Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests.

Although transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been used successfully for management of lepidopteran and coleopteran pest species, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. To overcome this limitation, we demonstrate that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12-aa pea aphid gut-binding peptide by adding to or replacing amino acids in one of three loops of the Bt cytolytic toxin, Cyt2Aa, resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture.

Immunolocalization of odorant-binding proteins on antennal chemosensilla of the peach aphid Myzus persicae (Sulzer).

The antennal sensilla of Myzus persicae were mapped using scanning and transmission electron microscopy. Placoid sensilla and coeloconic sensilla were found on the 6th segments, whereas 2 types of trichoid sensilla were present all through the length of the antenna. A single sensillum placoideum was located on the 5th segment, whereas alate aphids also presented placoid sensilla on the 3rd antennal segment. Immunocytochemical localization of odorant-binding proteins (OBPs) was performed on ultrathin sections of antennal chemosensilla. The antiserum against OBP7 intensively labeled all placoid sensilla from both primary and secondary rhinaria, with gold granules concentrated in the lymph surrounding the dendrite. OBP6 and OBP3 could also be detected in placoid sensilla, but less strongly than OBP7. Barely significant reaction or no reaction was observed with antibodies against OBP8.

Wax glands of the horned gall aphid, Schlechtendalia chinensis, at different stages.

The horned gall aphid, Schlechtendalia chinensis, inhabits the productive species of Chinese gallnuts, which have economic value. Aphid wax glands are crucial for the survival of the insects, since the secreted waterproofing wax is important to protect the aphids from predators, pathogens and honeydew contamination. In this study, we investigated the structure of wax glands and their role in different aphid stages using light and electron microscopy. Our results show that aphids of all stages except the newly hatched fundatrix possess six parallel dorsal lines and have a total of 56 wax gland plates. Although no wax glands occur on the dorsum of the newly hatched fundatrix (first instar), the glands do appear once a fundatrix enters the second instar. The wax gland plate is composed of 2-22 polygonal depressions, each of which corresponds to a secretory cell covered by cuticle. The wax glands of this aphid belong to the class 1 glands, which are formed by epidermal secretory cells. The structure of the wax glands varies in the different stages and these changes may be adaptive to the changeable microenvironments in which the aphids live.

Morphological analysis of sensilla on different organs in Pachyneuron aphidis, a hyperparasitoid of Myzus persicae.

Aphidius gifuensis is the main enemy of Myzus persieae. While its parasitic rate can be influenced by the hyperparasitoid, Pachyneuron aphidis. As important parts of insects to sense odors from various environments, study of sensilla can lay the foundation of the further study about the parasitic mechanisms, reduce the hyperparasitic rate, and make the most effect usage of A. gifuensis. Here, we give a fundamental study about the morphology of the sensilla on the whole body of male and female P. aphidis. We observed seven main types of sensilla on them totally by using scanning electron microscopy. Including Böhm bristle (BB), chaetica sensilla (ChS), basiconic sensilla (BS), trichoid sensilla (TS), and placodea sensilla (PS), coeleoconica sensilla (CoS), basiconic capitate peg sensilla (BCPS). In addition, TS on antennae can be divided into four subtypes, on wings can be divided into two subtypes. Sensilla were most abundant on the antennae. We observed all types of sensilla on antennae. TS4 was uniporous and PS was multiporous. The other sensilla were nonporous. We did not find sexual dimorphism with regards to sensilla on the antennae except for the location of CoS. In male, CoS situated on the fourth subsegment of flagellum, but on the eighth subsegment in female. In other organs, TS has the largest number. We also found BS on compound eyes and ovipositor, BB on thoracic legs. The possible roles of these sensilla played in life activities are discussed. Our study makes a contribution of the parasitic mechanism of hyperparasitoids.

Effects of Lecanicillium longisporum infection on the behaviour of the green peach aphid Myzus persicae.

