D. noxia has a reduced demand for endosymbiont-derived essential amino acids. Received: 11 September 1997 / Accepted: 23 September 1997     

Plasmid-Encoded Anthranilate Synthase (TrpEG) in Buchnera aphidicola from Aphids of the Family Pemphigidae

Buchnera aphidicola is an obligate intracellular symbiont of aphids. One of its proposed functions is the synthesis of essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. The genetic organization of the tryptophan pathway in Buchnera from proliferous aphids of the family Aphididae has previously been shown to reflect a capacity to overproduce this essential amino acid (C.-Y. Lai, L. Baumann, and P. Baumann, Proc. Natl. Acad. Sci. USA 91:3819–3823, 1994). This involved amplification of the genes for the first enzyme in the pathway, anthranilate synthase (TrpEG), on a low-copy-number plasmid. Here we report on the finding and molecular characterization of TrpEG-encoding plasmids in Buchnera from aphids of the distantly related family Pemphigidae. Buchnera from Tetraneura caerulescens contained a 3.0-kb plasmid (pBTc2) that carried a single copy of trpEG and resembled trpEG plasmids of Buchnera from the Aphididae. The second plasmid (pBPs2), isolated from Buchnera of Pemphigus spyrothecae, contained a different replicon. It consisted of a putative origin of replication containing iterons and an open reading frame, designated repAC, which showed a high similarity to the gene encoding the replication initiation protein RepA of the RepA/C replicon from the broad-host-range IncA/C group of plasmids. The plasmid population was heterogeneous with respect to the number of tandem repeats of a 1.8-kb unit carrying repAC₁, trpG, and remnants of trpE. The two principal forms consisted of either five or six copies of this repeat and a single-copy region carrying repAC₂, the putative origin of replication, and trpE. The unexpected finding of elements of the RepA/C replicon in previously characterized trpEG plasmids from Buchnera of the Aphididae suggests that a replacement of replicons has occurred during the evolution of these plasmids, which may point to a common ancestry for all Buchnera trpEG amplifications.     

The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): Genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae

The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R. maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units. Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway. The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host. The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication. Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome. The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA). In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II). Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages. Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids. Received: 22 August 1995 / Accepted: 6 September 1995     

Relating genotype and phenotype for tryptophan synthesis in an aphid–bacterial symbiosis

The symbiotic bacteria Buchnera provide their aphid hosts with tryptophan and other essential amino acids. Tryptophan production by Buchnera varied among 12 parthenogenetic clones of the pea aphid Acyrthosiphon pisum (Harris), as determined from both the incorporation of radioactivity from ¹⁴C‐anthranilate into tryptophan and the protein‐tryptophan growth rate of larval aphids on tryptophan‐free diet. The values of tryptophan production obtained for the two methods were correlated significantly with each other but not with the level of amplification of the Buchnera genes trpEG, which code for anthranilate synthase, a key enzyme in tryptophan biosynthetic pathway. This study provides the first direct demonstration of interclonal variation in production of any nutrient in an aphid–Buchnera symbiosis and indicates that a key aspect of Buchnera phenotype (tryptophan production) does not vary in a simple fashion with Buchnera genotype.     

Genetic Characterization of Plasmids Containing Genes Encoding Enzymes of Leucine Biosynthesis in Endosymbionts (Buchnera) of Aphids

The prokaryotic endosymbionts (Buchnera) of aphids are known to provision their hosts with amino acids that are limiting in the aphid diet. Buchnera from the aphids Schizaphis graminum and Diuraphis noxia have plasmids containing leuABCD, genes that encode enzymes of the leucine biosynthetic pathway, as well as genes encoding proteins probably involved in plasmid replication (repA1 and repA2) and an open reading frame (ORF1) of unknown function. The newly reported plasmids closely resemble a plasmid previously described in Buchnera of the aphid Rhopalosiphum padi [Bracho AM, Martínez-Torres D, Moya A, Latorre A (1995) J Mol Evol 41:67–73]. Nucleotide sequence comparisons indicate conserved regions which may correspond to an origin of replication and two promoters, as well as inverted repeats, one of which resembles a rho-independent terminator. Phylogenetic analyses based on amino acid sequences of leu gene products and ORF1 resulted in trees identical to those obtained from endosymbiont chromosomal genes and the plasmid-borne trpEG. These results are consistent with a single evolutionary origin of the leuABCD-containing plasmid in a common ancestor of Aphididae and the lack of plasmid exchange between endosymbionts of different aphid species. Trees for ORF1 and repA (based on both nucleotides and amino acids) are used to examine the basis for leu plasmid differences between Buchnera of Thelaxes suberi and Aphididae. The most plausible explanation is that a single transfer of the leu genes to an ancestral replicon was followed by rearrangements. The related replicon in Buchnera of Pemphigidae, which lacks leuABCD, appears to represent the ancestral condition, implying that the plasmid location of the leu genes arose after the Pemphigidae diverged from other aphid families. This conclusion parallels previously published observations for the unrelated trpEG plasmid, which is present in Aphididae and absent in Pemphigidae. Recruitment of amino acid biosynthetic genes to plasmids has been ongoing in Buchnera lineages after the infection of aphid hosts. Received: 9 March 1998 / Accepted: 18 May 1998.     

Phylogenetic congruence between Mollitrichosiphum (Aphididae: Greenideinae) and Buchnera indicates insect–bacteria parallel evolution

We wanted to test whether Mollitrichosiphum, an aphid genus with life cycles on subtropical woody host plants, and Buchnera, the primary endosymbiont of aphids, evolve in parallel. We used three aphid genes (mitochondrial COI, cytochrome oxidase subunit I and Cytb, cytochrome b; nuclear EF1α, translation elongation factor 1 alpha) and two Buchnera genes (16S rDNA; gnd, gluconate‐6‐phosphate dehydrogenase) to reconstruct phylogenies. The congruence between the phylogenetic trees of aphids and Buchnera was then measured. The results present phylogenetic evidence for the parallel evolution of Mollitrichosiphum and Buchnera at the intraspecific as well as the interspecific levels. Our results support the possibility of using endosymbiont genes to study host evolutionary history and biogeographical patterns. We also investigated the usability of the Buchnera gnd gene as a barcoding marker for aphid identification.     

Evolutionary relationships of Pemphigus and allied genera (Hemiptera: Aphididae: Eriosomatinae) and their primary endosymbiont,Buchnera aphidicola

Aphids harbor primary endosymbionts, Buchnera aphidicola, in specialized cells within their body cavities. Aphids and Buchnera have strict mutualistic relationships in nutrition exchange. This ancient association has received much attention from researchers who are interested in endosymbiotic evolution. Previous studies have found parallel phylogenetic relationships between non‐galling aphids and Buchnera at lower taxonomic levels (genus, species). To understand whether relatively isolated habitats such as galls have effect on the parallel relationships between aphids and Buchnera, the present paper investigated the phylogenetic relationships of gall aphids from Pemphigus and allied genera, which induce pseudo‐galls or galls on Populus spp. (poplar) and Buchnera. The molecular phylogenies inferred from three aphid genes (COI, COII and EF‐1α) and two Buchnera genes (gnd, 16S rRNA gene) indicated significant congruence between aphids and Buchnera at generic as well as interspecific levels. Interestingly, both aphid and Buchnera phylogenies supported three main clades corresponding to the galling locations of aphids, namely leaf, the joint of leaf blade and petiole, and branch of the host plant. The results suggest phylogenetic conservatism of gall characters, which indicates gall characters are more strongly affected by aphid phylogeny, rather than host plants.     

Altered tRNA characteristics and 3' maturation in bacterial symbionts with reduced genomes

Translational efficiency is controlled by tRNAs and other genome-encoded mechanisms. In organelles, translational processes are dramatically altered because of genome shrinkage and horizontal acquisition of gene products. The influence of genome reduction on translation in endosymbionts is largely unknown. Here, we investigate whether divergent lineages of Buchnera aphidicola, the reduced-genome bacterial endosymbiont of aphids, possess altered translational features compared with their free-living relative, Escherichia coli. Our RNAseq data support the hypothesis that translation is less optimal in Buchnera than in E. coli. We observed a specific, convergent, pattern of tRNA loss in Buchnera and other endosymbionts that have undergone genome shrinkage. Furthermore, many modified nucleoside pathways that are important for E. coli translation are lost in Buchnera. Additionally, Buchnera’s A + T compositional bias has resulted in reduced tRNA thermostability, and may have altered aminoacyl-tRNA synthetase recognition sites. Buchnera tRNA genes are shorter than those of E. coli, as the majority no longer has a genome-encoded 3'' CCA; however, all the expressed, shortened tRNAs undergo 3′ CCA maturation. Moreover, expression of tRNA isoacceptors was not correlated with the usage of corresponding codons. Overall, our data suggest that endosymbiont genome evolution alters tRNA characteristics that are known to influence translational efficiency in their free-living relative.     

