Anas Cherqui

Identification of aphid salivary proteins: a proteomic investigation of Myzus persicae

The role of insect saliva in the first contact between an insect and a plant is crucial during feeding. Some elicitors, particularly in insect regurgitants, have been identified as inducing plant defence reactions. Here, we focused on the salivary proteome of the green peach aphid, Myzus persicae. Proteins were either directly in‐solution digested or were separated by 2D SDS‐PAGE before trypsin digestion. Resulting peptides were then identified by mass spectrometry coupled with database investigations. A homemade database was constituted of expressed sequence tags from the pea aphid Acyrtosiphon pisum and M. persicae. The databases were used to identify proteins related to M. persicae with a nonsequenced genome. This procedure enabled us to discover glucose oxidase, glucose dehydrogenase, NADH dehydrogenase, α‐glucosidase and α‐amylase in M. persicae saliva. The presence of these enzymes is discussed in terms of plant–aphid interactions.

Local and systemic responses induced by aphids in Solanum tuberosum plants

The aphids Macrosiphum euphorbiae (Thomas) and Myzus persicae (Sulzer) (Homoptera: Aphididae) are serious pests of potato (Solanum tuberosum L.) (Solanaceae), notably in transmitting several plant viruses. Heterospecific interactions may occur between these two species as they are often seen at the same time on the same potato plant in the field. As aphid infestation is known to induce both local and systemic changes, we conducted experiments to determine the effect of previous infestation on probing behaviour and feeding‐related parameters. We used the DC electrical penetration graph technique to characterize the influence of previous infestation by conspecific M. persicae or by heterospecific Ma. euphorbiae on M. persicae feeding behaviour at both local and systemic levels, i.e., on previously infested leaves and on non‐previously infested leaves of infested plants, respectively. Conspecific and heterospecific infestation led to similar modification of M. persicae feeding activities. However, the effects of previous infestation occurring at the local level were opposite to those observed at the systemic level. Myzus persicae food acceptance was slightly enhanced on previously infested leaves, whereas it was inhibited on non‐infested leaves of infested plants, which indicated an induced resistance mechanism. Our results advance the understanding of the mechanisms involved in aphid–host plant acceptance and colonization processes on potato plants in conspecific and heterospecific situations.

Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linné) and in vitro.

With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid population’s growth. Artificial diets were used to provide active (1, 10, 100 and 500 μg ml−1) and inactive (500 μg ml−1) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day population’s doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.

Deadly venom of Asobara japonica parasitoid needs ovarian antidote to regulate host physiology.

Asobara japonica (Braconidae) is an endophagous parasitoid developing in Drosophila larvae. The present study shows that A. japonica was never encapsulated in Drosophila melanogaster, and that it caused an overall inhibition of the host encapsulation reaction since injected foreign bodies were never encapsulated in parasitized hosts. Both the number of circulating hemocytes and the phenoloxidase activity decreased in parasitized larvae, and the hematopoietic organ appeared highly disrupted. We also found that A. japonica venom secretions had atypical effects on hosts compared to other braconid wasps. A. japonica venom secretions induced permanent paralysis followed by death of D. melanogaster larvae, whether injected by the female wasp during an interrupted oviposition, or manually injected into unparasitized larvae. More remarkably, these effects could be reversed by injection of ovarian extracts from female wasps. This is the first report that the venom of an endophagous braconid parasitoid can have a deadly effect on hosts, and moreover, that ovarian extracts can act as an antidote to reverse the effects of the wasps venom. These results also demonstrate that A. japonica secretions from both venom gland and ovary are required to regulate synergistically the host physiology for the success of the parasitoid.

