Genome-wide analysis identifies gain and loss/change of function within the small multigenic insecticidal Albumin 1 family of Medicago truncatula.

BACKGROUND: Albumin 1b peptides (A1b) are small disulfide-knotted insecticidal peptides produced by Fabaceae (also called Leguminosae). To date, their diversity among this plant family has been essentially investigated through biochemical and PCR-based approaches. The availability of high-quality genomic resources for several fabaceae species, among which the model species Medicago truncatula (Mtr), allowed for a genomic analysis of this protein family aimed at i) deciphering the evolutionary history of A1b proteins and their links with A1b-nodulins that are short non-insecticidal disulfide-bonded peptides involved in root nodule signaling and ii) exploring the functional diversity of A1b for novel bioactive molecules. RESULTS: Investigating the Mtr genome revealed a remarkable expansion, mainly through tandem duplications, of albumin1 (A1) genes, retaining nearly all of the same canonical structure at both gene and protein levels. Phylogenetic analysis revealed that the ancestral molecule was most probably insecticidal giving rise to, among others, A1b-nodulins. Expression meta-analysis revealed that many A1b coding genes are silent and a wide tissue distribution of the A1 transcripts/peptides within plant organs. Evolutionary rate analyses highlighted branches and sites with positive selection signatures, including two sites shown to be critical for insecticidal activity. Seven peptides were chemically synthesized and folded in vitro, then assayed for their biological activity. Among these, AG41 (aka MtrA1013 isoform, encoded by the orphan TA24778 contig.), showed an unexpectedly high insecticidal activity. The study highlights the unique burst of diversity of A1 peptides within the Medicago genus compared to the other taxa for which full-genomes are available: no A1 member in Lotus, or in red clover to date, while only a few are present in chick pea, soybean or pigeon pea genomes. CONCLUSION: The expansion of the A1 family in the Medicago genus is reminiscent of the situation described for another disulfide-rich peptide family, the "Nodule-specific Cysteine-Rich" (NCR), discovered within the same species. The oldest insecticidal A1b toxin was described from the Sophorae, dating the birth of this seed-defense function to more than 58 million years, and making this model of plant/insect toxin/receptor (A1b/insect v-ATPase) one of the oldest known.

Effects of plant protease inhibitors, oryzacystatin I and soybean Bowman-Birk inhibitor, on the aphid Macrosiphum euphorbiae (Homoptera, Aphididae) and its parasitoid Aphelinus abdominalis (Hymenoptera, Aphelinidae).

Transgenic plants expressing protease inhibitors (PIs) have emerged in recent years as an alternative strategy for pest control. Beneficial insects such as parasitoids may therefore be exposed to these entomotoxins either via the host or by direct exposure to the plant itself. With the objective of assessing the effects of PIs towards aphid parasitoids, bioassays using soybean Bowman-Birk inhibitor (SbBBI) or oryzacystatin I (OCI) on artificial diet were performed on Macrosiphum euphorbiae-Aphelinus abdominalis system. OCI significantly reduced nymphal survival of the potato aphid M. euphorbiae and prevented aphids from reproducing. This negative effect was much more pronounced than with other aphid species. On the contrary, SbBBI did not affect nymphal viability but significantly altered adult demographic parameters. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of Aphelinus abdominalis predominantly relies on serine proteases and especially on chymotrypsin-like activity. Immunoassays suggested that OCI bound to aphid proteins and accumulated in aphid tissues, whereas SbBBI remained unbound in the gut. Bioassays using M. euphorbiae reared on artificial diets supplemented with both OCI and SbBBI showed a fitness impairment of Aphelinus abdominalis that developed on intoxicated aphids. However, only SbBBI was detected in parasitoid larvae, while no PI could be detected in adult parasitoids that emerged from PI-intoxicated aphids. The potential impact of PI-expressing plants on aphid parasitoids and their combined efficiency for aphid control are discussed.

Toxicity to the pea aphid Acyrthosiphon pisum of anti-chymotrypsin isoforms and fragments of Bowman-Birk protease inhibitors from pea seeds.

