Two Odorant-Binding Proteins Mediate the Behavioural Response of Aphids to the Alarm Pheromone (E)-?-farnesene and Structural Analogues

Background

Aphids are agricultural pests of great economical interest. Alternatives to insecticides, using semiochemicals, are of difficult applications. In fact, sex pheromones are of little use as aphids reproduce partenogenetically most of the time. Besides, the alarm pheromone, (E)-ß-farnesene for a great number of species, is difficult to synthesize and unstable in the environment. The search for novel semiochemicals to be used in population control can be efficiently approached through the study of the olfactory system at the biochemical level. Recently odorant-binding proteins (OBPs) have been shown to play a central role in olfactory recognition, thus becoming the target of choice for designing new semiochemicals.

Methodology/Principal Findings

To address the question of how the alarm message is recognised at the level of OBPs, we have tested 29 compounds, including (E)-ß-farnesene, in binding assays with 6 recombinant proteins and in behaviour experiments. We have found that good repellents bind OBP3 and/or OBP7, while non repellents present different spectra of binding. These results have been verified with two species of aphids, Acyrthosiphon pisum and Myzus persicae, both using (E)-ß-farnesene as the alarm pheromone.

Conclusions

Our results represent further support to the idea (so far convincingly demonstrated only in Drosophila) that OBPs are involved in decoding the chemical information of odorants and pheromones, and for the first time provide such evidence in other insect species and using wild-type insects. Moreover, the data offer guidelines and protocols for the discovery of potential alarm pheromones, using ligand-binding assays as a preliminary screening before subjecting selected compounds to behaviour tests.     

Has the Attraction of Predatory Coccinellids to Cornicle Droplets Constrained Aphid Alarm Signaling Behavior?

When attacked by a predator, aphids of many species secrete cornicle droplets, containing an alarm pheromone, that results in the dispersal of nearby conspecifics. As females are parthenogenetic, alarm signaling functions to enhance the survival of clone-mates. Enigmatically, however, aphids are physically able to, but usually do not emit alarm pheromone when initially detecting a predator, but rather signal only when captured by a predator. We hypothesized that cornicle droplets may be attractive to natural enemies and result in an increased risk of predation for the signaler, thereby selecting for prudent alarm signalers. We tested this hypothesis by investigating the olfactory cues that the multicolored Asian ladybird beetle, Harmonia axyridis Pallas, uses to locate pea aphids, Acyrthosiphon pisum. In choice tests, H. axyridis were attracted to odors from pea aphid colonies, whether feeding or not feeding on a host plant leaf, but were not attracted to cornicle droplets containing alarm pheromone. Further, individual pea aphids emitting cornicle droplets were not located more often or in a shorter period of time by beetles than aphids not emitting cornicle droplets. Thus, the cost of emitting early alarm signals is not prohibitively high in regards to the attraction of predators such as H. axyridis.     

Structural Response of Soybean Leaf to Elevated CO２ Concentration

The effects of CO2 concentration on the morphological and anatomical characters of soybean (Glycine max) leaf were investigated by means of light microscopy and SEM. It was noticed that exomorphology did not show dramatic change, while stomatal density decreased with increasing CO2 concentration. Under SEM, no epicuticular wax was observed on both abaxial and adaxial sides of the control group as well as on adaxial side of the treatment group. However, leaf surface of abaxial side was noticed to be densely covered with microasterisk epicuticular wax when they were exposed to CO2-enriched environment. The epicuticular wax deposition was present in equal abundance on both stomatal and nonstomatal areas. Furthermore, leaf thickness increased significantly due largely to the origin of an extra layer of palisade in the treatment group. The results confirmed that CO2 enrichment might enhance cell division and induce greater quantity of epicuticular wax.     

Does Gray Squirrel (Sciurus carolinensis) Response to Heterospecific Alarm Calls Depend on Familiarity or Acoustic Similarity?