Host‐plant mediated interactions between two aphid species

Herbivory induces numerous defence reactions in plants, which can in turn alter the plant quality for insects. The potato aphid, Macrosiphum euphorbiae (Thomas), and the green peach aphid, Myzus persicae (Sulzer) (both Hemiptera: Aphididae), are two important sympatric potato pests in northern France. The objective of this study was to evaluate the effect of a previous infestation of a potato plant, Solanum tuberosum L. (Solanaceae), by M. persicae or M. euphorbiae on the host attractiveness, feeding behaviour, and biological performance of M. euphorbiae subsequently colonising the plant. The preference of aphids was studied with a dual‐choice olfactometer and their feeding behaviour was monitored using the electrical penetration graph technique. Their biological performance was assessed by an in planta bioassay. Non‐infested plants were significantly more attractive to M. euphorbiae than plants pre‐infested by conspecific individuals. Aphids showed a strong reduction in the time spent ingesting phloem sap when feeding on pre‐infested plants. The biological performance of M. euphorbiae was not affected by previous conspecific infestation. Conversely, M. euphorbiae feeding behaviour was not affected on plants previously infested by M. persicae but aphids were more attracted to and had a faster population build‐up on those plants. Our results show that plant response and its effect on M. euphorbiae differed depending on the aphid species previously feeding on the potato plant. This variability in plant response could lead to competition or facilitation between aphids temporally and spatially separated, and promote dispersal under field conditions.

Effects of potato plants expressing the nptII-gus fusion marker genes on reproduction, longevity, and host-finding of the peach-potato aphid, Myzus persicae

Transgenesis developed in the last 20 years offers new possibilities for crop protection. The transgenic process, however, requires the use of marker fusion genes to select and visualize the transformed tissues. Although the expression products of these marker genes are stably expressed in crops, little attention has been given to assess the eventual risks of these recombinant proteins on phytophage populations. Three independent transgenic potato (Solanum tuberosum) clones from the cultivar Désirée (DG5, DG18, and DG20) carrying the commonly used nptII‐gus gene construct and exhibiting different β‐glucuronidase activity (0.843 ± 0.011, 0.576 ± 0.096, and 0.002 ± 0.000 pmol min−1.mg−1, respectively) were evaluated to determine the impact of the encoded proteins on the behaviour, development, reproduction, and demography of the peach‐potato aphid, Myzus persicae, under laboratory‐controlled light and temperature. Our results revealed that the transgenic event can alter aphid physiology or behaviour. Experiments showed a probiotic effect of one transgenic line, the DG5, resulting in reduced prereproductive period and mortality, and enhanced daily fecundity, which was expressed in a greater population growth potential (rm = 0.205 vs. rm = 0.174 of the control). In contrast, aphids fed with the DG18 line exhibited reduced adult survival and reproductive period but no alteration of their demographic parameters (rm = 0.176). Finally, no physiological alteration was induced in aphids fed on a DG20 diet (rm = 0.170). Behavioural experiments conducted in a 4‐choice olfactometer demonstrated that insects were significantly more attracted by the odour of transgenic DG18 potato plant than that of Désirée non‐transformed plant, spending twice as much time in the DG18 plant odour. The two other transformed clones (DG5 and DG20) were as attractive as the non‐transformed cultivar. It is concluded that the β‐glucuronidase expression in potato plants might be responsible for the probiotic effect measured on the feeding aphids, whereas alteration of the foliage odour would result from a pleiotropic effect.

Components of Asobara venoms and their effects on hosts.

Hymenoptera of the Asobara genus are endophagous parasitoids of Drosophila larvae. In these apocrita insects whose venom gland is associated with the female reproductive tract, the wasp venom is injected into the host along with the parasitoid egg during oviposition. We conducted a comparative study of the venom apparatuses from three Asobara spp.: the European Asobara tabida, the Asiatic A. japonica and the African A. citri. Light and electron microscopy of venom glands, together with the biochemical analysis of their contents, revealed important differences between Asobara spp. In addition, the physiological effects of female wasps venom injected into Drosophila larvae differed greatly between the tested Asobara spp.

