David Martínez-Torres

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.

Discovery and molecular characterization of a plasmid localized in Buchnera sp. bacterial endosymbiont of the aphid Rhopalosiphum padi

We have identified and completely sequenced a novel plasmid isolated from the aphid Rhopalosiphum padi. Evidence which suggests that the plasmid occurs localized within the bacterial endosymbionts is presented. The plasmid contains the four genes that constitute the entire leucine operon. This fact makes it really unique since most plasmids are dispensable and lack genes that encode essential anabolic functions. Four more phloem-feeding aphid species also seem to contain homologous plasmids.Although further work is necessary, we hypothesize that this plasmid has appeared during the evolution of the symbiotic association between the aphid and the bacterial endosymbiont. The fact that this plasmid contains the entire leucine operon can be related to physiological evidence showing that the aphid hosts diet of plant phloem is deficient in essential amino acids.

Molecular characterization of cyclic and obligate parthenogens in the aphid Rhopalosiphum padi (L.)

Holocyclic clones of the aphid Rhopalosiphum padi (L.) reproduce by cyclic parthenogenesis, whereas anholocyclic individuals are obligate parthenogens. Mitochondrial DNA (mtDNA) and random amplified polymorphic DNA markers in R. padi as well as plasmid DNA markers of its bacterial endosymbiont, Buchnera aphidicola, were examined to determine the extent of genetic divergence between clones with these differing breeding systems. These analyses revealed that cyclically parthenogenetic lineages possessed differing mtDNA and plasmid haplotypes than most obligately asexual clones. The extent of sequence divergence between these maternally inherited molecules suggests a relatively ancient origin of asexuality. Our work also identified a random amplified polymorphic DNA marker linked to the life-cycle variation in R. padi. This marker not only permits the rapid diagnosis of breeding system but sets the stage for studies to identify the gene(s) controlling this variation in mode of reproduction.

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.

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.

Morphological stasis of two species belonging to the L-morphotype in the Brachionus plicatilis species complex

Detection and characterization of sibling species complexes in zooplankton are critical to understanding their ecological responses and patterns of evolution. The taxon Brachionus plicatilis is a complex of at least 14 species with three major, deeply diverged clades, which are morphologically distinct. We studied morphometric differences between two species – B. plicatilis sensu stricto and B. ‘Manjavacas’– which belong to the L-(large) morphotype and often co-occur in ponds or lakes. B. plicatilis s.s. was on average 6% longer than B. ‘Manjavacas’. They differed significantly in the measurements related to lorica spines. A significant discriminant function relating spine measurements was found, however, individuals from each species showed extensive overlap. Our morphometric data provide additional evidence for the species status of B. plicatilis s.s. and B. ‘Manjavacas’. Since these are ancient species, our results support that a morphological stasis occurs in these taxa. We identified COI restriction sites for PvuII and KpnI which are diagnostic for B. ‘Manjavacas’ and B. plicatilis s.s., respectively. We conclude that morphometry is not useful in classifying the two species. At present, this can only be done reliably using molecular methods.

Identification and characterization of circadian clock genes in the pea aphid Acyrthosiphon pisum

The molecular basis of circadian clocks is highly evolutionarily conserved and has been best characterized in Drosophila and mouse. Analysis of the Acyrthosiphon pisum genome revealed the presence of orthologs of the following genes constituting the core of the circadian clock in Drosophila: period (per), timeless (tim), Clock, cycle, vrille, and Pdp1. However, the presence in A. pisum of orthologs of a mammal‐type in addition to a Drosophila‐type cryptochrome places the putative aphid clockwork closer to the ancestral insect system than to the Drosophila one. Most notably, five of these putative aphid core clock genes are highly divergent and exhibit accelerated rates of change (especially per and tim orthologs) suggesting that the aphid circadian clock has evolved to adapt to (unknown) aphid‐specific needs. Additionally, with the exception of jetlag (absent in the aphid) other genes included in the Drosophila circadian clock repertoire were found to be conserved in A. pisum. Expression analysis revealed circadian rhythmicity for some core genes as well as a significant effect of photoperiod in the amplitude of oscillations.

Assessing the nucleotide diversity of three aphid species by RAPD

A method is presented for the estimation of nucleotide diversity and genetic structure of populations from RAPD (random amplified polymorphic DNA) data. It involves a modification of the technique developed by Lynch and Crease (1990) for the case of restriction sites as survey data. As new elements the method incorporates (i) dominance correction, (ii) values of asexual reproduction of the populations sampled, and (iii) an analytical variance of the number of nucleotide substitutions per site.

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.

Genetic variation in natural populations of the aphid Rhopalosiphum padi as revealed by maternally inherited markers

A survey on 148 clones of the aphid Rhopalosiphum padi from 11 widespread localities has been carried out to study the genetic structure of populations of this species as revealed by mitochondrial DNA restriction site and length polymorphisms as well as by restriction site analysis of a maternally inherited plasmid carried by the aphid eubacterial endosymbiont Buchnera aphidicola. Our results support the existence in the area under study of two main aphid maternal lineages strikingly coincidental with the two main reproductive categories displayed by this species. Those aphid clones possessing an incomplete life cycle that lacks the sexual phase (anholocyclic or androcyclic clones) show mitochondrial DNA (mtDNA) haplotype I and plasmid haplotype I, whereas those clones displaying the complete life cycle (holocyclic clones) posses some other distinct mtDNA haplotypes closely related to each other and plasmid haplotype II. While restriction‐site analysis of maternally inherited markers points to a relatively ancient origin of anholocycly/androcycly (between 460 000 and 1 400 000 years) followed by interrupted gene flow with respect to the ancestral holocyclic population, mtDNA size variation also suggests that historical stochastic processes have a different effect on the evolution of both main aphid lineages. Evidence of occasional nuclear gene flow between lineages and its consequences on the correspondence between maternally inherited haplotypes and life cycle are also presented and discussed.