The effects of the entomopathogenic fungus Lecanicillium longisporum (Zimmerman) Zare & Gams on three parameters of behaviour (feeding, reproduction and movement) of the green peach aphid Myzus persicae (Homoptera: Aphididae) were investigated in the laboratory. Visual analysis of video tapes established that honeydew excretion events of mycosed aphids gradually declined from 2 d post inoculation and reproduction rate was significantly reduced 2 d prior to death (which occurred on day 6); both parameters were stable in controls over the same period. A detailed comparison was made between mobility of aphids during infection with two isolates of L. longisporum, using image analysis of video recordings. Both isolates caused an increase in activity at the beginning of mycosis (during fungal germination and cuticle invasion) though the intensity and the duration of this behaviour varied with the isolate. The possibility that increased movement in early mycosis helps disseminate disease is discussed in the light of the observation that saprophytic surface growth occurs on living M. persicae as it does in at least some other Lecanicillium spp-insect interactions.

Leaf surface factors of transgenic Bt cotton associated with the feeding behaviors of cotton aphids: a case study on non-target effects.

The present paper reports case study results of the risk assessment of transgenic Bt cotton on a non-target pest, cotton aphid, Aphis gossypii. Several types of techniques, i.e., electrical penetration graph (EPG), light and electron microscopy, bioassays and chemical analysis, were applied to investigate physical and chemical leaf factors of 2 transgenic Bt cotton lines (GK12 and GK19) and their parental non-Bt cotton line (Simian3) associated with searching and feeding behaviors of cotton aphids on leaves or leaf extracts of cotton plants. EPG results showed that there were some differences among behaviors of cotton aphids on 2 Bt cotton and 1 non-Bt cotton lines. Cotton aphids performed similarly to leaf surface extracts from 3 cotton lines; and leaf surface chemicals, mainly volatiles and waxes, were almost identical in the components and concentrations among the cotton lines. However, three cotton lines were quite different from each other in the densities of certain kinds of covering trichomes. Therefore, the relationships between the physical characteristics and the searching behaviors of cotton aphids on the three cotton lines were constructed as the regression equations. Glandular trichomes and covering trichomes with 5 branches influenced the cotton aphids' searching behaviors effectively; and other trichomes with other branches affected aphids in varying ways. These results demonstrated that leaf surface physical factors of transgenic Bt cotton lines different from their parental non-Bt line could affect the penetration behaviors of non-target cotton aphids. Cotton aphids penetrate and feed more easily on two Bt cotton lines than on the non-Bt cotton line.

A comparative morphological revision of the aphid genus Myzaphis van der Goot, 1913 (Insecta: Hemiptera: Aphididae) revealed a new genus and three new species.

The aphid genus Myzaphis van der Goot, 1913 from the tribe Macrosiphini is revised to include eight species. Apterous and alate viviparous females, known fundatrices and known sexual morphs (oviparous females and males) of Myzaphis bucktoni, M. juchnevitschae, M. rosarum, M. tianshanica and M. turanica are re-described and illustrated. Lectotype and paralectotypes of Myzaphis bucktoni and M. turanica are designated. The status of M. komatsubarae nomen dubium is discussed. Myzaphis avariolosa is regarded as a species belonging to the genus Ericaphis. Three new species: M. oezdemirae Kanturski & Barjadze sp. nov., M. tuatayae Kanturski & Barjadze sp. nov. from Turkey and M. rezwanii Kanturski & Barjadze sp. nov. from Iran are described and illustrated. Myzaphis bucktoni is recorded from Portugal for the first time. Diagnosis of the genus Myzaphis van der Goot, 1913 is redefined and a new genus Richardsaphis Kanturski & Barjadze gen. nov. is erected with the type species R. canadensis (Richards) comb. nov. Richardsaphis is for the first time recorded from the USA and hitherto unknown oviparous female and alate male are described and illustrated. Original keys to species of the genus Myzaphis and aphid genera of the tribe Macrosiphini with 2-2-2 first tarsal chaetotaxy are also provided.

Perianal structures in myrmecophilous subterranean aphids (Insecta: Hemiptera: Aphididae) - Comparative morphology of trophobiotic organ with its first description in Lachninae.

Scanning electron microscopy (SEM) and light stereoscopic microscopy (LSM) were used for the first time to elucidate the external morphology of the so called "trophobiotic organ" on the end of abdomen of apterous viviparous females of six aphid species (Insecta: Hemiptera: Aphididae), representatives of the myrmecophilous, subterranean aphids from the subfamilies Anoeciinae (Anoecia furcata), Eriosomatinae (Forda formicaria, Geoica utricularia, Tetraneura ulmi), and Lachninae (Protrama flavescens, Trama troglodytes). We examined and compared the external morphology in the parthenogenetic generation living on roots of deciduous plants. FE-SEM images based on HMDS preparation techniques revealed great similarity of perianal structures even between not closely related groups. Rectangular, vertically positioned anal plate, extremely shortened cauda and setae around the anus seem to be common features of these aphids. However, some differences in the number and length of setae, their arrangement and inclination of anal plate may be observed. The discussion focuses on the adaptive importance of such modifications, with respect to underground life mode and myrmecophily, but with reservations concerning living in galls by representatives of Eriosomatinae, which is hypothesized to be a factor driving the development of such modifications of perianal structures in this group of aphids.

Mouthpart structure in the woolly apple aphid Eriosoma lanigerum (Hausmann) (Hemiptera: Aphidoidea: Pemphigidae).

Mouthparts are important sensory and feeding structures in insects and differences in mouthpart structure reflect differences among lineages in feeding strategy and behavior. The woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann), is an important pest of apple orchards worldwide, causing direct damage through feeding by the highly specialized piercing-sucking mouthparts. To obtain a better understanding of feeding, the morphology of mouthparts of the WAA was examined using scanning electron microscopy (SEM). The mouthparts of E. lanigerum are similar to those of previously studied aphid species in most aspects and composed of a cone-shaped labrum, a tube-like, four-segmented labium with a deep groove on the anterior side, and a stylet fascicle consisting of two mandibular and two maxillary stylets. The sculpturing on the lateral margin of the distal extremity of the maxillary stylets and a dentate protuberance at the very sharp tip are newly observed features that distinguish E. lanigerum from other aphids and Auchenorrhyncha. Also, there is a common duct in E. lanigerum as based on SEM. Two types of sensilla trichodea and three types of sensilla basiconica occur at different locations on the labium; the labial tip has eight pairs of small sensilla basiconica. The morphology of the mouthparts and the distribution of sensilla located on the labium in E. lanigerum are discussed with respect to their possible taxonomic and functional significance.

Effect of Eupatorium adenophorum Spreng leaf extracts on the mustard aphid, Lipaphis erysimi Kalt: a scanning electron microscope study.

Scanning electron microscopic study revealed some remarkable adverse effects of Eupatorium adenophorum Spreng leaf extract on cuticular and antennal sensilla of the mustard aphid, Lipaphis erysimi Kalt. The study appears to be the first of its kind in providing evidence on toxic property of the plant on certain agricultural pests, although the indigenous tribes of the region know the insecticidal property of the plant for a long time. The study further provides indications regarding the neurotoxic nature of the leaf extract since it has caused abnormalities in the sensory structures. The presence of prominent swellings in some sensilla and shrinkage in others suggests that the leaf extract affects different systems through different mechanisms. The possible use of the plant as a potential biopesticide against certain insect pests is discussed.

Occurrence of nitric oxide synthase in Megoura viciae Buckton (Homoptera, Aphididae): an histochemical and immunohistochemical localisation.

