Olfactory receptors on the maxillary palps of small ermine moth larvae: evolutionary history of benzaldehyde sensitivity

In lepidopterous larvae the maxillary palps contain a large portion of the sensory equipment of the insect. Yet, knowledge about the sensitivity of these cells is limited. In this paper a morphological, behavioral, and electrophysiological investigation of the maxillary palps of Yponomeuta cagnagellus (Lepidoptera: Yponomeutidae) is presented. In addition to thermoreceptors, CO2 receptors, and gustatory receptors, evidence is reported for the existence of two groups of receptor cells sensitive to plant volatiles. Cells that are mainly sensitive to (E)-2-hexenal and hexanal or to (Z)-3-hexen-1-ol and 1-hexanol were found. Interestingly, a high sensitivity for benzaldehyde was also found. This compound is not known to be present in Euonymus europaeus, the host plant of the monophagous Yponomeuta cagnagellus, but it is a prominent compound in Rosaceae, the presumed hosts of the ancestors of Y. cagnagellus. To elucidate the evolutionary history of this sensitivity, and its possible role in host shifts, feeding responses of three Yponomeuta species to benzaldehyde were investigated. The results confirm the hypothesis that the sensitivity to benzaldehyde evolved during the ancestral shift from Celastraceae to Rosaceae and can be considered an evolutionary relict, retained in the recently backshifted Celastraceae-specialist Y. cagnagellus.
Introduction
Recently, progress has been made in our understanding of speciation in herbivorous insects, and mathematical models demonstrate that adaptive speciation driven by selection on ecological traits is theoretically plausible (Kondrashov and Kondrashov 1999; Dickman and Doebeli 1999; Doebeli 2005). However, as yet not much work has been done on the actual nature of these traits. The theoretical results make clear that host shifts and concomitant changes in host acceptance behaviour are important factors in adaptive speciation, but the underlying changes in the neural system are poorly studied and understood. Only recently work addressing this type of question is starting to appear (cf. Olsson et al. 2005a, b) .
Although host acceptance in phytophagous insects is usually viewed as solely governed by the adult (“mother knows best”), it should be noted that in many species larval host acceptance is equally important. Lepidopterous larvae in particular often display striking food preferences, which are based on a small set of chemoreceptors (Schoonhoven and van Loon 2002), thus making caterpillars promising models to study the neural basis of host plant recognition, host shifts, and the evolution of insect–plant relations. However, such an approach requires detailed knowledge about the responses of the involved receptors, information that is almost completely lacking.
External chemoreceptors in caterpillars are located on the antennae, on the maxillae, and in the epipharynx. The gustatory sensilla styloconica on the galea are important in host acceptance behaviour, and a large body of data is available about their sensitivity (see Schoonhoven and van Loon 2002; Schoonhoven et al. 2005 for review). The other sets of receptors, the epipharyngeal organs on the inside of the labrum, and the sensilla on the antennae and the maxillary palps, are more difficult to study and as a consequence less well known. The maxillary palp receptors in particular have received very little attention. Except for the pioneering studies of Schoonhoven and Dethier (1966), recordings from the maxillary nerve of the silkworm by Hirao et al. (1976, and a recent paper on gustatory receptors in the palp of Choristoneura fumiferana (Albert 2003), no electrophysiological data on the maxillary palps of Lepidoptera appears to be available. However, behavioural work indicates that the influence of the palps on feeding can be as significant as that of the taste sensilla on the galea (Hanson and Dethier 1973; Glendinning et al. 1998; de Boer 1993, 2006).
The small ermine moth genus Yponomeuta Latreille (Lepidoptera: Yponomeutidae) consists of some 75 species (Gershenson and Ulenberg 1998). Most are specialised on Celastraceae, and phylogenetic analysis (Menken 1996; Turner H, Lieshout N, van Ginkel W, Menken SBJ, submitted; Ulenberg, personal communication) strongly suggest that this association is the ancestral state in the genus. Moreover, mapping host use upon the phylogeny indicates an intriguing evolutionary scenario: the genus most likely originated in the Far East and dispersed to the western Palearctic concomitant with a (probably single) shift to Rosaceae and further to Salicaceae (Menken 1996). Interestingly, one lineage within this clade, Y. cagnagellus (Hübner), shifted back to the ancestral host, so this species is secondarily associated with Celastraceae. Yponomeuta plumbellus on the other hand is present in Western Europe, but this species is more closely related to the clades in the Far East, and retained the ancestral relation with Celastraceae.
