Identification of the prothoracicotropic hormone (Ptth) coding gene and localization of its site of expression in the pea aphid Acyrthosiphon pisum.

Insect hormones control essential aspects of physiology, behaviour and development in insects. The majority of insect hormones are peptide hormones that perform a highly diverse catalogue of functions. Prothoracicotropic hormone (PTTH) is a brain neuropeptide hormone whose main function is to stimulate the secretion of ecdysone (the moulting hormone) by the prothoracic glands in insect larvae thus playing a key role in the control of moulting and metamorphosis. Moreover, both PTTH release or blockade have been reported to act as a switch to terminate or initiate larval and pupal diapauses. In insects, diapause is a prevalent response often regulated by the photoperiod. It has been shown that PTTH participates as an output of the circadian clock and a role in photoperiodic processes is suggested in some insect species. Aphids (Hemiptera: Aphididae) reproduce by cyclical parthenogenesis with a sexual phase, induced by short photoperiods, that leads to the production of diapausing eggs. With the availability of the pea aphid (Acyrthosiphon pisum) genome, efforts to identify and characterize genes relevant to essential aspects of aphid biology have multiplied. In spite of its relevance, several genomic and transcriptomic studies on aphid neuropeptides failed to detect aphid PTTH amongst them. Here we report on the first identification of the aphid PTTH coding gene and the neuroanatomical localization of its expression in the aphid brain.

Identification, characterization and analysis of expression of genes encoding arylalkylamine N-acetyltransferases in the pea aphid Acyrthosiphon pisum.

Most organisms exhibit some kind of rhythmicity in their behaviour and/or physiology as an adaptation to the cyclical movements of the Earth. In addition to circadian rhythms, many organisms have an annual rhythmicity in certain activities, such as reproduction, migration or induction of diapause. Current knowledge of the molecular basis controlling seasonal rhythmicity, especially in insects, is scarce. One element that seems to play an essential role in the maintenance of both circadian and seasonal rhythms in vertebrates is the hormone melatonin. In vertebrates, the limiting enzyme in its synthesis is the arylalkylamine N-acetyltransferase (AANAT). Melatonin is also present in insects but the precise biochemical pathway and the enzymes involved in its synthesis are unknown. Insects possess phylogenetically distant arylalkylamine N-acetyltransferases but their involvement in melatonin synthesis still needs to be fully demonstrated. Aphids have a seasonally rhythmical life cycle, reproducing parthenogenetically by viviparity in favourable seasons but, in unfavourable seasons, they produce a single generation of sexual individuals. The length of the photoperiod is the main environmental factor that controls the mode of reproduction in aphids. Taking advantage of the availability of the genome of the aphid Acyrthosiphon pisum, we searched for genes encoding aphid arylalkylamine N-acetyltransferase homologues that could be candidates for participation in seasonal rhythmicity. We identified four AANAT genes, of which at least two (Ap-AANAT1 and Ap-AANAT3) showed highly significant variation in transcription levels depending on the photoperiod conditions. These results are discussed in the context of how seasonality can be controlled in aphids.

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.

Sex versus parthenogenesis: a transcriptomic approach of photoperiod response in the model aphid Acyrthosiphon pisum (Hemiptera: Aphididae).

Most aphids develop a cyclic parthenogenesis life-cycle. After several generations of viviparous parthenogenetic females, it 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 is a key factor inducing the sexual response. With the survey here reported we aimed at identifying a collection of candidate genes to participate at some point in the cascade of events that lead to the sexual phenotypes. Following a suppression subtractive hybridization methodology (SSH) on the model aphid Acyrthosiphon pisum, we built and characterised two reciprocal cDNA libraries (SDU and SDD) enriched respectively in genes up-regulated or down-regulated by short photoperiod conditions that lead to the sexual response in this aphid species. A total of 557 ESTs were obtained altogether representing 223 non-overlapping contigs. 29% of these were new sequences not present in previous aphid EST libraries. BLAST searches allowed putative identification of about 54% of the contigs present in both libraries. Relative quantification of expression through real-time quantitative PCR demonstrated the differential expression in relation with the photoperiod of 6 genes (3 up-regulated and 3 down-regulated by shortening the day length). Among these, expression of a tubulin gene, two cuticular proteins and a yet unidentified sequence along the day-night cycle was further investigated. Implications for current studies on gene regulation of the dichotomy sex vs. parthenogenesis in aphids are discussed.

Seasonal photoperiodism regulates the expression of cuticular and signalling protein genes in the pea aphid.

Seasonal photoperiodism in aphids is responsible for the spectacular switch from asexual to sexual reproduction. However, little is known on the molecular and physiological mechanisms involved in reproductive mode shift through the action of day length. Earlier works showed that aphid head, but not eyes, directly perceives the photoperiodic signal through the cuticle. In order to identify genes regulating the photoperiodic response, a 3321 cDNA microarray developed for the pea aphid, Acyrthosiphon pisum was used to compare RNA populations extracted from heads of short- and long-day reared aphids. Microarray analyses revealed that 59 different transcripts were significantly regulated, among which a majority encoded cuticular proteins and several encoded proteins involved in cellular signalling or signal transduction. These results were confirmed by quantitative RT-PCR experiments on two cuticular and three signalling protein genes. Complementary experiments eliminated moulting and circadian rhythms as putative confounding effects. Quantitative RT-PCR performed at additional developmental stages demonstrated the regulation of expression of cuticular and signalling protein genes during the whole process of photoperiod shortening. This suggests that photoperiodic changes could affect cuticle structure and cell to cell communication in the head of aphids in relation with the switch of reproductive modes.