In habitats in which multiple species are prey to the same predators, individuals can greatly benefit from recognizing information regarding predators that is provided by other species. Past studies have demonstrated that various mammals respond to familiar heterospecific alarm calls, but whether acoustic similarity to a familiar call can prompt a mammal's recognition of an unfamiliar call has yet to be shown. We presented alarm calls to free‐ranging eastern gray squirrels (Sciurus carolinensis) and recorded behavioral changes in vigilance and antipredatory response. Playbacks included alarm calls of a sympatric bird (American robin, Turdus migratorius), an allopatric bird with a call structure similar to that of the robin (common blackbird, Turdus merula), and an allopatric bird with a distinct call structure (New Holland honeyeater, Phylidonyris novaehollandiae). Squirrels responded significantly more frequently to squirrel alarm calls (positive control) than to robin song (negative control) or honeyeater calls. Squirrel response to robin and blackbird alarm calls was statistically similar to their response to squirrel alarm calls, indicating that squirrels responded to those alarm calls as if they provided information about the presence of predators. However, squirrel response to robin song was not statistically different from response to any of the other avian calls, including the robin and blackbird alarms, suggesting that squirrels neither respond to blackbird alarms as if they clearly signify danger, nor as if they clearly do not signify danger, perhaps reflecting some ambiguity in interpretation of the calls. These results suggest that squirrel responses to alarm calls are generally based on call familiarity, but that acoustic similarity of an unfamiliar allopatric call to a familiar call also can elicit antipredator behavior. The lack of response to honeyeater alarm calls also supports the hypothesis that call recognition by gray squirrels is dependent on familiarity, rather than simply detection of an acoustic feature common to alarm calls across a variety of avian species.     

Aphid parasites (Hym., Aphidiidæ) and their relationship to aphid attending ants,with respect to biological control

The author's results of the research on the relationship of aphid parasites to aphid attending ants based both on the study of taxonomy and ecology of the parasites (Hym., Aphidiidæ) have shown that the parasites are disregarded by the aphid attending ants. The ant attendance of the host aphid does not influence the parasite specificity. In biological control praxis, in case of experiments on introduced parasites of pest aphid establishment, the ant attendance, however, is but recommended to be also considered.     

Dine or Dash?: Ontogenetic Shift in the Response of Yellow Perch to Conspecific Alarm Cues

During their first year of growth yellow perch, Perca flavescens, undergo an ontogenetic niche shift from invertebrate feeding to piscivory. They also undergo a similar shift in their response to heterospecific alarm cues, switching from anti-predator to foraging behaviour. We conducted laboratory trials to determine whether yellow perch experience a comparable ontogenetic shift in their response to conspecific alarm cues. When exposed to either young-of-year (YOY) or adult perch skin extract, YOY perch responded with decreased time in motion and number of feeding attempts as well as increased time spent with spines erect and latency to first feeding attempt, all of which are indicative of an anti-predator response. Adult perch, when exposed to the same cues, responded with increased time spent moving and number of feeding attempts as well as decreased time spent with spines erect and latency to first feeding attempt, indicative of a foraging response. These data suggest that yellow perch undergo an ontogenetic niche shift in response to conspecific alarm cues.     

(E)-β-Farnesene synthase genes affect aphid (Myzus persicae) infestation in tobacco (Nicotiana tabacum)

Aphids are major agricultural pests which cause significant yield losses of the crop plants each year. (E)-β-farnesene (EβF) is the alarm pheromone involved in the chemical communication between aphids and particularly in the avoidance of predation. In the present study, two EβF synthase genes were isolated from sweet wormwood and designated as AaβFS1 and AaβFS2, respectively. Overexpression of AaβFS1 or AaβFS2 in tobacco plants resulted in the emission of EβF ranging from 1.55 to 4.65 ng/day/g fresh tissues. Tritrophic interactions involving the peach aphids (Myzus persicae), predatory lacewings (Chrysopa septempunctata) demonstrated that the transgenic tobacco expressing AaβFS1 and AaβFS2 could repel peach aphids, but not as strongly as expected. However, AaβFS1 and AaβFS2 lines exhibited strong and statistically significant attraction to lacewings. Further experiments combining aphids and lacewing larvae in an octagon arrangement showed transgenic tobacco plants could repel aphids and attract lacewing larvae, thus minimizing aphid infestation. Therefore, we demonstrated a potentially valuable strategy of using EβF synthase genes from sweet wormwood for aphid control in tobacco or other economic important crops in an environmentally benign way.     

