Beatriz Sabater-Muñoz

Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea

To elucidate the co‐evolutionary relationships between phloem‐feeding insects and their secondary, or facultative, bacterial symbionts, we explore the distributions of three such microbes — provisionally named the R‐type (or PASS, or S‐sym), T‐type (or PABS), and U‐type — across a number of aphid and psyllid hosts through the use of diagnostic molecular screening techniques and DNA sequencing. Although typically maternally transmitted, phylogenetic and pairwise divergence analyses reveal that these bacteria have been independently acquired by a variety of unrelated insect hosts, indicating that horizontal transfer has helped to shape their distributions. Based on the high genetic similarity between symbionts in different hosts, we argue that transfer events have occurred recently on an evolutionary timescale. In several instances, however, closely related symbionts associate with related hosts, suggesting that horizontal transfer between distant relatives may be rarer than transmission between close relatives. Our findings on the prevalence of these symbionts within many aphid taxa, along with published observations concerning their effects on host fitness, imply a significant role of facultative symbiosis in aphid ecology and evolution.

Extreme genome reduction in Buchnera spp.: Toward the minimal genome needed for symbiotic life

Buchnera is a mutualistic intracellular symbiont of aphids. Their association began about 200 million years ago, with host and symbiont lineages evolving in parallel since that time. During this coevolutionary process, Buchnera has experienced a dramatic decrease of genome size, retaining only essential genes for its specialized lifestyle. Previous studies reported that genome size in Buchnera spp. is very uniform, suggesting that genome shrinkage occurred early in evolution, and that modern lineages retain the genome size of a common ancestor. Our physical mapping of Buchnera genomes obtained from five aphid lineages shows that the genome size is not conserved among them, but has been reduced down to 450 kb in some species. Here we show evidence of six species with a genome size smaller than Mycoplasma genitalium, the smallest bacterial genome reported thus far (580 kb). Our findings strongly suggest that the Buchnera genome is still experiencing a reductive process toward a minimum set of genes necessary for its symbiotic lifestyle.

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.

PHYLOGENETIC EVIDENCE FOR HYBRID ORIGINS OF ASEXUAL LINEAGES IN AN APHID SPECIES

Abstract.— Understanding the mode of origin of asexuality is central to ongoing debates concerning the evolution and maintenance of sexual reproduction in eukaryotes. This is because it has profound consequences for patterns of genetic diversity and ecological adaptability of asexual lineages, hence on the outcome of competition with sexual relatives both in short and longer terms. Among the possible routes to asexuality, hybridization is a very common mechanism in animals and plants. Aphids present frequent transitions from their ancestral reproductive mode (cyclical parthenogenesis) to permanent asexuality, but the mode of origin of asexual lineages is generally not known because it has never been thoroughly investigated with appropriate molecular tools. Rhopalosiphum padi is an aphid species with coexisting sexual (cyclically parthenogenetic) and asexual (obligately parthenogenetic) lineages that are genetically distinct. Previous studies have shown that asexual lineages of R. padi are heterozygous at most nuclear loci, suggesting either that they have undergone long‐term asexuality (under which heterozygosity tends to increase) or that they have hybrid origins. To discriminate between these alternatives, we conducted an extensive molecular survey combining the sequence analysis of alleles of two nuclear DNA markers and mitochondrial DNA haplotypes in sexual and asexual lineages of R. padi. Both nuclear and cytoplasmic markers clearly showed that many asexual lineages have hybrid origins, the first such demonstration in aphids. Our results also indicated that asexuals result from multiple events of hybridization between R. padi and an unknown sibling species, and are of recent origin (contradicting previous estimates that asexual R. padi lineages were of moderate longevity). This study constitutes another example that putatively ancient asexual lineages are actually of much more recent origin than previously thought. It also presents a robust approach for testing whether hybrid origin of asexuality is also a common phenomenon in aphids.

Tracking medfly predation by the wolf spider, Pardosa cribata Simon, in citrus orchards using PCR-based gut-content analysis.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), which is often controlled chemically, is a major citrus pest in Spain; however, alternative biological control strategies such as those based on the conservation of polyphagous predators should be developed. The wolf spider, Pardosa cribata Simon, is an abundant predator found in citrus orchards in eastern Spain. In this study, we have evaluated polymerase chain reaction (PCR)-based techniques as a means of detecting C. capitata DNA remains in P. cribata specimens. To do so, two pairs of C. capitata species-specific primers were designed and tested. Primer specificity was tested on species closely related to C. capitata and with other pests and natural enemies present in citrus orchards. Medfly DNA was detectable in 100% of P. cribata from 0 to 12 h post ingestion for both primer pairs, decreasing to 37% at 96 h after prey ingestion for one pair of primers. DNA detectability half-lives were of 78.25 h and 78.08 h for each pair of primers but no statistical differences were found between them. Pardosa cribata specimens were field-collected daily after sterile C. capitata pupae had been deployed in the citrus orchard. Afterwards, the wolf spiders were analyzed and DNA remains of C. capitata were detected in 5% of them, with a peak of 15% coinciding with maximum C. capitata emergence. This study is the first to reveal the potential use of DNA markers to track medfly predation by P. cribata in citrus orchards and provides a new tool to estimate the potential role of this spider in biological-control conservation programs.