Molecular and biochemical analysis of an aspartylglucosaminidase from the venom of the parasitoid wasp Asobara tabida (Hymenoptera: Braconidae).

The most abundant venom protein of the parasitoid wasp Asobara tabida was identified to be an aspartylglucosaminidase (hereafter named AtAGA). The aim of the present work is the identification of: 1) its cDNA and deduced amino acid sequences, 2) its subunits organization and 3) its activity. The cDNA of AtAGA coded for a proalphabeta precursor molecule preceded by a signal peptide of 19 amino acids. The gene products were detected specifically in the wasp venom gland (in which it could be found) under two forms: an (active) heterotetramer composed of two alpha and two beta subunits of 30 and 18 kDa respectively and a homodimer of 44 kDa precursor. The activity of AtAGA enzyme showed a limited tolerance toward variations of pH and temperatures. Since the enzyme failed to exhibit any glycopeptide N-glycosidase activity toward entire glycoproteins, its activity seemed to be restricted to the deglycosylation of free glycosylasparagines like human AGA, indicating AtAGA did not evolve a broader function in the course of evolution. The study of this enzyme may allow a better understanding of the functional evolution of venom enzymes in hymenopteran parasitoids.

Transgenic potato plants expressing the nptII-gus marker genes affect survival and development of the Colorado potato beetle

Despite the routine use of marker genes in genetically engineered plants, little is known about the influence of the marker proteins produced by transgenic plants on phytophagous insects. Among the few available marker genes, the nptII gene encoding the neomycin phosphotransferase II and the uid A (gus) gene, encoding for the β-glucuronidase activity, are commonly used. Control untransformed plants and three independent transgenic lines of potato plants from the cultivar Desiree carrying only one insert of the gene construct nptII-gus and exhibiting different β-glucuronidase activity were used to breed Colorado potato beetles, Leptinotarsa decemlineata. Our results show that consumption of foliage of some of these transgenic plants leads to physiological alterations of the beetle feeding. Post-embryonic development of beetles fed with the foliage of the nptII-gus transformed line exhibiting the higher β-glucuronidase activity is shortened by more than 2 days. Imagos emerged from such larvae show higher weight (more than 10%) and size (at least 0.15 mm more) and survive on average 6 days more in complete starvation. Moreover, the number of modified biological parameters and the intensity of the modifications are closely related with the β-glucuronidase activity level of these transgenic lines. It is suggested that β-glucuronidase expression in potato plants affects development and survival of this phytophagous beetle.

When Parasitoids Lack Polydnaviruses, Can Venoms Subdue the Hosts? The Case Study of Asobara Species

Host regulation has been described as the many effects-- mostly physiological changes--that parasitoids cause in their host which benefit their own development (Vinson and Iwantsch, 1980). It evokes developmental disruption usually via hormonal or neurohormonal pathways, like the endocrine signaling which coordinates development of the parasitoid with that of the host so that the two partners molt in synchrony (Beckage and Gelman, 2004). It also includes all the effects on the host immune system (Strand and Pech, 1995; Schmidt et al., 2001; Pennacchio and Strand, 2006; Carton et al., 2008; Eslin et al., 2009), the first physiological barrier that endophagous parasitoids encounter after they enter the hemocoel of their host. In order to regulate their hosts immunity and physiology, parasitoids produce and release active factors in the host hemocoel. These factors may come from either the female wasps reproductive apparatus and its associated glands, or the parasitic egg or larva itself. In many species of the ichneumonid and braconid families (Ichneumonoidea), symbiotic polydnaviruses (PDVs) or virus-like particles (VLPs) (Schmidt and Schumann-Feddersen, 1989; Strand and Pech, 1995; Beckage, 1998; Drezen et al., 2003; Beckage and Gelman, 2004; Pennacchio and Strand, 2006; Bezier et al., 2009) can act as infecting agents. PDVs multiply in the calyx cells of the female wasps ovaries while