Aphids feed on a protein-poor diet and are insensitive to several serine protease inhibitors. However, among the Bowman-Birk family of plant trypsin inhibitors (BBI), some members display significant toxicity to the pea aphid Acyrthosiphon pisum. A BBI isoform purified from pea seeds (PsTI-2) displays an IC50 of 41 microM and a LC50 of 48 microM at 7 days. Our data show that the chymotrypsin-directed active site from these bifunctional inhibitors is responsible for this activity, and that artificial cyclic peptides bearing the Bowman-Birk anti-chymotrypsin head induce much greater toxicity and growth inhibition than their anti-trypsin counterparts. The toxic syndrome included a rapid behavioural response of aphids on diets containing the toxic peptides, with induced restlessness after only 1 h of exposure to the chymotrypsin inhibitor. Nevertheless, chymotrypsin activity was not detected in aphid guts, using two chromogenic chymotrypsin substrates, and the physiological target of the chymotrypsin inhibitor remains unknown. These data show for the first time that plant chymotrypsin inhibitors, still widely unexplored, may act as paradoxical toxicants to aphids and serve as defensive metabolites for phloem-feeding insects.

Fate of dietary sucrose and neosynthesis of amino acids in the pea aphid, acyrthosiphon pisum, reared on different diets

The fate of sucrose, the major nutrient of an aphid's natural food, was explored by radiolabeling in the pea aphid Acyrthosiphon pisum. To investigate the influence of nitrogen quality of food on amino acid neosynthesis, pea aphids were reared on two artificial diets differing in their amino acid composition. The first (diet A) had an equilibrated amino acid balance, similar to that derived from analysis of aphid carcass, and the other (diet B) had an unbalanced amino acid composition similar to that of legume phloem sap. Aphids grown on either diet expired the same quantity of sucrose carbon as CO(2), amounting to 25-30 % of the ingested sucrose catabolized in oxidation pathways. On diet A, the aphids excreted through honeydew about twice as much sucrose carbon as on diet B (amounting to 12.6 % of the ingested sucrose for diet A and 8.4 % for diet B), while amounts of sucrose carbons incorporated into exuviae were almost identical (1.9 % of the ingested sucrose on diet A and 2.7 % on diet B). There was also no difference in the amounts of sucrose carbon incorporated into the aphid tissues, which represented close to 50 % of the ingested sucrose. Sucrose carbons in the aphid tissues were mainly incorporated into lipids and the quantities involved were the same in aphids reared on either diet. On diet B, we observed neosynthesis of all protein amino acids from sucrose carbons and, for the first time in an aphid, we directly demonstrated the synthesis of the essential amino acids leucine, valine and phenylalanine. Amino acid neosynthesis from sucrose was significantly higher on diet B (11.5 % of ingested sucrose carbons) than on diet A (5.4 %). On diet A, neosynthesis of most of the amino acids was significantly diminished, and synthesis of two of them (histidine and arginine) was completely suppressed. The origin of amino acids egested through honeydew was determined from the specific activity of the free amino acid pool in the aphid. Aphids are able to adjust to variation in dietary amino acids by independent egestion of each amino acid. While more than 80 % of excreted nitrogen was from food amino acids, different amino acids were excreted in honeydew of aphids reared on the two diets. The conversion yields of dietary sucrose into aphid amino acids determined in this study were combined with those obtained previously by studying the fate of amino acids in pea aphids reared on diet A. The origin of all the amino acid carbons in aphid tissues was thus computed, and the metabolic abilities of aphid are discussed from an adaptive point of view, with respect to their symbiotic status.

Characteristics and development of the tyrosine-rich protein granules in the adipose tissue of the Curculionid beetle Sitophilus oryzae.