Deciphering reproductive polyphenism in aphids

Summary Polyphenism, which allows one given genotype to produce several discrete phenotypes, is an extreme case of phenotypic plasticity and is mainly found in arthropods. Social insects are the canonical example of polyphenism with the development of castes in the colonies. However, aphids display one of the largest range of polyphenisms, notably by producing winged or wingless, as well as asexual or sexual forms, depending on environmental conditions. During spring and summer, aphids reproduce by viviparous parthenogenesis, whereas in autumn they enter sexual reproduction. This switch in reproductive mode is triggered by changes in photoperiod and temperature. Here, the data accumulated since the 1960s on the identification of photoperiodic clocks, counter and putative neural photoreceptors that participate in this reproductive shift are reviewed. After perception, the photoperiodic signal is transduced through the secretion of hormones (juvenile hormones may well be involved) which, in turn, may act on the target cells, namely the oocytes. In short-day conditions, oocytes enter meiosis and produce haploid eggs which develop a 2n embryo after fertilisation. By contrast, in long-day conditions, a single maturation division produces 2n oocytes which immediately enter parthenogenetic embryogenesis. A physiological model of the determination of sexual vs. asexual reproduction in aphids is proposed and viewed from the perspective of newly initiated molecular studies.

Annotated expressed sequence tags and xenobiotic detoxification in the aphid Myzus persicae (Sulzer)

Aphids (Hemiptera: Aphididae) are phytophagous insects that are important agricultural pests. The enormous negative economic impacts caused by aphids worldwide are well known, and are mostly due to their high multiplication rate and the transmission of phytopathogenic viruses. Aphid management strategies mainly involve chemical treatments which are pollutants and are increasingly inefficient, since aphids have developed multiple insecticide‐resistant mechanisms. Among the most economically important species is the green peach aphid Myzus persicae Sulzer (Aphididae: Macrosiphini), which is able to colonize a wide range of host plants belonging to many different families, and transmits numerous plant viruses. Because of its large prevalence, M. persicae has been the target of massive insecticide treatments; consequently, it has evolved several insecticide‐resistant mechanisms. In this work, a collection of expressed genes from M. persicae is presented in order to identify putative genes involved in xenobiotic detoxification. After cDNA cloning and sequencing, 959 expressed sequence tags (EST) were annotated. Most sequences matched known genes corresponded to metabolism proteins (26%), ribosomal proteins (23%) and structural proteins (8%). Among them, several sequences corresponded to proteins putatively involved in sensing, degradation or detoxification of plant xenobiotic products.

Comparative molecular evolution of primary (Buchnera) and secondary symbionts of aphids based on two protein-coding genes.

Abstract. A+T content, phylogenetic relationships, codon usage, evolutionary rates, and ratio of synonymous versus non-synonymous substitutions have been studied in partial sequences of the atpD and aroQ/pheA genes of primary (Buchnera) and secondary symbionts of aphids and a set of selected non-symbiotic bacteria, belonging to the five subdivisions of the Proteobacteria. Compared to the homologous genes of the last group, both genes belonging to Buchnera behave in a similar way, showing a higher A+T content, forming a monophyletic group, a loss in codon bias, especially in third base position, an evolutionary acceleration and an increase in the number of non-synonymous substitutions, confirming previous results reported elsewhere for other genes. When available, these properties have been partly observed with the secondary symbionts, but with values that are intermediate between Buchnera and free living Proteobacteria. They show high A+T content, but not as high as Buchnera, a non-solved phylogenetic position between Buchnera, and the other γ-Proteobacteria, a loss in codon bias, again not as high as in Buchnera and a significant evolutionary acceleration in the case of the three atpD genes, but not when considering aroQ/pheA genes. These results give support to the hypothesis that they are symbionts at different stages of the symbiotic accommodation to the host.

A Novel Molecular Approach to Assess Mating Success of Sterile Ceratitis capitata (Diptera: Tephritidae) Males in Sterile Insect Technique Programs

Abstract Areawide sterile insect technique (SIT) programs against Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), are increasingly implemented worldwide. A key issue in SIT is to assess mating success of released sterile males, which could be currently estimated by egg hatchability and by stored sperm head measurements. We report here on a novel molecular approach that would allow detecting the presence of Mediterranean fruit fly sterile male sperm in the female spermathecae under field conditions, as a precise marker to assess mating performance. The simplicity (only two polymerase chain reactions) and reliability of this method, jointly with the capability to detect Vienna sperm in wild Mediterranean fruit fly maintained in monitoring traps for 7 d under field conditions, suggest that it could be an efficient tool when coupled with areawide SIT programs.

Alternatives to ginger root oil aromatherapy for improved mating performance of sterile Ceratitis capitata (Diptera: Tephritidae) males

Autocidal control of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) is an environmentally friendly and safe practice employed successfully worldwide. A key component in the Sterile Insect Technique (SIT) is to release sterile males that can compete with wild males in nature. Exposure of sterile males to ginger root oil (GRO) in the form of aromatherapy prior to release has been shown to significantly improve male competitiveness and performance. However, alternatives to GRO exposure are desired to (i) reduce personnel costs and handling‐time requirements and (ii) find new aromatherapeutic compounds. In this study, alternatives to GRO aromatherapy were investigated, such as incorporating GRO into the post‐teneral diet, or employing other molecules (limonene and linalool) occurring naturally in citrus release areas. Our results reveal that both the GRO‐supplemented diet and linalool aromatherapy were equal to the GRO aromatherapy control in improving mating performance in sterile males. In contrast, exposure to limonene did not afford a mating advantage to sterile mates when compared to the control. In addition, sterile male survival was not affected by any of the treatments tested. Implications for how both new approaches could be implemented in SIT programmes in citrus production areas are discussed.