Alex C. C. Wilson

SSCP is not so difficult: the application and utility of single‐stranded conformation polymorphism in evolutionary biology and molecular ecology

All genetic markers are estimators of DNA nucleotide sequence variation. Rather than obtaining DNA sequence data, it is cheaper and faster to use techniques that estimate sequence variation, although this usually results in the loss of some information. SSCP (single‐stranded conformation polymorphism) offers a sensitive but inexpensive, rapid, and convenient method for determining which DNA samples in a set differ in sequence, so that only an informative subset need be sequenced. In short, most DNA sequence variation can be detected with relatively little sequencing. SSCP has been widely applied in medical diagnosis, yet few studies have been published in population genetics. The utility and convenience of SSCP is far from fully appreciated by molecular population biologists. We hope to help redress this by illustrating the application of a single simple SSCP protocol to mitochondrial genes, nuclear introns, microsatellites, and anonymous nuclear sequences, in a range of vertebrates and invertebrates.

Aphid Thermal Tolerance Is Governed by a Point Mutation in Bacterial Symbionts

Symbiosis is a ubiquitous phenomenon generating biological complexity, affecting adaptation, and expanding ecological capabilities. However, symbionts, which can be subject to genetic limitations such as clonality and genomic degradation, also impose constraints on hosts. A model of obligate symbiosis is that between aphids and the bacterium Buchnera aphidicola, which supplies essential nutrients. We report a mutation in Buchnera of the aphid Acyrthosiphon pisum that recurs in laboratory lines and occurs in field populations. This single nucleotide deletion affects a homopolymeric run within the heat-shock transcriptional promoter for ibpA, encoding a small heat-shock protein. This Buchnera mutation virtually eliminates the transcriptional response of ibpA to heat stress and lowers its expression even at cool or moderate temperatures. Furthermore, this symbiont mutation dramatically affects host fitness in a manner dependent on thermal environment. Following a short heat exposure as juveniles, aphids bearing short-allele symbionts produced few or no progeny and contained almost no Buchnera, in contrast to aphids bearing symbionts without the deletion. Conversely, under constant cool conditions, aphids containing symbionts with the short allele reproduced earlier and maintained higher reproductive rates. The short allele has appreciable frequencies in field populations (up to 20%), further supporting the view that lowering of ibpA expression improves host fitness under some conditions. This recurring Buchnera mutation governs thermal tolerance of aphid hosts. Other cases in which symbiont microevolution has a major effect on host ecological tolerance are likely to be widespread because of the high mutation rates of symbiotic bacteria and their crucial roles in host metabolism and development.

Horizontal Gene Transfer from Diverse Bacteria to an Insect Genome Enables a Tripartite Nested Mealybug Symbiosis

The smallest reported bacterial genome belongs to Tremblaya princeps, a symbiont of Planococcus citri mealybugs (PCIT). Tremblaya PCIT not only has a 139 kb genome, but possesses its own bacterial endosymbiont, Moranella endobia. Genome and transcriptome sequencing, including genome sequencing from a Tremblaya lineage lacking intracellular bacteria, reveals that the extreme genomic degeneracy of Tremblaya PCIT likely resulted from acquiring Moranella as an endosymbiont. In addition, at least 22 expressed horizontally transferred genes from multiple diverse bacteria to the mealybug genome likely complement missing symbiont genes. However, none of these horizontally transferred genes are from Tremblaya, showing that genome reduction in this symbiont has not been enabled by gene transfer to the host nucleus. Our results thus indicate that the functioning of this three-way symbiosis is dependent on genes from at least six lineages of organisms and reveal a path to intimate endosymbiosis distinct from that followed by organelles.

Genomic insight into the amino acid relations of the pea aphid, Acyrthosiphon pisum, with its symbiotic bacterium Buchnera aphidicola.

The pea aphid genome includes 66 genes contributing to amino acid biosynthesis and 93 genes to amino acid degradation. In several respects, the pea aphid gene inventory complements that of its symbiotic bacterium, Buchnera aphidicola (Buchnera APS). Unlike other insects with completely sequenced genomes, the pea aphid lacks the capacity to synthesize arginine, which is produced by Buchnera APS. However, consistent with other insects, it has genes coding for individual reactions in essential amino acid biosynthesis, including threonine dehydratase and branched‐chain amino acid aminotransferase, which are not coded in the Buchnera APS genome. Overall the genome data suggest that the biosynthesis of certain essential amino acids is shared between the pea aphid and Buchnera APS, providing the opportunity for precise aphid control over Buchnera metabolism.

Microevolution, low clonal diversity and genetic affinities of parthenogenetic Sitobion aphids in New Zealand

In sharp contrast to their southeast Asian and European counterparts, Sitobion miscanthi and S. near fragariae aphids in Australia exhibit a complete absence of sexual reproduction. This demands an explanation within the context of the evolution and maintenance of sex and parthenogenesis. Accordingly, we executed a genetic analysis of the two species in neighbouring New Zealand. Microsatellites and single‐stranded conformation polymorphism/sequence analysis of the nuclear gene elongation factor 1α were used to identify aphid clones and confirm species identification, respectively. Karyotypic variation was also investigated. The New Zealand fauna showed few (nonrecombining) genotypes and appears to have received migrants from both Australia and Asia. Other genotypes have apparently arisen in situ in New Zealand, exhibiting stepwise mutation of microsatellite alleles and also karyotypic change. Thus, these data represent rare evidence of evolution within wild‐living parthenogenetic lineages. Karyotypic changes appear to occur at a rate even greater than that of microsatellite evolution. Strong geographical partitioning of genotypes/karyotypes was found, with certain ones predominating over large areas. These data suggest that clonal selection could be important in the distribution and patterning of genetic variation. We present a model to explain the genetic patterns, with particular reference to the absence of sexual reproduction in Sitobion aphids in New Zealand and Australia.