The striking case of tryptophan provision in the cedar aphid Cinara cedri

Buchnera aphidicola BCc has lost its symbiotic role as the tryptophan supplier to the aphid Cinara cedri. We report the presence of a plasmid in this endosymbiont that contains the trpEG genes. The remaining genes for the pathway (trpDCBA) are located on the chromosome of the secondary endosymbiont “Candidatus Serratia symbiotica.” Thus, we propose that a symbiotic consortium is necessary to provide tryptophan.     

Intracellular Bacterial Symbionts of Aphids Possess Many Genomic Copies per Bacterium

Although Buchnera, the endosymbiotic bacteria of aphids, are close relatives of Escherichia coli, their genome size is only a seventh that of E. coli. In this study, we estimated the genomic copy number of Buchnera by dot-blot hybridization and fluorimetry using a video-intensified microscope photon-counting system and obtained convincing evidence that each cell of these bacteria contains an average of 120 genomic copies. Thus, the Buchnera symbiont, with many copies of a small-sized genome, is reminiscent of cell organelles such as mitochondria and chloroplasts. Received: 25 November 1998 / Accepted: 25 December 1998     

Happens in the best of subfamilies: establishment and repeated replacements of co‐obligate secondary endosymbionts within 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. We propose 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.     

The gnd gene of Buchnera as a new,effective DNA barcode for aphid identification

DNA barcoding uses a standard DNA sequence to facilitate species identification. Although the COI gene has been adopted as the standard, COI alone is imperfect due to several shortcomings. The primary endosymbiont of aphids, Buchnera, has higher evolutionary rates and interspecies divergence than its co‐diverging aphid hosts, making it a potential tool for resolving the ambiguities in aphid taxonomy. We compared the effectiveness of employing two different DNA regions, gnd and COI, for the discrimination of over 100 species of aphids. The mean interspecific divergence of the gnd region was significantly higher than the mean intraspecific variation; there were nearly nonoverlapping distributions between the intra‐ and interspecific samples. In contrast, COI showed a lower interspecific divergence, which led to difficulties in identifying closely related species. Our results show that gnd can identify species in the Aphididae, which suggests that the gnd region of Buchnera is a potentially effective barcode for aphid species identification. We also recommend the 2‐locus combination of gnd + COI as the aphid barcode. This will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of aphids.     

Faster evolutionary rates in endosymbiotic bacteria than in cospeciating insect hosts

The hypothesis of a universal molecular clock holds that divergent lineages exhibit approximately constant rates of nucleotide substitution over evolutionary time for a particular macromolecule. We compare divergences of ribosomal DNA for aphids (Insecta) and Buchnera, the maternally transmitted, endosymbiotic bacteria that have cospeciated with aphids since initially infecting them over 100 million years ago. Substitution rates average 36 times greater for Buchnera than for their aphid hosts for regions of small-subunit rDNA that are homologous for prokaryotes and eukaryotes. Aphids exhibit 18S rDNA substitution rates that are within the range observed in related insects. In contrast, 16S rDNA evolves about twice as fast in Buchnera as in related free-living bacterial lineages. Nonetheless, the difference between Buchnera and aphids is much greater, suggesting that rates may be generally higher in bacteria. This finding adds to evidence that molecular clocks are only locally rather than universally valid among taxonomic groups. It is consistent with the hypothesis that rates of sequence evolution depend on generation time.     