(E,E)-α-Farnesene as a host-induced plant volatile that attracts Apanteles taragamae (Hymenoptera: Braconidae) to host-infested cucumber plants

In tritrophic interactions between cucumber plants, the cucumber moth Diaphania indica Saunders (Lepidoptera: Crambidae) and a larval parasitoid Apanteles taragamae Viereck (Hymenoptera: Braconidae), female A. taragamae may use herbivore-induced plant volatiles (HIPVs) to locate their host. However, the specific compound or blend of chemicals attracting A. taragamae remains unknown. In this study, differences in volatiles released from uninfested, mechanically damaged and host-infested cucumber plants were examined by the headspace volatile collection method. Responses of the larval parasitoid A. taragamae to the volatile extracts were examined in a four-arm olfactometer. We also investigated the attraction of female A. taragamae to a single compound identified as an HIPV from host-infested cucumber plants. Parasitoids discriminated between the volatiles from uninfested, host-infested and mechanically damaged plants. Chemical analysis of headspace volatiles from host-infested cucumber plants showed that (E,E)-α-farnesene was released as a major component (73.1%). When (E,E)-α-farnesene was tested alone in the range of 1.7–170?ng, female parasitoids responded to 17?ng only. Therefore, tritrophic interactions between A. taragamae and D. indica appear to be partly mediated by (E,E)-α-farnesene.     

Leg Cramps (Systremma) and “Restless Legs” Syndrome — Response to Vitamin E (Tocopherol)

A clinical investigation into the therapeutic value of vitamin E (tocopherol) in certain dermatological conditions of obscure etiology led to the incidental observation that this compound produced beneficial effects in some of the patients who were suffering from frequent and severe nocturnal leg cramps. Nearly all of the patients with leg cramps received prompt and gratifying relief from their symptoms while taking vitamin E in the form of d, alpha-tocopheryl acetate, 100 I.U. three times a day before meals. The group included 24 private patients with leg cramps and two with the “restless legs” syndrome, probably a related condition. One of the patients with leg and foot cramps also had severe nocturnal rectal cramps which were also relieved.Nocturnal leg cramps constitute a relatively common complaint in the general practice of medicine and may be very distressing to the patient. Not only is the cause obscure and the treatment relatively unsatisfactory, but even its proper medical name, systremma (anything twisted up together), is unknown to most physicians.     

Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown planthopper (Nilaparvata lugens Stål) in transgenic rice

Proteinase inhibitors are widely distributed in animals, plants and microorganisms and their roles in plants are associated with defense against pests. The utilization of proteinase inhibitors for crop protection has been actively investigated with a variety of proteinase inhibitors. Soybean Kunitz trypsin inhibitor (SKTI), one of the major seed storage protein, is synthesized for a short period during seed development. To investigate the role of SKTI in a plant's defense system against insect predation, a recombinant plasmid containing the full-length cDNA of SKTI under control of the CaMV 35S promoter was introduced into rice protoplasts by using the PEG direct gene transfer method and a large number of transgenic rice plants were regenerated. The integration, expression, and inheritance of this gene was demonstrated in R1 and R2 generations by Southern, northern, and western analyses. Accumulation levels (0.05–2.5% of soluble proteins) of SKTI protein were detected in R1 and R2 plants. Bioassay with R1 and R2 transgenic plants revealed that transgenic plants are more resistant to destructive insect pest of rice, brown planthopper (Nilaparvata lugens Stål), than the control plants. Thus, introduction of SKTI into rice plants can be used to control insect pests.     

Do common cuckoos (Cuculus canorus) possess an optimal laying behaviour to match their own egg phenotype to that of their Oriental reed warbler (Acrocephalus orientalis) hosts?

Optimality theory suggests that parasitic cuckoos should evolve an optimal laying behaviour aiming to positively select host nests in which the eggs match the phenotype of their own eggs, thus minimizing the rejection risk from hosts and, in turn, maximizing the cuckoos' fitness. We tested this hypothesis by investigating cuckoo‐egg matching between parasitized and nonparasitized nests in a common cuckoo (Cuculus canorus) host, the Oriental reed warbler (Acrocephalus orientalis), by use of Vorobyev–Osorio and Nature‐Pattern‐Match models to quantify the matching of egg colour and pattern from avian vision, respectively. The results of our study indicated that cuckoo‐egg matching in parasitized nests was no better than that in nonparasitized nests, and thus we found no support for the optimal laying hypothesis in cuckoos. The mixed conclusions from all previous studies, including the present study, may be explained by (1) the parallel coevolution in different cuckoo–host systems; (2) the inappropriate methodology; and (3) the deficiency of the assumption itself. We suggest that a better methodology should be developed to solve the puzzle of whether cuckoos choose to lay eggs matching those of the host.     

Do bumblebees (Hymenoptera: Apidae) really forage close to their nests?

This paper questions whether bumblebees really forage as close to their nests as has commonly been assumed in the bumblebee literature. Three experiments are described that involved marking and reobservation bumblebees. None of these experiments showed any tendency for bumblebees to concentrate their foraging close to (e.g., within 50 m from) the nest. Rather, the results suggested that bumblebees may prefer to forage at some distance from their nest. Further, a closer review of the bumblebee literature showed that similar findings were quite common. Some possible explanations to the observed behavior patterns are given as outlines for further research.     