In the Curculionid beetle Sitophilus oryzae, the fat body is composed of one type of adipocyte, interstitial cells and oenocytes. Synthesis and storage of tyrosine-rich-protein granules (TRPG) in adipocytes are observed during all the larval and prepupal stages (except the first larval instar which has not been studied). They appear first in the posterior part of the fat body, around the nucleus of adipocytes. They progressively invade the cytoplasm. In the young pupa, TRPG are present in every part of the body, including the head and the appendages in formation. TRPG grow in size by fusing together. Their mean diameter is 6 microm, but some of them reach up to 50 microm. They present a basic core and an acidic periphery. Their charge in tyrosine increases until the prepupa. They are APS and lipid negative and contain no RNA. During metamorphosis they take on a reticulated structure, evoking a golf ball, and disintegrate into small granules, the tyrosine content of which diminishes drastically, especially in contact with epidermal cells, whence tyrosine is probably transferred. TRPG in S. oryzae contain 16 different insoluble proteins. Five of them are tyrostaurins characterized by their very high content in tyrosine (up to 27%) and their strict insolubility in aqueous solution. Arylphorin-like proteins have not been detected in S. oryzae granules.

A putative insect intracellular endosymbiont stem clade, within the Enterobacteriaceae, infered from phylogenetic analysis based on a heterogeneous model of DNA evolution.

Insect intracellular symbiotic bacteria (intracellular endosymbionts, or endocytobionts) were positioned within the gamma 3-Proteobacteria using a non-homogeneous model of DNA evolution, allowing for rate variability among sites, for GC content heterogeneity among sequences, and applied to a maximum likelihood framework. Most of them were found to be closely related within the Enterobacteriaceae family, located between Proteus and Yersinia. These results suggest that such a bacterial group might possess several traits allowing for insect infection and the stable establishment of symbiotic relationships and that this could represent a stem clade for numerous insect endocytobionts. Based on the estimations of the equilibrium GC content and branch lengths in the phylogenetic tree, we have made comparisons of the relative ages of these different symbioses.

Cloning and characterization of a gut-specific cathepsin L from the aphid Aphis gossypii.

We have characterized proteinase activities in gut extracts from the cotton-melon aphid (Aphis gossypii Glover), an insect feeding strictly on protein-poor phloem. The major, if not exclusive, intestinal proteinases of this aphid are of the cysteine type. A cDNA has been cloned from a gut library and codes for the cysteine proteinase AgCatL, a cathepsin L-like cysteine proteinase. The AgCatL protein shows high sequence similarity with mammalian and some arthropod cathepsin L-like proteinases, but can be reliably distinguished from the secreted (digestive) proteinases identified in other arthropods. AgCatL is widely expressed in aphid intestinal cells. Immunolocalization of AgCatL showed an intense signal at the level of the anterior 'stomach' part of the midgut, and especially at intracellular localization. Although the precise role of AgCatL in aphid midgut physiology is still unclear, this enzyme could be involved in the processing of exogenous ingested polypeptides.

Metabolic and symbiotic interactions in amino acid pools of the pea aphid, Acyrthosiphon pisum, parasitized by the braconid Aphidius ervi.

Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera, Aphididae). This parasitoid strongly redirects host reproduction and metabolism to favour nutrition and development of its juvenile stages. Parasite-regulated biosynthesis and mobilization of nitrogen metabolites determine a significant increase of host nutritional suitability. The aim of the present study was mainly to investigate the temporal changes of A. pisum amino acid pools, as affected by A. ervi parasitism, and to assess the role of the aphid bacterial endosymbiont Buchnera in determining the observed changes. In parasitized aphids, we observed a very significant increase in total free amino acids, compared with synchronous non-parasitized controls, starting from day 4 after parasitization (+51%). This trend culminated with more than doubling the control value (+152%) on day 6 after parasitization. However, a significant "parasitism" effect was observed only for 10 of the 28 amino acids detected. Tyrosine accumulation was the most prominent parasitoid-induced alteration, with a fourfold increase over control levels registered on day 6. In parasitized hosts, the amino acid biosynthetic capacity of Buchnera was unaltered, or even enhanced for the phenolic pool, and contributed greatly to the definition and maintainance of host free amino acid pools. The hypertyrosinemic syndrome was not dependent on food supply of the aromatic nucleus but was induced by parasitism, which likely enhanced the aromatic shuttle mediating phenylalanine transfer from bacteria to the host tissues, where tyrosine conversion occurs. This process is likely associated with a selective disruption of the host's functions requiring tyrosine, leading to the remarkable accumulation of this amino acid. The possible mechanisms determining these parasitism-induced host alterations, and their nutritional significance for the developing parasitoid larva, are discussed.