Temperature effect on the growth of <Emphasis Type="Italic">Buchnera</Emphasis> endosymbiont in <Emphasis Type="Italic">Aphis craccivora</Emphasis> (Hemiptera: Aphididae)

Effect of temperature on the growth of the primary endosymbiont Buchnera aphidicola in the cowpea aphid Aphis craccivora was studied by measuring quantitatively the copy number of 16S rDNA of this endosymbiont. A 1.5 kb segment of eubacterial 16S rDNA amplified by PCR from total DNA of Aphis craccivora was confirmed by RFLP analysis and sequence BLAST as that of Buchnera aphidicola. No secondary endosymbiont was detected in the aphid population studied. The relative levels of Buchnera ratio, quantified by real-time PCR, were higher in old nymphs than in young ones at temperatures between 10–30˚C, and this age-dependent difference was more pronounced at lower temperatures. Throughout the entire reproductive stage of Aphis craccivora, the relative levels of Buchnera ratio were higher at 10–25˚C than at 30˚C and 35˚C. A close relationship was found between these levels and the net reproductive rate (R ₀ ) of aphid, which was suppressed not only at 35˚C but also at 10˚C. The decoupling of Aphis craccivora and Buchnera response at low temperatures suggests that the cowpea aphid was more sensitive to low temperatures, while Buchnera was more sensitive to high temperatures.     

Levels of Buchnera aphidicola Chaperonin GroEL During Growth of the Aphid Schizaphis graminum

Buchnera aphidicola is the prokaryotic, intracellular symbiont found in the aphid Schizaphis graminum. Using an immunological approach, we have quantitated the amount of the B. aphidicola chaperonin, GroEL, present in aphid cell-free extracts during the growth cycle of S. graminum at 23°C. Our results indicate that the increase in GroEL approximately follows the increase in aphid weight and endosymbiont number for the first 12 days after birth of the aphid. A 9-day-old aphid contains 1.6 × 10⁵ molecules of GroEL per μm³ of cell volume. This number is similar to that found in Escherichia coli growing at 46°C, close to its maximal growth temperature, and a condition at which there is a major increase in the levels of chaperonins and other stress proteins. It is estimated that at 23°C, 10% of the B. aphidicola protein is GroEL. When S. graminum grown at 23°C was shifted to 33°C for 1 day and subsequently to 23°C, there was no change in the level of GroEL or the rate of growth. It is possible that the high level of GroEL in the endosymbiont masked an increase in the protein owing to a heat shock response.     

Aromatic amino acid biosynthesis in Buchnera aphidicola (Endosymbiont of aphids): Cloning and sequencing of a DNA fragment containing aroH-thrS-infC-rpmI-rplT

A 4.5-kilobase DNA fragment from Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, was cloned and sequenced. On the basis of homology to Escherichia coli, the following genes were found in the order listed: aroH-thrS-infC-rpmI-rplT. AroH corresponds to the E. coli tryptophan-inhibited 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase. Evidence was presented indicating that this is the sole gene for DAHP synthase in the B. aphidicola genome. This enzyme initiates the complex branched pathway leading to aromatic amino acid biosynthesis. The presence of aroH is consistent with past observations indicating that aphid endosymbionts are able to synthesize tryptophan for the aphid host. thrS, infC, rpmI, and rplT correspond to genes for threonine tRNA synthase, initiation factor-3, and large ribosome subunit proteins L35 and L20, respectively. Sequence comparisons indicate some differences and similarities between E. coli and B. aphidicola with respect to the possible regulation of synthesis of these proteins.     

Impact of a parasitoid on the bacterial symbiosis of its aphid host

Embryo production in aphids is absolutely dependent on the function of symbiotic bacteria, mainly Buchnera, and the growth and development of koinobiont parasitoids in aphids requires the diversion of nutrients from aphid embryo production to the parasitoid. The implication that the bacterial symbiosis may be promoted in parasitized aphids to support the growing parasitoid was explored by analysis of the number and biomass of mycetocytes, and the aphid cells bearing Buchnera, in the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) parasitized by the wasp Aphidius ervi Haliday (Hymenoptera: Braconidae). Aphids hosting a young larval parasitoid bore more mycetocytes of greater total biomass, and embryos of lower biomass than unparasitized aphids. Furthermore, one of the three aphid clones tested, which limited teratocyte growth (giant cells of parasitoid origin having a trophic role), bore smaller mycetocytes and larger embryos, than one or both of the two aphid clones with greater susceptibility to the parasitoid. These data suggest that susceptibility of the aphid‐Buchnera symbiosis to parasitoid‐mediated manipulation may, directly or indirectly, contribute to aphid susceptibility to parasitoid exploitation.