AphidBase: a centralized bioinformatic resource for annotation of the pea aphid genome

AphidBase is a centralized bioinformatic resource that was developed to facilitate community annotation of the pea aphid genome by the International Aphid Genomics Consortium (IAGC). The AphidBase Information System designed to organize and distribute genomic data and annotations for a large international community was constructed using open source software tools from the Generic Model Organism Database (GMOD). The system includes Apollo and GBrowse utilities as well as a wiki, blast search capabilities and a full text search engine. AphidBase strongly supported community cooperation and coordination in the curation of gene models during community annotation of the pea aphid genome. AphidBase can be accessed at http://www.aphidbase.com.

Genomic revelations of a mutualism: the pea aphid and its obligate bacterial symbiont

The symbiosis of the pea aphid Acyrthosphion pisum with the bacterium Buchnera aphidicola APS represents the best-studied insect obligate symbiosis. Here we present a refined picture of this symbiosis by linking pre-genomic observations to new genomic data that includes the complete genomes of the eukaryotic and prokaryotic symbiotic partners. In doing so, we address four issues central to understanding the patterns and processes operating at the A. pisum/Buchnera APS interface. These four issues include: (1) lateral gene transfer, (2) host immunity, (3) symbiotic metabolism, and (4) regulation.

Microsatellite isolation, linkage group identification and determination of recombination frequency in the peach-potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)

Fifteen polymorphic microsatellite markers were used to establish linkage groups and relative rates of recombination in male and female Myzus persicae (Sulzer) (Hemiptera: Aphididae) (peach-potato aphid). We cloned nine markers from M. persicae and for these we report primer sequences and levels of allelic diversity and heterozygosity in four Australian M. persicae populations. Of the remaining six loci, four loci, also cloned from M. persicae, were obtained from G. Malarky (Natural History Museum, London) and two loci from Sitobion miscanthi were used. Additionally, the primer sequences of locus M77, a locus monomorphic in M. persicae but polymorphic in the closely related Myzus antirrhinii, are presented. Eleven of the 15 polymorphic markers were autosomal and four were X-linked. A linkage analysis was performed on a European pedigree of aphids containing five families with between seven and 11 offspring each. There was no linkage between any loci in females. In males, several pairwise comparisons yielded no recombinant offspring. With the exception of locus M40, these observations were supported in a linkage analysis performed on larger families produced from Australian M. persicae crosses. Locus M40 showed segregation consistent with involvement in a translocation between autosomes 1 and 3 in European samples but not in the Australian samples. From the Australian crosses we report an absence of recombination in males but high recombination rates in females. One X chromosome and four autosomal linkage groups were identified and tentatively assigned to chromosomes. The relevance of achiasmate meiosis to the evolution of sex is discussed.

A dual-genome microarray for the pea aphid, Acyrthosiphon pisum, and its obligate bacterial symbiont, Buchnera aphidicola

BackgroundThe best studied insect-symbiont system is that of aphids and their primary bacterial endosymbiont Buchnera aphidicola. Buchnera inhabits specialized host cells called bacteriocytes, provides nutrients to the aphid and has co-speciated with its aphid hosts for the past 150 million years. We have used a single microarray to examine gene expression in the pea aphid, Acyrthosiphon pisum, and its resident Buchnera. Very little is known of gene expression in aphids, few studies have examined gene expression in Buchnera, and no study has examined simultaneously the expression profiles of a host and its symbiont. Expression profiling of aphids, in studies such as this, will be critical for assigning newly discovered A. pisum genes to functional roles. In particular, because aphids possess many genes that are absent from Drosophila and other holometabolous insect taxa, aphid genome annotation efforts cannot rely entirely on homology to the best-studied insect systems. Development of this dual-genome array represents a first attempt to characterize gene expression in this emerging model system.ResultsWe chose to examine heat shock response because it has been well characterized both in Buchnera and in other insect species. Our results from the Buchnera of A. pisum show responses for the same gene set as an earlier study of heat shock response in Buchnera for the host aphid Schizaphis graminum. Additionally, analyses of aphid transcripts showed the expected response for homologs of known heat shock genes as well as responses for several genes with unknown functional roles.ConclusionWe examined gene expression under heat shock of an insect and its bacterial symbiont in a single assay using a dual-genome microarray. Further, our results indicate that microarrays are a useful tool for inferring functional roles of genes in A. pisum and other insects and suggest that the pea aphid genome may contain many gene paralogs that are differentially regulated.

Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts

Significance Nutritional bacterial endosymbionts are housed in specialized host cells and are partitioned from the host cell cytoplasm by a host-derived symbiosomal membrane. This cellular organization isolates bacterial symbionts from nutrient pools in the host cell and makes possible host control of nutrient supply to bacterial symbionts. Here, using the aphid–Buchnera nutritional endosymbiosis, we demonstrate that the most active host glutamine (precursor) transporter, Acyrthosiphon pisum glutamine transporter 1, is competitively inhibited by arginine (a Buchnera-synthesized end product). We propose a model of endosymbiosis regulation in which precursor transport is regulated by a symbiont-synthesized end product. Thus, we provide insights into the molecular mechanism of host control of bacterial endosymbiont essential nutrient biosynthesis. Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid–Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses.