Structure of the dnaA region of the endosymbiont, Buchnera aphidicola, of aphid Schizaphis graminum

Buchnera aphidicola is an intracellular prokaryote (endosymbiont)that lives in the body cavity of the aphid. Phylogenetic studiesindicated that it is closely related to Escherichia coli andmembers of Enterobacteria. The gene order of the region containingthe dnaA gene is well conserved in many bacteria. Seven genesof the endosymbiont of the aphid Schizaphis graminum, gyrB,dnaN, dnaA, rpmH, rnpA, yidD, and 60K, were found to be homologousin sequence and relative location to those of E. coli. We havefurther sequenced the region downstream of the 60K gene to elucidatethe boundary of the conserved region, and found that one moregene, thdF , is conserved. The comparison of gene organizationsof the dnaA region of the related bacteria supported the closephylogenetic relationship of B. aphidicola to E. coli. In addition,we have identified groES and groEL genesnext to the thdF gene.GroEL protein was reported to be expressed at an elevated levelin the endosymbionts of aphids, and is considered to play animportant role in their association with the aphid host. Comparisonof the structure of the groE operon with that of the endosymbiontof the aphid Acyrthosiphon pisum revealed the conservation ofa sequence resembling the E. coli consensus heat shock promoter,and this sequence may be responsible for the high expressionof the groEL gene in aphid endosymbionts.     

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.     

Determining the relative roles of different aphid species as vectors of cucumber mosaic and bean yellow mosaic viruses in lupins

Glasshouse and field studies were done to determine the relative roles of different colonising and non-colonising aphid species as vectors of two non-persistently transmitted viruses, cucumber mosaic cucumovirus (CMV) and bean yellow mosaic potyvirus (BYMV) in narrow-leafed lupin (Lupinus angustifolius) crops in Australia. The abilities of nine different aphid species in transmitting CMV from infected to healthy lupins and BYMV from infected subterranean clover to healthy lupins were compared in the glasshouse using 5–10 min acquisition access feeds. The percentage transmission efficiencies found with lupin-colonising aphid species were (CMV/BYMV): Acyrthosiphon kondoi (6/15), Aphis craccivora (10/14) and Myzus persicae (11/77). With non-colonising species the respective efficiencies were: Brachycaudus rumexicolens (0.9/0), Lipaphis erysimi (4/8), Rhopalosiphum maidis (9/6), R. padi (5/5), Sitobion miscanthi (2/11) and Therioaphis trifolii (4/5). When flying aphids were trapped in the field in four successive years (1993–1996) on vertical nets downwind of virus-infected lupins, 13 different species were caught at a “wheatbelt” site and 18 at an urban irrigated site. Of 2833 aphids caught at the “wheatbelt” site, 64 transmitted CMV to lupin test plants. At the irrigated site, numbers of aphids transmitting CMV/numbers caught were 12/186 while the corresponding numbers for BYMV were 11/727. M. persicae, A. kondoi and R. padi transmitted both viruses, while additional vectors of CMV found were A. craccivora, Acyrthosiphon pisum, B. rumexicolens, L erysimi, R. insertum, T. trifolii and Toxoptera citricidus. Averaged over four years, A. kondoi accounted for 50% of CMV transmissions at the “wheatbelt” site, M. persicae for 16% and R. padi for 22%, and these three species were caught in the greatest numbers, comprising 28%, 13% and 37% respectively of the total catch. At the irrigated site R. padi accounted for half the CMV transmissions, while R. padi and A. kondoi together accounted for most of the BYMV transmissions. R. padi, A. kondoi, M. persicae and T. citridus were the most common aphid species at this site. These findings suggest that M. persicae, A. kondoi and R. padi are the aphid species likely to be most important as vectors of CMV and BYMV in narrow-leafed lupins grown in mediterranean-type climatic zones of southern Australia.     

Growth Kinetics of the Endosymbiont Buchnera aphidicola in the Aphid Schizaphis graminum

The aphid Schizaphis graminum is dependent on its prokaryotic endosymbiont, Buchnera aphidicola. As a means of determining B. aphidicola numbers during the growth cycle of the aphid we have used the quantitative PCR to measure the number of copies of rrs (the gene coding for 16S rRNA, which is present as one copy in the B. aphidicola genome). In addition we have measured the aphid wet weight and the DNA and protein content. The results indicate an approximately parallel (23- to 31-fold) increase of these properties during the period of aphid growth. A 1-day-old aphid (24 μg [wet weight]) has 0.2 × 10⁶ copies of rrs, while a 9-day-old aphid (497 μg [wet weight]) has 5.6 × 10⁶ copies. The coupling of endosymbiont and aphid growth is consistent with the requirement of the endosymbiont for growth and reproduction of the aphid.     