Nitric oxide (NO) is known to be involved in many physiological reactions of insects. We analysed NOS localisation in aphids of the species Megoura viciae by means of histochemical reaction for the NADPH-diaphorase activity and immunohistochemical methods for uNOS, nNOS and iNOS. The obtained data provided a complex and peculiar pattern of NOS distribution in cells and tissue of M. viciae. The histochemical reaction for NADPH-diaphorase was an indicative, but not exact marker of NOS localisation in aphids. The use of anti uNOS antiserum (frequently applied in insects) was of limited value in our specimens, whereas more satisfactory results were obtained with anti nNOS and iNOS antisera of human origin. The results of Western blot analysis confirmed the immunohistochemical ones, showing an aphid protein that reacted strongly with the polyclonal antibody anti-iNOS and anti-nNOS while a similar protein band was weakly immunoreactive with the polyclonal antibody anti-uNOS. Our results suggest that NO, prevalently synthesised by calcium/calmodulin-dependent isoform, plays important physiological roles both in adult and embryological stages of aphids. The data of principal interest was NOS presence in bacteriocytes, cells that host symbiotic prokaryotes belonging to the species Buchnera aphidicola, and in nuclei of adipocytes and gut cells.

Morphology of the European species of the aphid genus Eulachnus (Hemiptera: Aphididae: Lachninae) - A SEM comparative and integrative study.

Scanning electron microscopy (SEM) methods were used for the first time to elucidate the external morphology of the European species of the genus Eulachnus (Hemiptera: Aphididae: Lachninae), a representative genus of the conifer-feeding aphids tribe Eulachnini. We examined and compared the external morphology of apterous and alate viviparous females from the parthenogenetic generation as well as oviparous females and alate males belonging to the sexual generation. FE-SEM images based on HMDS and cryo-SEM preparation techniques revealed better image quality than the CPD technique in regard to surface tension and morphological signs of cell deteriorations (i.e., existence of depressions, drying artifacts and membrane blebs). Three morphologically different species groups "agilis", "brevipilosus" and "cembrae" were proposed due to the differences in head, antennae, legs and dorsal chaetotaxy as well as dorsal sclerotization. The most characteristic features and differences of representatives of these groups are presented and discussed.

Ultrastructural exploration on the histopathological change in Phenacoccus fraxinus infected with Lecanicillium lecanii.

The histopathological changes of the second instar nymph of the mealybug Phenacoccus fraxinus infected with Lecanicillium lecanii strain 3.4505 were investigated using light, scanning and transmission electron microscopy. The results demonstrated that L. lecanii 3.4505 could infect P. fraxinus in a short period. At 24 h post-inoculation, the conidia of L. lecanii 3.4505 adhered to the indented gloves or intersegmental folds of the insect body surface. Subsequently, the germinated conidia produced germ-tubes, appressoria and extended hyphae, which tightly adhered to the cuticle. Penetration of cuticle could be achieved either by peg form appressoria or directly by hyphae. Also, the conidia and hyphae could secrete massive mucilages causing visible damage to the host cuticle. After 48 h, the body wall, tissues and organs, including cuticle, trachea, fat body, muscle, Malpighian tubules and nerve ganglion, were destroyed by ramification of hyphae as a result of infection. The endoplasmic reticulum hypertrophied and formed obvious fingerprint agglomerates, and the mitochondria swelled and deformed in the haemocytes. Finally, the mycelium fully occupied the entire haemocoel. The entire bodies were wrapped in a white mycelium, with the mycelium extending radically outward.

Ultrastructure of antennal sensilla of the peach aphid Myzus persicae Sulzer, 1776.