Molecular systematics of aphids and their primary endosymbionts.

Aphids constitute a monophyletic group within the order Homoptera (i.e., superfamily Aphidoidea). The Aphidoidea originated in the Jurassic about 150 my ago from some aphidiform ancestor whose origin can be traced back to about 250 my ago. They exhibit a mutualistic association with intracellular bacteria (Buchnera sp.) related to Escherichia coli. Buchnera is usually considered the aphids' primary endosymbiont. The association is obligate for both partners. The 16S rDNA-based phylogeny of Buchnera from four aphid families showed complete concordance with the morphology-based phylogeny of their aphid hosts, which pointed to a single original infection in a common ancestor of aphids some 100-250 my ago followed by cospeciation of aphids and Buchnera. This study concentrated on the molecular phylogeny of both the aphids and their primary endosymbionts of five aphid families including for the first time representatives of the family Lachnidae. We discuss results based on two Buchnera genes (16S rDNA and the beta subunit of the F-ATPase complex) and on one host mitochondrial gene (the subunit 6 of the F-ATPase complex). Although our data do not allow definitive evolutionary relationships to be established among the different aphid families, some traditionally accepted groupings are put into question from both bacterial and insect data. In particular, the Lachnidae and the Aphididae, which from morphological data are considered recently evolved sister groups, do not seem to be as closely related as is usually accepted. Finally, we discuss our results in the light of the proposed parallel evolution of aphids and their endosymbionts.

A sodium channel point mutation is associated with resistance to DDT and pyrethroid insecticides in the peach-potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae).

The voltage-gated sodium channel is the primary target site of DDT and pyrethroid insecticides, and point mutations in the domain II region of the channel protein have been implicated in the knockdown resistant (kdr ) phenotype of several insect species. Here, we report that one of these mutations, a leucine-to-phenylalanine replacement in transmembrane segment IIS6, is also found in certain insecticide-resistant clones of the peach-potato aphid, Myzus persicae. The mutation was present in four clones with amplified E4 esterase genes, but was absent from both susceptible clones and those with amplified FE4 genes. The inferred presence of kdr-type resistance in the four E4 clones was subsequently confirmed by bioassays that showed this to be the primary mechanism of resistance to deltamethrin and DDT, although the esterase-based mechanism also contributes to the overall level of deltamethrin resistance. The kdr mutation on its own conferred 35-fold resistance to deltamethrin and this was enhanced up to 540-fold when it was present in a high (E4) esterase background. The esterase (FE4) mechanism was far less effective without the kdr mutation, conferring just 3-4-fold resistance to deltamethrin. These findings, and the linkage disequilibrium of the kdr mutation within clones overproducing the E4 esterase, have important implications for the evolution of resistance in this insect and for the use of pyrethroid sprays in the management of M. persicae populations in the field.

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.

Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s.

Pyrethroid-impregnated bednets are playing an increasing role for combating malaria, especially in stable malaria areas. More than 90% of the current annual malaria incidence (c. 500 million clinical cases with up to 2 million deaths) is in Africa where the major vector is Anopheles gambiae s.s. As pyrethroid resistance has been reported in this mosquito, reliable and simple techniques are urgently needed to characterize and monitor this resistance in the field. In insects, an important mechanism of pyrethroid resistance is due to a modification of the voltage-gated sodium channel protein recently shown to be associated with mutations of the para-type sodium channel gene. We demonstrate here that one of these mutations is present in certain strains of pyrethroid resistant A. gambiae s.s. and describe a PCR-based diagnostic test allowing its detection in the genome of single mosquitoes. Using this test, we found this mutation in six out of seven field samples from West Africa, its frequency being closely correlated with survival to pyrethroid exposure. This diagnostic test should bring major improvement for field monitoring of pyrethroid resistance, within the framework of malaria control programmes.

Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides.

We report the isolation of cDNA clones containing the full 6.3-kb coding sequence of the para-type sodium channel gene of the housefly, Musca domestica. This gene has been implicated as the site of knockdown resistance (kdr), an important resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. The cDNAs predict a polypeptide of 2108 amino acids with close sequence homology (92% identity) to the Drosophila para sodium channel, and around 50% homology to vertebrate sodium channels, Only one major splice form of the housefly sodium channel was detected, in contrast to the Drosophila para transcript which has been reported to undergo extensive alternative splicing. Comparative sequence analysis of housefly strains carrying kdr or the more potent super-kdr factor revealed two amino acid mutations that correlate with these resistance phenotypes. Both mutations are located in domain II of the sodium channel. A leucine to phenylalanine replacement in the hydro-phobic IIS6 transmembrane segment was found in two independent kdr strains and six super-kdr strains of diverse geographic origin, while an additional methionine to threonine replacement within the intracellular IIS4-S5 loop was found only in the super-kdr strains. Neither mutation was present in five pyrethroid-sensitive strains. The mutations suggest a binding site for pyrethroids at the intracellular mouth of the channel pore in a region known to be important for channel inactivation.

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 and (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 the maternally inherited molecules suggest 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.

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 host's diet of plant phloem is deficient in essential amino acids.