Comparative analysis of two genomic regions among four strains of Buchnera aphidicola, primary endosymbiont of aphids

Preliminary analysis of two selected genomic regions of Buchnera aphidicola BCc, the primary endosymbiont of the cedar aphid Cinara cedri, has revealed a number of interesting features when compared with the corresponding homologous regions of the three B. aphidicola genomes previously sequenced, that are associated with different aphid species. Both regions exhibit a significant reduction in length and gene number in B. aphidicola BCc, as it could be expected since it possess the smallest bacterial genome. However, the observed genome reduction is not even in both regions, as it appears to be dependent on the nature of their gene content. The region fpr-trxA, that contains mainly metabolic genes, has lost almost half of its genes (45.6%) and has reduced 52.9% its length. The reductive process in the region rrl-aroK, that contains mainly ribosomal protein genes, is less dramatic, since it has lost 9.3% of genes and has reduced 15.5% of its length. Length reduction is mainly due to the loss of protein-coding genes, not to the shortening of ORFs or intergenic regions. In both regions, G+C content is about 4% lower in BCc than in the other B. aphidicola strains. However, when only conserved genes and intergenic regions of the four B. aphidicola strains are compared, the G+C reduction is higher in the fpr-trxA region.     

Symbionin,an aphid endosymbiont-specific protein—III: Symbionin present in the male,ovipara and fundatrix

Electron microscopic observations demonstrated that the male of the kondo aphid, A. kondoi harbors intracellular symbionts different in shape from those in the viviparous female. Two-dimensional gel electrophoresis indicated that the endosymbiont in the male is less active in synthesizing symbionin, an aphid endosymbiont-specific protein than that in the viviparous female. Symbionin was also found in the winter egg though it was much less in amount than proteins related to the yolk formation. In the fundatrix which hatches out of the fertilized winter egg, symbionin was the most abundant protein.     

基于线粒体COⅠ基因序列的小萤叶甲属部分种类分子系统学研究（鞘翅目：叶甲科：萤叶甲亚科）

本文目的是通过对小萤叶甲属部分种类的线粒体COⅠ基因进行比较,探讨小萤叶甲属昆虫进化与寄主植物之间的关系,同时对几种分类地位模糊的昆虫进行分析和归类。测定了我国菱角萤叶甲Galerucella birmanica Jacoby和褐背小萤叶甲Galerucella grisescens Joannis以及小猿叶甲Phaedon brassicae Baly线粒体COⅠ基因720 bp序列,并调用GenBank中小萤叶甲属等其他8种昆虫的同源序列,对序列的碱基组成、转换颠换、遗传距离等进行了分析。并以小猿叶甲为外群,分别采用邻接法（NJ）、最大简约法（MP）和贝叶斯推论法（BI）建立这些种的分子系统发育关系。序列分析结果表明：小萤叶甲属昆虫COⅠ基因A+T含量平均为71.8％,存在较强的A+T含量偏向性,氨基酸的变异率为18.3％; 小萤叶甲属与外群之间的遗传距离(0.169～0.198)远远大于属内种间的距离(0.001～0.134)。依据分子系统树结果我们推测小萤叶甲属昆虫的进化与寄主植物之间有着显著的关系,在传统分类学上曾隶属于其他属的几种昆虫与小萤叶甲昆虫有着更近的亲缘关系。     

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     

Fungi attacking the blue-green lucerne aphid in New Zealand

Abstract

The blue-green lucerne aphid, Acyrthosiphon kondoi, is attacked on lucerne in New Zealand by the entomophthoraceous fungi Entomophthora aphidis and E. ignobilis (= E. thaxteriana). E. aphidis appeared to be the most pathogenic of the two species, causing an epizootic in one of the sampled fields which was severely infested by the aphid.     

Occurrence and Transmission of Facultative Endosymbionts in Aphids

The occurrence of a secondary bacterial symbiont (PASS) of pea aphid, Acyrthosiphon pisum (Harris), was detected by polymerase chain reaction (PCR) with specific nucleotide primers based on PASS 16S rDNA nucleotide sequences from over 80% (50/57) of clones of pea aphid collected from widely separated locations in California. PASS was also detected by PCR in both red and green phenotypes of rose aphid, Macrosiphum rosae (L.), but not in six other species of aphids examined, including blue alfalfa aphid (A. kondoi Shinji). The nucleotide sequences of the PCR-amplified, partial 16S rDNAs (1060 bp) from pea aphid and rose aphid were identical and 99.9% similar to the published 16S rDNA of PASS. PASS and a recently described new rickettsia of pea aphid (PAR) were transmitted by needle injection of hemolymph from positive pea aphid clones into negative clones and into blue alfalfa aphids. Both PASS and PAR were maintained in the offspring of some of the injected mother aphids via high rate of maternal transmission. Received: 18 September 1996 / Accepted: 30 September 1996     