Response to novel food in infant chimpanzees: Do infants refer to mothers before ingesting food on their own?

We investigated infant response toward novel food in captive chimpanzees under the condition in which they can explore such items freely together with their mother. Infants first approached novel foods rather than familiar ones when presented simultaneously. However, they did not ingest novel food immediately, but always sniff-licked it first. Infants tended to pay attention to their mothers before mouthing or ingesting novel foods themselves, but never did so with familiar ones. In response to the infant's activity, mother chimpanzees were tolerant rather than actively interfering. Those results imply that chimpanzee infants respond to novel foods in a neophobic way and refer to their mother for some kind of cue before attempting to ingest them.     

Electrophysiological and Behavioral Responses of Female Beet Armyworm Spodoptera exigua (Hübner) to the Conspecific Female Sex Pheromone

Electroantennogram (EAG) recordings showed that female Spodoptera exigua can detect their own sex pheromones (two single components and their mixture), displaying a similar dose–response pattern to that of males, although intensities of female responses were much less at all doses compared with males. Furthermore, the female calling behavior was inhibited and late-shifted by the presence of the female sex pheromone. When the pheromone components were presented, the calling female proportion in the peak calling period was significantly reduced and the calling peak time and calling termination time postponed, compared with controls. Although the calling behavior was inhibited, the pheromone titer of treated females was not different to the control, implying a reduced pheromone biosynthesis in the pheromone glands of treated moths. However, observations during the olfactometer experiments revealed that there were no obvious behavioral responses of females exposed to sex pheromone stimuli including whole gland extracts, 0.1, 1 or 10 μg binary pheromone mixtures.     

Effects of Bacillus thuringiensis δ-Endotoxins on the Pea Aphid (Acyrthosiphon pisum)