The antennal sensilla of alate Myzus persicae were mapped using transmission electron microscopy and the ultrastructure of sensilla trichoidea, coeloconica, and placoidea are described. Trichoid sensilla, located on the tip of the antennae, are innervated by 2-4 neurons, with some outer dendrites reaching the distal end of the hair. Coeloconic sensilla in primary rhinaria are of two morphological types, both equipped with two dendrites. Dendrites of Type II coeloconic sensilla are enveloped in the dendrite sheath, containing the sensillum lymph. In sensilla coeloconica of Type I, instead, dendrites are enclosed by an electron opaque solid cuticle, with no space left for the sensillum lymph. The ultrastructure of big placoid sensillum reveals the presence of three groups of neurons, with 2-3 dendrites in each neuron group, while both small placoid sensilla are equipped with a single group of neurons, consisting of three dendrites. Both large and small placoid sensilla bear multiple pores on the outer cuticle. The function of these sensilla is also discussed.

Spatial organization of the glucosinolate-myrosinase system in brassica specialist aphids is similar to that of the host plant.

Secondary metabolites are important in plant defence against pests and diseases. Similarly, insects can use plant secondary metabolites in defence and, in some cases, synthesize their own products. The paper describes how two specialist brassica feeders, Brevicoryne brassicae (cabbage aphid) and Lipaphis erysimi (turnip aphid) can sequester glucosinolates (thioglucosides) from their host plants, yet avoid the generation of toxic degradation products by compartmentalizing myrosinase (thioglucosidase) into crystalline microbodies. We propose that death, or damage, to the insect by predators or disease causes disruption of compartmentalized myrosinase, which results in the release of isothiocyanate that acts as a synergist for the alarm pheromone E-beta-farnesene.

Binding of the insecticidal lectin Concanavalin A in pea aphid, Acyrthosiphon pisum (Harris) and induced effects on the structure of midgut epithelial cells.

Concanavalin A (lectin from Canavalia ensiformis L., ConA) has previously been shown to act as a feeding inhibitor for Acyrthosiphon pisum, the pea aphid. In the present study a range of histochemical and biochemical techniques were used to elucidate the target tissues and binding sites of the lectin in the aphid. Diet uptake was evaluated using a radioactive tracer (14C-methylated inulin) and demonstrated that adults were capable of ingesting high quantities of the toxin (approx. 1 microg over a 48 h period). Electophoretic analysis and enzyme-linked immuno-sorbent assay of honeydew samples confirmed these results and further demonstrated that only small levels of ConA were excreted. Histofluorescence and immunolocalisation studies on nymphs revealed that the stomach was the primary target for ConA. At concentrations up to 400 microg ml(-1), lectin binding only occurred in the stomach region, however, at high concentrations (800 microg ml(-1)) the whole digestive tract was stained, although there was no evidence of binding in either the oesophagus or rectum. In addition to binding, there was evidence to suggest that ConA was also causing systemic effects in that the lectin appeared to cross the intestinal epithelial barrier. Immunohistochemical and electron microscopy studies revealed that ConA induced severe cellular swelling of the epithelial cells, accompanied by hypersecretion and a progressive detachment of the apical membrane; however, the striated border itself did not appear to be directly affected. Furthermore, there was no lysis of the epithelium, nor loss of integrity of the epithelial cells themselves. Our results suggest that ConA interacts with glycosylated receptors at the surface of the stomach epithelial cells, interfering with normal metabolism and cell function, resulting in a rapid feedback response on feeding behaviour. Whilst our results provide a much greater understanding regarding the modes of action of ConA in insects, they suggest that different lectins, including other mannose binding lectins, have different modes of action at the cellular levels, and thus generalizations should be treated with caution.

Probiotic effects of beta-glucuronidase on the peach-potato aphid Myzus persicae (Aphididae).

beta-glucuronidase (GUS) is a reporter protein commonly expressed in transgenic plants allowing the visualization of the transformed individuals. In our recent work, we showed that consumption of transformed potato plants expressing this GUS enzyme improves performance of the phloem feeding aphid Myzus persicae. Those results led us to the conclusion that the expression of GUS in potato plants might be responsible for the probiotic effect measured in feeding aphids. In the present paper, artificial diets were used to provide active GUS (10 and 500 microg ml(-1)), inactivated heated GUS (500 microg ml(-1)), glucuronic acid (10, 100 and 500 microg ml(-1)), and bovine serum albumin (500 microg ml(-1)) to M. persicae. Our results reveal that these chemicals provided as food intake might influence the biological parameters of this aphid. Experiments showed a probiotic effect of 500 microg ml(-1) GUS diet, resulting in reduced larval mortality, and increased adult reproduction period and fecundity, which led to an increased population growth potential (r(m)=0.17+/-0.01 versus r(m)=0.12+/-0.03 for aphids fed on control diet). A lower amount of added GUS led to fewer variations, biological parameters being only slightly altered (r(m)=0.14+/-0.03). Statistically similar alterations of the biological parameters were obtained when comparing aphids fed on the diet added with inactivated GUS or the non-structural bovine serum albumin protein (r(m)=0.15+/-0.02 and 0.14+/-0.03, respectively). Feeding assays conducted with glucuronic acid supplemented diets enhanced longevity and nymph production of the adult aphids and reduced larval mortality, resulting in r(m)=0.15+/-0.02 for the highest dose (500 microg ml(-1)). Although 100 microg ml(-1) glucuronate diet did not induce any effect on M. persicae (r(m)=0.12+/-0.03), aphids fed on 10 microg ml(-1) glucuronate diet exhibited unexpected reduced demographic parameters (r(m)=0.10+/-0.03). Immuno-histological analysis showed GUS labeling along the whole digestive epithelium of adults and in various tissues including embryos and bacteriocytes. These results suggest that GUS crosses through the digestive tract. Western blots performed with protein extracts of transformed potato plants expressing the gus gene showed a unique band of molecular weight 76 kDa. On the contrary, in extracts from aphids fed on transgenic potato plants or bred on GUS 500 microg ml(-1) artificial diet, several proteins of lower molecular weight were hybridized, revealing proteolysis of ingested GUS. It is concluded that GUS protein, and more precisely GUS activity, is responsible for the probiotic effects on aphid feeding. The possible pathways of induction of such physiological alterations by GUS are discussed.

An electron microscopic study of flight muscle breakdown in an aphid Megoura viciae.

Within two days of settling to feed on a host plant degenerative changes can be detected in the flight muscles of aphids at the ultrastructural level. The thick myosin filaments and the M Lines and later the Z bands disappear leaving the thin actin filaments in the cytoplasm. The mitochondria change their configuration and become enveloped in cytosegresomes. As mitochondria become reduced in number the numbers of lysosomes and residual bodies in the cells build up. The cells then appear to become dispersed as finger-like processes protrude from them and are pinched off.

Endosymbiotic microorganisms in Adelges (Sacchiphantes) viridis (Insecta, Hemiptera, Adelgoidea: Adelgidae): Molecular characterization, ultrastructure and transovarial transmission.

The aim of this paper was to identify endosymbiotic microorganisms living in the body cavity of a Polish population of an aphid, Adelges (Sacchiphantes) viridis, as well as to describe their ultrastructure and mode of transmission between generations. Molecular data (amplification and sequencing of 16S rRNA genes) indicated that endosymbionts of A. (S.) viridis are Betaproteobacteria of the species "Candidatus Vallotia virida". Endosymbiotic bacteria are rod-shaped and localized in the cytoplasm of specific cells, termed bacteriocytes, of host insects. Endosymbionts sharing the same bacteriocytes differ in the density of their cytoplasm. There are two morphotypes of endosymbiotic bacteria: with electron-dense cytoplasm and electron-translucent cytoplasm. Since only bacteria containing electron-dense cytoplasm were observed in the binary fusion stage, differences in density of the cytoplasm are probably due to changes in the cytoskeleton of bacteria during division. Endosymbionts of A. (S.) viridis are transovarially (i.e. via oocytes) transmitted from the mother to the offspring.