Cospeciation Between the Primary Endosymbionts of Mealybugs and Their Hosts

Mealybugs have an association with prokaryotic endosymbionts that are located in specialized cells called bacteriocytes. In order to compare the phylogeny of the host with that of the previously published phylogeny of the endosymbionts, 3.1 to 3.2 kilobase DNA fragments containing mitochondrial cytB (part), nd1,16S ribosomal DNA(rDNA), and 12S rDNA (part) were amplified and sequenced. A phylogenetic analysis of the data and a comparison with the trees obtained from endosymbiont genes and host 18S and 28S rDNA indicated that all the trees were similar. This result is consistent with an infection of a mealybug ancestor with a precursor of the endosymbiont followed by the vertical transmission of the endosymbiont to progeny. Comparison of the guanine + cytosine (G + C) contents of the mealybug mitochondrial genes with the same genes from other members of Sternorrhyncha and Arthropoda indicated that the mealybug genes had unusually low G + C contents in their DNAs (10.2 to 11.1 mol%).     

Nucleotide sequence and presumed secondary structure of the 28S rRNA of pea aphid implication for diversification of insect rRNA

Determination of the entire nucleotide sequence of the aphid 28S ribosomal RNA gene (28S rDNA) revealed that it is 4,147 by in length with a G + C content of 60.3%. Based on the nucleotide sequence, we constructed a presumed secondary-structure model of the aphid 28S rRNA which indicated that the aphid 28S rRNA is characterized by the length and high G + C content of its variable regions. The G + C content of the aphid's variable regions was much higher than that of the entire sequence of the 28S rRNA, which formed a striking contrast to those ofDrosophila with the G + C content much lower than the entire 28S molecule. In this respect, the aphid 28S rRNA somewhat resembled those of vertebrates. This is the third report of a complete large-subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a nondipterous insect. Correspondence to: H. Ishikawa     

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.     

Molecular phylogeny reveals the existence of two sibling species in the aphid pest Brachycaudus helichrysi (Hemiptera: Aphididae)

Piffaretti, J., Vanlerberghe‐Masutti, F., Tayeh, A., Clamens, A.‐L., C?ur d’Acier, A. & Jousselin E. (2012). Molecular phylogeny reveals the existence of two sibling species in the aphid pest Brachycaudus helichrysi (Hemiptera: Aphididae). —Zoologica Scripta, 41, 266–280. Brachycaudus helichrysi is a worldwide polyphagous aphid pest that seriously damages its primary hosts (Prunus spp.) and the various cultivated plants among its secondary hosts (e.g. sunflower). A recent study of the Brachycaudus genus suggested that this species might encompass two differentiated lineages. We tested this hypothesis, by carrying out a phylogenetic study of this aphid pest based on worldwide sampling and the evaluation of mitochondrial, nuclear and Buchnera aphidicola DNA markers. We show that this species is actually an amalgamation of two sibling taxa, B. helichrysi H1 and B. helichrysi H2, that seem to have overlapping geographic ranges and herbaceous host plant preferences. These two taxa displayed levels of genetic divergence as great as those generally found between sister species in the Brachycaudus genus, suggesting that they actually correspond to two distinct species. Our phylogenetic reconstructions revealed a degree of incongruence between the topologies obtained with the aphid gene data set and with data for a DNA marker from its primary endosymbiont. We identified possible reasons for this observation and discuss the ecological and genotypic data suggesting that B. helichrysi H1 and B. helichrysi H2 have different life cycles.     

Molecular evolution of bat color vision genes

The two suborders of bats, Megachiroptera (megabats) and Microchiroptera(microbats), use different sensory modalities for perceivingtheir environment. Megabats are crepuscular and rely on a well-developedeyes and visual pathway, whereas microbats occupy a nocturnalniche and use acoustic orientation or echolocation more thanvision as the major means of perceiving their environment. Inview of the differences associated with their sensory systems,we decided to investigate the function and evolution of colorvision (opsin genes) in these two suborders of bats. The middle/longwavelength (M/L) and short wavelength (S) opsin genes were sequencedfrom two frugivorous species of megabats, Haplonycteris fischeriand Pteropus dasymallus formosus, and one insectivorous speciesof microbat, Myotis velifer. Contrary to the situation in primates,where many nocturnal species have lost the functional S opsingene, both crepuscular and strictly nocturnal species of batsthat we examined have functional M/L and S opsin genes. Surprisingly,the S opsin in these bats may be sensitive to UV light, whichis relatively more abundant at dawn and at dusk. The M/L opsinin these bats appears to be the L type, which is sensitive tored and may be helpful for identifying fruits among leaves orfor other purposes. Most interestingly, H. fischeri has a recentduplication of the M/L opsin gene, representing to date theonly known case of opsin gene duplication in non-primate mammals.Some of these observations are unexpected and may provide insightsinto the effect of nocturnal life on the evolution of opsingenes in mammals and the evolution of the life history traitsof bats in general.     

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.     

Influence of the 3'-UTR-length of mKIAA cDNAs and their Sequence Features to the mRNA Expression Level in the Brain

We have previously described the sequence features of 1500 mouseKIAA (mKIAA) genes in comparison with those of human KIAA genes(Okazaki, N., Kikuno, R., Inamoto, S., Hara, Y., Nagase, T.,Ohara, O., and Koga, H. 2002, DNA Res., 9, 179–188; Okazaki,N., Kikuno, R., Ohara, R., Inamoto, S., Aizawa, H., Yuasa, S.,Nakajima, D., Nagase, T., Ohara, O., and Koga, H. 2003, DNARes., 10, 35–48; Okazaki, N., Kikuno, R., Ohara, R., Inamoto,S., Koseki, H., Hiraoka, S., Saga, Y., Nagase, T., Ohara, O.,and Koga, H. 2003, DNA Res., 10, 167–180; and Okazaki,N., F-Kikuno, R., Ohara, R., Inamoto, S., Koseki, H., Hiraoka,S., Saga, Y., Seino, S., Nishimura, M., Kaisho, T., Hoshino,K., Kitamura, H., Nagase, T., Ohara, O., and Koga, H. 2004,DNA Res., 11, 205–218). To validate the orthologous relationshipbetween mKIAA and KIAA genes in detail, we examined their chromosomalpositions and evolutionary rate of synonymous substitutionsand confirmed that >93% of the mKIAA/KIAA gene pairs areorthologous. During the sequence analysis of mKIAA genes, wefound that 3'-untranslated region (3'-UTR) lengths of mKIAAand KIAA genes are extremely long. In the meanwhile, we havealso examined the tissue-specific expression of 1700 mKIAA genesusing cDNA microarray and verified predominantly their expressionin adult brain (Koga, H., Yuasa, S., Nagase, T., Shimada, K.,Nagano, M., Imai, K., Ohara, R., Nakajima, D., Murakami, M.,Kawai, M., Miki, F., Magae, J., Inamoto, S., Okazaki, N., Ohara,O. 2004, DNA Res., 11, 293–304). To connect these twoevidences, we statistically analysed the relationship betweenthem by using the mKIAA genes. Consequently, a positive correlationwas observed between the 3'-UTR lengths and the relative expressionintensities in adult brain. Furthermore, we searched sequenceelements in the 3'-UTR possibly related with their expressionand found some candidates regarding the brain-specific expression.     

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.     

Interspecific variation in the escape responses of aphids: effect on risk of predation from foliar-foraging and ground-foraging predators

A series of laboratory experiments was conducted to determine the effect of interspecific differences on prey defensive behavior on the susceptibility of two aphid species (Acyrthosiphon pisum and A. kondoi) to a ground-foraging predator, Harpalus pennsylvanicus, and a foliar-foraging predator, Coccinella septempunctata. These organisms are representative of a biologically and economically important predator/prey system in alfalfa. The primary defensive behavior of both aphid species toward C. septempunctata was to “drop” from the plant. Both aphid species were significantly more likely to drop from the plant in the presence of C. septempunctata. However, when C. septempunctata was present, a significantly lower proportion of A. kondoi individuals dropped (0.42 ± 0.07) compared to A. pisum (0.73 ± 0.08). As a result of their lower propensity to drop from the plant A. kondoi individuals are significantly more likely to be consumed by C. septempunctata. Conversely, the higher propensity of A. pisum individuals to drop increased their susceptibility to ground-foraging predators. When A. pisum was the prey species, ground-foraging predators made a significant contribution to overall aphid suppression and there was a significant synergistic interaction between ground and foliar-foraging predators. When A. kondoi was the prey there was no interaction between the predator species. As either a cause or consequence of its higher propensity to drop, A. pisum seems to be more adapted for survival and dispersal off the plant. In comparison to A. kondoi individuals, A. pisum individuals relocate plants more quickly (63 ± 41 s vs. 164 ± 39 s), disperse farther (18 ± 1.7 cm vs. 13 ± 0.66 cm), and survive longer (37 ± 2.0 h vs. 25 ± 2.0) off the plant. This study demonstrates the importance of prey defensive behavior in determining the susceptibility of a prey species to a multiple-predator complex. Received: 24 February 1997 / Accepted:17 December 1997