Four Bacillus thuringiensis δ-endotoxins, Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa, were found to exhibit low to moderate toxicity on the pea aphid, Acyrthosiphon pisum, in terms both of mortality and growth rate. Cry1Ab was essentially nontoxic except at high rates. To demonstrate these effects, we had to use exhaustive buffer-based controls.Many species of aphids are important sucking-insect pests that feed on plant vascular fluids. Their feeding mechanism makes these insects excellent vectors for many plant pathogens, especially viruses, yet less amenable to standard, nonsystemic chemical control by insecticides. Minor effects on the survival and fecundity of aphids reared on Bacillus thuringiensis (Bt) crops have been noted in some studies but not in others (1, 3, 6). However, the sensitivity of aphids to Bt toxins, or the lack thereof, has not been previously tested through artificial-diet bioassays with exhaustive buffer-based controls.Bt δ-endotoxins Cyt1A, Cry4A/Cry4B, and Cry11, obtained from three recombinant strains of B. thuringiensis subsp. israelensis, as well as Cry1Ab and Cry3A, obtained from recombinant Escherichia coli, were purified by ultracentrifugation in a discontinuous sucrose gradient as described previously (9). Cry proteins were solubilized in solubilization buffer (50 mM Na₂CO₃, 100 mM NaCl, pH 10) with dithiothreitol (10 mM) added before use. Cyt1A was first solubilized on 10 mM Na₂CO₃ (pH 11) buffer and then neutralized at pH 7.5 to 8 with 10 μl HCl (1 N). Both solubilized and trypsin-digested samples (1:30 over toxin weight) were used at different concentrations (32, 125, and 500 μg/ml; trypsin-activated toxin concentrations were calculated on the basis of the preactivation concentrations of the protoxins) to supplement the AP3 aphid synthetic diet (7) used to feed Acyrthosiphon pisum (LL01 green clone). Ampicillin (100 μg/ml), an ineffective antibiotic for A. pisum or its obligate symbiont Buchnera, was added to the medium to avoid bacterial growth. For each concentration, 30 nymphs (10 nymphs/box and three repetitions) were bioassayed at 20°C and under a 16:8 (light-dark) photoperiod. Survival time was calculated from aphid deposition on the test diet (day 0). Mortality was surveyed daily, and body weights of survivors were noted at day 7. ST₅₀ (median survival time after challenge) was calculated by using an actuarial survival analysis (Statview) with censoring values of survivors at the end of the experiments. The approximate concentrations resulting in a 50% decrease in mean body weight (IC₅₀) and killing of 50% of the insects tested (LC₅₀) were calculated at the end of the experiments from the growth reduction and mortality data, respectively, derived with the three doses by using Statview and the censoring values of survivors.All of the Cry δ-endotoxins tested were lethal to A. pisum and retarded the growth of survivors (Fig. ​(Fig.11 and ​and2).2). Mortalities ranged from only 25% (Cry1Ab) to 100% (Cry4 and Cry11) after 3 to 6 days of exposure to 500 μg/ml of solubilized protein (Fig. ​(Fig.1).1). When significant mortalities were achieved (Cry3A, Cry4, and Cry11), trypsin activation enhanced toxicity. Activation of Cry4 at the intermediate concentration tested (125 μg/ml) resulted in a twofold increase in mortality (Fig. ​(Fig.1D).1D). ST₅₀s were calculated for both solubilized protoxins and activated Cry3A, Cry4, and Cry11. The ST₅₀s (at 500 μg/ml) ranged from 1.8 ± 0.14 days for solubilized Cry4 and Cry11 to 3.7 ± 1.2 days for trypsin-activated Cry3A (Table ​(Table1).1). Control aphids fed buffer all survived for >8 days. The LC₅₀ of Cry1Ab was not calculated, since mortality associated with Cry1Ab reached a plateau at 500 μg/ml. The LC₅₀ of Cry4 was estimated to be 70 to 100 μg/ml (data not shown).Open in a separate windowFIG. 1.Mortality assays over the nymphal life stage of the pea aphid, A. pisum, upon ingestion of artificial diets containing purified Bt toxins after either solubilization (open symbols) or solubilization and trypsin activation (solid symbols). The toxins used were Cry1Ab (circles), Cry3A (squares), a mixture of Cry4A and Cry4B (diamonds), and Cry11A (triangles). The soluble-toxin doses used were low at 32 μg/ml (blue), intermediate at 125 μg/ml (violet), and high at 500 μg/ml (red). Assays were carried out with 30 initial neonate insects in three batches of 10 individuals.Open in a separate windowFIG. 2.Growth inhibition assays with purified Bt toxins Cry3A, Cry4, and Cry 11 (A) and Cry1Ab and Cyt1A (B) on the pea aphid, A. pisum. Toxins were added to the diet either after solubilization (open symbols) or after solubilization and trypsin activation (solid symbols). Error bars show the standard errors (SE) of individual weights at day 7 of experiments, standardized by the control group mean weight (toxin dose, 0; initial number, 30). Color coding of toxins: Cry3A, red squares; Cry4A and Cry4B mixture, violet diamonds; Cry11A, blue triangles; Cry1Ab, green circles; Cyt1A, yellow squares. In the experiment with Cry1Ab (B), the toxin was purified by high-performance liquid chromatography and activated toxin was provided as a salt-free lyophilisate by W. Moar (Auburn University, Auburn, AL).

TABLE 1.

ST₅₀s of pea aphids feeding on solubilized Cry toxins and solubilized Cry toxins activated with trypsin

Die Geißelorgane des Geißelskorpions Mastigoproctus giganteus (Lucas) (Arachnida: Uropygi): Transportepithel

Zusammenfassung Die Geißelorgane (Kaudalorgane) liegen jeweils proximal an der Ventralseite der meisten Geißelglieder. Da die Cuticula über diesen Organen wesentlich dünner ist, sind sie bereits lichtmikroskopisch als hellere, ovale Regionen erkennbar. Die Untersuchung der Ultrastruktur zeigt ein Epithel, das durch apikale und basale Einfaltungen der Zellmembran, zahlreiche Mitochondrien und konzentrische Komplexe von glattem endoplasmatischem Reticulum gekennzeichnet ist. Diese ultrastrukturellen Merkmale sprechen für transepitheliale Transportprozesse. Die mögliche Funktion der Geißelorgane wird unter Berücksichtigung experimenteller Befunde diskutiert.

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The whip organs of the whip scorpion mastigoproctus giganteus (Lucas): Transporting-epithelia

Summary The whip organs are situated proximally on the ventral side of most segments of the whip scorpion's whip. As the cuticular layer covering these organs is much thinner than the usual cuticle, the whip organs may be recognized easily under the light microscope as oval regions. The study of the ultrastructure reveals an epithelium characterized by apical and basal invaginations of the cell membrane with numerous mitochondria and prominent whorls of smooth endoplasmic reticulum. Thus, the whip organs possess the characteristics required for transepithelial transport processes. The possible function is discussed, based on experimental study.

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Für die Anfertigung der Zeichnung danken wir Herrn P. Adam