The feeding of the Western European Yponomeuta species, and in particular Y. cagnagellus, is relatively well studied. Both the behaviour (Gerrits-Heybroek et al. 1978; van Drongelen 1980; Kooi and van de Water 1988; Peterson et al. 1990) and the underlying sensory physiology (van Drongelen 1979; Roessingh et al. 1999) have been addressed. The results demonstrate a clade of predominantly monophagous species with well-adapted sensory systems displaying high sensitivity for sugar alcohols characteristic for the host–plant families, as well as reduced sensitivities for host-specific deterrents. However, this whole body of work is focused on gustatory responses of styloconic sensilla on the galea that may provide only part of the relevant sensory inputs (de Boer 2006).
Here we present a morphological, behavioural, and electrophysiological survey of chemoreceptors on the maxillary palps in the larvae of Yponomeuta cagnagellus. In addition, we performed a comparative study into the effects of benzaldehyde on feeding behaviour of three related Yponomeuta species. Benzaldehyde strongly stimulated sensory cells in the maxillary palp of Y. cagnagellus, but its role in feeding behaviour is unknown. This compound is of particular interest because it is prominent in Rosaceae such as Prunus spinosa (Humpf and Schreier 1992), the host of Y. padellus (L), but absent from Euonymus europaeus, the host of Y. cagnagellus and Y. plumbellus (Denis and Schiffermüller) (Fig. 1). In this comparison we take advantage of the fact that the phylogenetic tree indicates that Y. plumbellus, although present in Western Europe, belongs to a clade that still displays the original ancestral association with Celastraceae. Yponomeuta padellus is in the middle of the clade that shifted to the benzaldehyde containing Rosaceae. Yponomeuta cagnagellus on the other hand is now feeding on the Celastraceous E. europaeus, that does not contain benzaldehyde, but this species most likely originated from an ancestor that was feeding on Rosaceae. An analysis of the phylogenetic pattern of the response to benzaldehyde in these three species will therefore contribute to our understanding of the flexibility of sensory systems and the role of sensory stimuli in host shifts and adaptive speciation.
Fig. 1Phylogeny of Yponomeuta, based on the internal transcribed spacer region (ITS-1) and on 16S rDNA (16S) and cytochrome oxidase (COII) mitochondrial genes, using maximum parsimony (Turner H, Lieshout N, van Ginkel W, Menken SBJ, submitted). Host plant use is plotted on the tree and the presence of benzaldehyde in the host and feeding responses to this compound are indicated for the three tested species
Materials and methods
Insects
Egg batches of Y. cagnagellus and Y. padellus were collected in the field in The Netherlands from their host plants: Y. cagnagellus from E. europaeus and Y. padellus from P. spinosa and Crataegus spp. Yponomeuta plumbellus larvae were collected as fourth instars. Egg batches were kept at 4°C and 80% relative humidity until needed. Larvae were reared in 10 cm wide Petri dishes (at 25°C, ambient humidity, L18:D6 h photoperiod) on host foliage from potted plants grown in the greenhouse (E. europaeus for Y. cagnagellus and Y. plumbellus; P. spinosa for Y. padellus). During the rearing period larvae were fed ad libitum. For the electrophysiological experiments. 1- to 4-day-old fifth instars were used that were starved for 2–3 h. In feeding experiments the caterpillars were used during the whole duration of the fifth stadium. Between feeding experiments, caterpillars were kept at 4°C.
Morphology
The morphology of the palps was studied using scanning EM. Fixation and drying often induce shrinking and cause collapse of soft tissue parts. We avoided these problems by using Cryo-SEM. Larvae were used directly after moulting into the fifth stadium. The preparation was stabilized by quick immersion in nitrogen slush (60 K), coated with a 200 Å thick layer of gold and viewed in the frozen state using a Philips 535 SEM equipped with a Hexland CT1000/CP2000 and a Jeol 35C SEM equipped with an EMSCOPE SP2000A cryo preparation assembly.
Host plant odour composition
One of the problems in determining the function of unknown sensory organs is the choice of the stimuli to be tested. To identify a set of relevant substances, the chemical composition of the headspace of E. europaeus, the host of Y. cagnagellus, was characterized. Branches (30 cm long) of E. europaeus growing in the field in Wageningen, the Netherlands, were cut with sharp garden scissors and transferred to the lab. About 2 kg of branches was placed in a 20-l stainless steel vessel within 30 min after cutting. Care was taken not to damage the leaves. With a membrane pump air was passed through KOH pellets, molecular sieves 5A and 3×, active charcoal, and a tenax pre-trap. This cleaned air flowed for 7–15 h at a rate of 70 ml/min through the stainless steel vessel and subsequently through the tenax-TA sample trap. The collected volatiles were released from the tenax by the TCT technique (Thermo-desorption cold trap; Dicke et al. 1990). The TCT unit (Chrompack, Middelburg, The Netherlands) was heated (250°C) for 3 min and desorbed compounds were collected on a Sil5CB coated, fused silica capillary at −100°C. Flash heating of the cold trap provided sharp injection of the compounds into the GC/MS unit (Chrompack Sil19CB column, 25 m × 0.25 mm, df 0.25 μm). Electron impact ionisation was carried out at 70 eV on a VG MM 7070F mass spectrometer.
Electrophysiological recording and odour stimuli
A diagram of the recording set-up is presented in Fig. 2. An isolated caterpillar head was mounted on a silver wire loop and connected via a 10-mm connector to the input terminal of a laboratory-built amplifier (Ri > 109 Ω, Ci < 1 pF, Ib < 1 pA). The sensory cells of the sensilla on the maxillary palp are located below the base of the terminal segment (Schoonhoven and Dethier 1966; Albert 1980). To gain access to these cells, the connecting membrane between the two most distal segments was pierced with a sharpened tungsten electrode. The actual single cell recordings were made using a glass capillary electrode filled with 3 M KCl (impedance about 40 MΩ) inserted through the hole and connected to ground (so actually serving as the indifferent electrode). The position of the glass electrode was carefully adjusted until action potentials could be recorded. The inside of the palp is probed with the tip until activity is picked up, analogous to the way in which recordings from the CNS are made. It should be noted that although this procedure has the advantage of allowing the recoding from single cells with good S/N ratios, and easy identification of separate cells, the drawback is that the position of the tip (and therefore the identity of the recorded cell) is not known, and not reproducible between recordings.
Fig. 2Schematic diagram of the electrophysiological set-up used to measure responses to both gustatory and olfactory stimuli. Open arrows indicate airflow, black arrows electrical signals. Note that in contrast to standard tip recording the stimulus pipette is electrically isolated
The preparation was placed in a continuous stream of clean, moistened air (80 ml/s, 25 cm/s). A second (control) air stream (2 ml/s) was injected in the main stream through an empty pasteur pipette. Odour stimuli were delivered by switching this second stream for 1 s through a pasteur pipette containing the stimulus. For stimulation with CO2, a single injected stream of commercial available bottled CO2 (Hoekloos, the Netherlands) was switched on. This injected stream could be adjusted to produce CO2 concentrations in the main stream between 0.1 and 10%. Olfactory stimuli were diluted in paraffin oil (10−3, 10−2, and 10−1 v/v). Twenty-five microlitres of these solutions was applied to filter paper strips (6.0 × 0.5 cm) and was placed in pasteur pipettes. The following compounds (99% pure) were used: 1-hexanol and benzaldehyde (Fluka, Switzerland); (Z)-3-hexenyl acetate and (E)-2-hexenal (Z)-3-hexen-1-ol (Roth, West Germany); hexanal (98% pure) (Merck, USA); limonene, citral (97% pure), and geraniol (98% pure) (Aldrich, Germany). All stimuli were applied in random order with at least 30 s between them. Longer intervals were used after strong stimuli. Preparations were discarded after 60 min and were changed after a successful recording. Odour stimuli were stored at 4°C between recording sessions, and replaced every few days.
The AC amplified action potentials from the sensory cells were recorded on tape (Racal FM taperecorder). To facilitate computer-assisted analysis, the valve signal and a pre-pulse 1 s earlier were recorded on an additional channel.
Analysis of recordings
The recordings contained activity from one to four sensory cells but usually not more than two. Recordings with more than three cells were discarded. Multi-cellular responses were analysed with the help of an interactive computer program based on the methods described in Roessingh et al. (1992). Spikes were characterized primarily by amplitude. Amplitude boundaries for different cells were taken from an amplitude versus time plot. The separation of the cells is an interactive process in which spike amplitude and spike interval distributions are used as criteria to judge the separation. After the separation, responses for each cell were calculated as the difference in action potential frequency in the last second before the onset of stimulation and the first reaction second. A cell was assumed to respond to a stimulus if an increase of at least ten spikes could be evoked. Cells with weaker reactions were assumed to be sensitive to untested stimuli and were not further analysed. To test the significance of observed differences in spike frequency, analysis of variance was used on log transformed response data.
No-choice feeding assay
A new no-choice feeding assay was designed to investigate the effect of benzaldehyde odour on larval feeding. The experimental setup is illustrated in Fig. 3. A clean air stream was split into two branches, with airflow meters to control the flow. Each branch was connected to a cluster of 18 Petri dishes, each dish containing a feeding caterpillar. In the control group, clean air was passed over the feeding larvae. The air for the experimental group was passed through a small bottle of benzaldehyde diluted in paraffin oil. A bypass with a needle valve allowed mixing in clear air and fine-tuning of the concentration of benzaldehyde (i.e. 50% flow through the bypass reduces the final concentration with a factor 2. Six different dilutions (v/v) of benzaldehyde in paraffin oil were used in the experiments, viz. 0.5 × 10−4, 0.5 × 10−3, 0.2 × 10−2, 0.5 × 10−2, 1 × 10−2, and 0.5 × 10−1. The total airflow was approximately 50 ml/s, resulting in a flow of approximately 1.4 ml/s through each Petri dish.
Fig. 3Feeding assay air flow diagram. Both the control group and treatment group consisted of 18 Petri dishes
A leaf disc with a diameter of 11 mm made from fresh host leaves was placed in each Petri dish on top of a piece of filter paper slightly wetted with distilled water. To reduce baseline palatability and detect feeding stimulation and/or inhibition, the leaf discs were extracted in alcohol and then washed in distilled water for 10 min. The duration of extraction needed to induce a weak feeding response depending upon both the host plants and leaf quality. For Y. cagnagellus, E. europaeus leaf discs were soaked in 96% EtOH for 24 h. Prunus spinosa leaf discs for Y. padellus were soaked for 2 h in 70% EtOH. Yponomeuta plumbellus appears later in the season than the other two species and feeds on slightly older E. europaeus foliage. For these older leaves extraction of leaf discs for 1 h in EtOH was sufficient to reduce palatability. The feeding experiments lasted 4–5 h. Usually 1 or 2 h after the start of the experiment, caterpillars started eating. The experiment was terminated after at least two third of the insects had been eating for more than 3 h.
To quantify the amount of feeding, the remainders of the leaf discs were scanned with an EPSON perfection 1250 scanner. The surface area was measured on a Macintosh computer using the open source program NIH Image version 1.62 (developed at the U.S. National Institutes of Health and available on the Internet at http://www.rsb.info.nih.gov/nih-image/). The differences in consumption between treatment and control groups were evaluated using a Mann–Whitney test in SPSS (SPSS Inc. 2001). For graphical display a feeding index (C − T)/(C + T) × 100% (ranging from −100 to 100%), was calculated, in which C and T denote, respectively, the mean of the remaining leaf surface in the control group and in the treatment group.
Results
Morphology
The palps of Y. cagnagellus are more elongated than in most other species of Lepidoptera, however, the sensory equipment does conform to the general pattern of ditrysian species as outlined by Grimes and Neunzig (1986). These authors divide the eight sensilla on the apex in a medial group (M1, M2), an apical group (A1, A2, A3), and a lateral group (L1, L2, L3). These groups can also be recognised in Y. cagnagellus, although the position of the sensilla is more linear than in the generalised pattern (4c).
On the apex seven sensilla basiconica and one sensillum styloconicum can be distinguished. On the side of the segment, a digitiform sensillum and at least one campaniform sensillum are visible (4b). With the exception of the styloconic sensillum, which has a prominent base, the sensilla on the apex are simple blunt cones. The lateral and medial sensilla, for which a gustatory function has been suggested, seem to be slightly more tapered (Fig. 4e, f) but the difference is only slight. Structural details like grooved surfaces or apical pores were not observed.
Fig. 4Cryo-SEM micrographs of Yponomeuta cagnagellus.a Ventral view of the head. b Maxillary palp showing the digitiform sensillum (leftmost arrow) and a large campaniform sensillum (rightmost arrow). c Overview of the tip of the most distal segment. Note the linear arrangement of the sensilla. d Detail of the digitiform sensillum. e On the apex of the palp, seven sensilla basiconica and one sensillum styloconicum are present. From left to right, L1, L2, L3, A1, a styloconic A2, A3, a partly hidden M1, and M2 can be seen (terminology after Grimes and Neunzig 1986). f High magnification of the lateral side of the apex. The two leftmost sensilla belong to the lateral group. The slightly more blunt sensillum on the right is A1. Note the absence of surface structures
Host plant odour composition
The compounds used for olfactory stimulation were chosen on the basis of the headspace analysis of E. europaeus. Whole undamaged plants release only limited amounts of volatiles (Buttery et al. 1985; van Poecke and Dicke 2004), and indeed small quantities were found in the present study. The only compounds that could be detected consistently were C6-based “green odours” (Visser and Avé 1978). Other compounds (limonene, beta-ocimene, alpha-copaene, beta-bourbonene, benzaldehyde) could not be detected reliably. Based on this result (Z)-3-hexenyl acetate (E)-2-hexenal, 1-hexanal (Z)-3-hexen-1-ol, hexanol, limonene, citral, geraniol, and benzaldehyde were chosen as olfactory stimuli. Benzaldehyde is not known from E. europaeus, but was nevertheless included in the analysis because it is a major compound in the Rosaceous hosts of some other Yponomeuta species that are closely related to Y. cagnagellus.
Electrophysiology
In experiments on 69 preparations, a total of 85 single cells were analysed. Twenty-four cells did not respond to any of the applied stimuli. Of the remaining receptors, 22 cells showed no response to odours, but responded to a 100 mM KCl solution. However, these gustatory responses were only weak (21 ± 4 spikes maximum) so the adequate stimulus was most likely not KCl. Gustatory responses will therefore not be considered here any further. Twenty-two other cells responded strongly to C6 compounds and to benzaldehyde (Fig. 5). For 19 of those cells more or less complete response spectra could be obtained (Fig. 6). At least two groups could be distinguished. Cells one to five responded to the aldehydes (E)-2-hexenal and hexanal. The other cells respond to the leaf alcohol (Z)-3-hexen-1-ol, to 1-hexanol, and to benzaldehyde. No cells were found that responded strongly to (Z)-3-hexen-1-yl acetate, or to the terpenoids limonene (6 cells tested), citral (3 cells tested) and geraniol (5 cells tested).
Fig. 5I Responses of a (E)-2 hexenal cell in the maxillary palp of Yponomeuta cagnagellus to plant odour compounds. A Hexanal 10−1, B Hexanal 10−2. C (Z)-3-hexen-1-ol 10−1. D (Z)-3-hexen-1-ol 10−2. The stimulus period (1 s) is indicated by the horizontal bar. II Responses of a (Z)-3-hexen-1-ol best cell in the maxillary palp of Yponomeuta cagnagellus to plant odour compounds. A (Z)-3-hexen-1-ol 10−1. B (Z)-3-hexen-1-ol 10−2, C (Z)-3-hexen-1-ol 10−3. D (E)-2-hexenal 10−2, E hexanal 10−2, F benzaldehyde 10−2. Note in D and E the presence of an aldehyde-sensitive cell in the noise (arrow). The stimulus duration (1 s) is indicated by the horizontal barFig. 6Response spectra for 19 cells sensitive to plant odour compounds in the maxillary palps of Yponomeuta cagnagellus at a concentration of 10−2. Response intensity is indicated by the diameter of the circles. Empty spots are missing observations
Dose response relations for green odours in the two main cell groups are given in Fig. 7. In Fig. 7a, the responses of the (E)-2-hexenal sensitive cells to the other green odours are plotted. The same is done in Fig. 7b for the (Z)-3-hexen-1-ol cells. In the alcohol-sensitive group, several cells responded to both (Z)-3-hexen-1-ol and 1-hexanol (cells 6–12) but cells that reacted mainly to one of the compounds were also found (cells 13–19).
Fig. 7Dose–response relations of olfactory receptor cells in the cells of Yponomeuta cagnagellus. a (E)-2-hexenal sensitive cells (data from cell 1 to 5 in Fig. 6, plus three additional cells). b (Z)-3-hexen-1-ol sensitive cells (data from cell 6 to 19 in Fig. 6). c. benzaldehyde sensitive cells (data from cell 6 to 16 in Fig. 6). The number of sensory cells actually recorded for each datapoint is given between brackets, vertical bars indicate the SE
The (Z)-3-hexen-1-ol and 1-hexanol cells were often also highly sensitive to benzaldehyde (Fig. 7c). Evidence indicating that this sensitivity could be attributed to another cell (e.g. superpositions or irregular spike interval histograms) was not observed. The “aldehyde” cells in our sample did not show this sensitivity and only two cells responded weakly to benzaldehyde.
Although the sensitivity to benzaldehyde was often associated with that for the leaf alcohols, examples of both benzaldehyde insensitive cells (cell 14–15) and apparent benzaldehyde specialists (cells 18–19) were found.
Fourteen cells responded selectively to CO2 (Fig. 8). The dose–response curve (Fig. 9) is almost linear from 0.2 to 2.5% CO2. No significant response to other stimuli was found for these cells. All tested C6 compounds sometimes inhibited spike activity in CO2 cells, but only at high concentrations (10−1 v/v). The terpenoids limonene, citral, and geraniol never inhibited the CO2 cells (n = 14).
Fig. 8Response of a CO2 sensitive cell in the maxillary palp of Yponomeuta cagnagellus. a to e are responses to, respectively, 0.26, 1.0, 1.5, 2.5, and 10% CO2. The time marker indicates 1 sFig. 9Dose–response relationship of 12 CO2 sensitive cells in the palp of Yponomeuta cagnagellus stimulated with CO2 or clean air. Vertical bars indicate 95% confidence interval
Three cells did not react to chemical stimuli but responded to changes in temperature induced by a hot or cold metal rod in the vicinity of the palp. A drop in temperature evoked a strong response. An increase in temperature inhibited the cells (Fig. 10), i.e. these cells functioned as cold receptors.
Fig. 10Response of a cold receptor in the maxillary palp of Yponomeuta cagnagellus. a Stimulation with a cold metal rod at an upwind distance of about 10 mm. b Stimulation with a warm metal rod at an upwind distance of about 10 mm. Stimulus bar 2.5 s
Feeding behaviour modulated by benzaldehyde
Feeding assays were conducted for Y. cagnagellus at 5 different concentrations of benzaldehyde on 72 insects per treatment (Fig. 11). Benzaldehyde had a positive feeding index at concentrations of 0.5 × 10−3, 0.2 × 10−2, and 0.5 × 10−2. However, the stimulating effect of benzaldehyde was significant only at the concentration of 0.2 × 10−2 M, with a feeding index of about 12% (percentage leaf disk left on benzaldehyde and control was, respectively, 60.9 ± 5.3 and 76.8 ± 4.4%, Mann–Whitney U = 427, P = 0.013). At higher concentrations, benzaldehyde tended to inhibit the feeding of Y. cagnagellus larvae. For example, the feeding index was −3% at the concentration of 0.5 × 10−1 but feeding inhibition was not significant compared to the control group.
Fig. 11Feeding index of three Yponomeuta species. FI = (C − T)/(C + T) (ranging from −100 to 100%). C and T denote, respectively, the mean of the remaining leaf surface in the control group and in the treatment (benzaldehyde) group. Feeding indexes with P < 0.05 are indicated with an asterisk
The feeding response of Y. padellus was tested at four concentrations, i.e. 0.5 × 10−4, 0.5 × 10−2, 1 × 10−2, and 0.5 × 10−1 (Fig. 11). Benzaldehyde significantly stimulated the feeding of caterpillars at all concentrations except at the lowest concentration (percentage leaf disk left on benzaldehyde and control at 0.5 × 10−2 was, respectively, 76.5 ± 3.7 and 84.5 ± 3.4%, Mann–Whitney U 375, P = 0.003, At 1 × 10−2 76.6 ± 3.8 and 90.4 ± 1.7%, Mann–Whitney U = 321, P = 0.001 and at 0.5 × 10−1 70 ± 3.8% and 82.8 ± 3.3% Mann–Whitney U = 408, P < 0.007). In Y. plumbellus, no effect of benzaldehyde on food intake was found, and the insects appeared to be fully indifferent to the compound (Fig. 11).
Discussion
Morphology
A description of the external structure of the maxillary palps of ditrysian larvae is given by Grimes and Neunzig (1986). From their results it is clear that location, size, and shape of palpal sensilla are constant in lepidopterous larvae. Eight sensilla are always found on the apex. In addition, a digitiform sensillum and up to four campaniform sensilla or pore plates are present in the wall of the most distal segment. Grimes and Neunzig (1986) make a distinction between the more primitive, endophagous feeders, and larger, exophagous feeders. In endophagous species, the walls of the A sensilla are smooth, and A2 is often two-tiered (styloconic). In the more advanced, exophagous groups A2 is basiconic and the A sensilla are wrinkled or pockmarked. The Yponomeutidae are regarded as relatively primitive Ditrysia (Kristensen and Skalski 1999), and indeed a styloconic A2 and smooth A sensilla are found (Fig. 4; see also van Drongelen 1979). On morphological grounds a gustatory function for L and M sensilla has been suggested (Grimes and Neunzig 1986), and for Choristoneura fumiferana this was confirmed by recordings (Albert 2003). In Helicoverpa armigera, however, some sensilla show ultrastructural features characteristic for both olfaction and taste (Keil 1996) and it is therefore difficult to assign a function based on mophology alone. Although we recorded responses to gustatory as well as olfacory stimuli, we stimulated all eight sensilla simultaneously and this method precludes assignment of the observed responses to particular sensilla. The wall of the most distal segment of the palp bears at least one campaniform sensillum. This sensillum is probably equivalent to the large plate sensillum described in C. fumiferana (Albert 2003), Euxoa messoria (Devitt and Smith 1982), and H. amigera (Keil 1996). The plate sensilla are multiporous, multiple innervated, and possess branching dendrites. If this is also true of Y. cagnagellus, these sensilla are presumably olfactory organs where some of the neural responses reported in this paper could originate.
In the wall of the most distal segment, a digitiform sensillum is present which is normally innervated by one neuron (Schoonhoven and Dethier 1966; Albert 1980; Devitt and Smith 1982; Keil 1996). In E. messoria and Heliotis amigera this sensillum appears to possess a laminated outer dendritic segment. Such laminated dendrites have been associated with cold receptors (see Altner and Loftus 1985), as well as with CO2 receptors (Chu-wang et al. 1975; McIver and Siemicki 1984; Lee et al. 1985; Bogner et al. 1986). In Y. cagnagellus both cold receptors and CO2 receptors were found. The digitiform sensillum of Y. cagnagellus might therefore house a CO2 receptor as suggested by Keil (1996) but without further electrophysiological evidence cold reception cannot be excluded.
Carbon dioxide reception
The ecological function of the CO2 receptor in Y. cagnagellus is not clear. CO2 reception is well known in soil dwelling organisms that must locate plant roots (Johnson and Gregory 2006). CO2 sensitivity is also common in adult lepidoptera (Bogner 1990; Stange and Stowe 1999). Helicoverpa armigera for instance is in principle capable of detecting modulation of CO2 concentrations normally occurring in the environment (Stange 1992). The CO2 receptor of Y. cagnagellus and other lepidopterous larvae is clearly less sensitive than that of adult Lepidoptera, however, CO2 receptors with a similar low sensitivity range have been described in the tick Amblyomma varigatum (Steullet and Guerin 1992). During the day, CO2 levels near the surface of green leaves will be 0.038% or lower, while this level may rise to a maximum of 1.2% during nocturnal respiration (see Bogner 1990 for references). Although the latter concentration is just within the range of the larval receptor, it seems unlikely that the CO2 receptor is used to assess subtle changes in the respiratory status of the leaf (as a messure of leaf quality). Alternative hypotheses are that the receptor is used to monitor CO2 levels in the communal nest during periods of tight clustering, or that it facilitates aggregation behaviour. However, as yet experimental data to support these ideas is lacking.
Temperature perception
Only qualitative data was collected from the three observed cold receptors. The actual drop in temperature induced by the hot and cold metal rods used, as stimulus is not known. The cold receptors might be used for monitoring the microclimate (Schoonhoven 1967) and in principle could be used to determine smaller temperature variations associated with leaf quality (Dethier and Schoonhoven 1968).
Plant odour responses
Stimulation with plant odours yielded cells of at least two spectral types: aldehyde-sensitive cells and alcohol-sensitive cells. The two groups are clearly separated from each other. However, within each group responses to several chemically very different stimuli were found. A single receptor cell that reacts to different types of compounds used to be interpreted as a reflection of the presence of several receptor “sites” on the membrane (Kaissling 1976; Selzer 1984; Hansen 1978; Wiezoreck and Wolff 1989). With the identification of the actual receptor genes and advanced labeling techniques it was suggested that normally only a single receptor protein is expressed in a particular sensory cell, the “one receptor–one neuron” rule (see Gaillard et al. 2004 for review). Recently however, it was shown that in Drosophila olfactory organs this rule does not always hold, and coexpression of different receptor proteins was observed (Goldman et al. 2005). In C. elegans coexpression must also occur, simply because there are too many receptor genes to express for the limited number of sensory neurons. We observed single cells that responded to structurally different compounds such as (Z)-3-hexen-1-ol and benzaldehyde, but also cells that responded to only one of these two compounds. These observations can in each case still be explained by the expression of a single receptor protein per cell, with a different binding specificity (i.e. a cell that responds to several compounds expresses a receptor with a broad specificity, while a cell that responds to only a single compound expresses a narrowly tuned receptor protein). Alternatively, cells might be broadly tuned because they express an assembly of several narrowly tuned receptor proteins with different sensitivity spectra. In either case, following this line of reasoning a simple quantitative shift in the numbers of receptor proteins could in principle result in a qualitative change in host-acceptance behaviour and host shifts, processes that were postulated as major factors in the speciation process in Yponomeuta species (Menken et al. 1992; Menken and Roessingh 1998).
From Fig. 6 is clear that although at least two groups of cells with comparable spectral responses can be distinguished (cells 1–5, cells 6–19), however, there are no sharp boundaries between these classes (cf. cell 5), and considerable variation does exist. Since care was taken to record only from cells that were reliable and reproducibly responding, it is likely that this variation does reflect natural variability in the receptor cell population. The separation in aldehyde- and alcohol sensitive cells should therefore at present merely be considered as a convenient way to summarise the data. Until more recordings are made, it cannot be excluded that a continuum from more specialised to, more generalised cells exists.
In the present study, no cells with a primary response to (Z)-3-hexenyl acetate were found in the maxillary palps of Y. cagnagellus, but receptors sensitive to this compound have been found on the antennae of this species (Roessingh, unpublished results) as well as on the antennae of Pieris brassicae larvae (Visser and de Jong 1988). In this respect it is interesting to note that in Manduca sexta fibers from the maxillary nerve travel into a core of neuropile in the suboesophageal ganglion that also receives antennal axons (Kent and Hildebrand 1987).
The maxillary palps of caterpillars contain 15–30 chemoreceptors (Schoonhoven and Dethier 1966; Albert 1980; Devitt and Smith 1982). If the 85 cells in the present study represent a random sample from the population in the palps, then the proportion of neurons sensitive to each of the tested stimuli could be estimated. However, it must be stressed that the position of the electrode was not reproducable between recordings. As a consequence, the recordings by no means represent a random sample. In spite of this limitation it can be concluded that receptors for the tested terpenoids and for (Z)-3-hexen-1-yl acetate are relatively scarce, since no cells primarily sensitive to these stimuli were encountered.
The role of benzaldehyde in feeding behaviour
Because benzaldehyde is absent from E. europaeus, Euonymus feeders such as Y. plumbellus and Y. cagnagellus are not expected to be sensitive to this compound, or use it as a repellent. The electrophysiological results clearly show that benzaldehyde is stimulating the sensory cells in the maxillary palp of Y. cagnagellus; the straightforward interpretation would be that in Y. cagnagellus benzaldehyde signals a non-host compound, and therefore low acceptability. On the other hand, as benzaldehyde is prominently present in Rosaceae, it can be expected to be a feeding stimulant for Rosaceae feeders like Y. padellus, a species that is closely related to Y. cagnagellus.
From a phylogenetic perspective a different hypothesis can thus be formulated. Yponomeuta cagnagellus is located within the European clade that feeds mostly on Rosaceae, which do contain benzaldehyde. Considering that the most recent common ancestor of this clade was presumably a Rosaceae feeder, the sensitivity of Y. cagnagellus to benzaldehyde could be viewed as a maintained sensitivity to a feeding stimulant from the ancestral host.
Under this hypothesis both Y. cagnagellus and Y. padellus are expected to be stimulated by benzaldehyde. Yponomeuta plumbellus, a Celastraceae feeder outside the European clade, is expected to be unresponsive because it has never been associated with Rosaceae nor have its ancestors. The results from the feeding experiment obtained from the three species are consistent with the latter scenario. Benzaldehyde stimulated feeding in Y. cagnagellus and Y. padellus and increased food intake by 6–12%. Yponomeuta plumbellus did not respond to benzaldehyde at all. The sensory response to benzaldehyde in Y. cagnagellus is therefore probably an evolutionary relict of the association with Rosaceae of the ancestral lineage of this species. In a previous study (van Drongelen 1979), dulcitol sensitivity was found in Y. evonymellus and Y. padellus, species feeding on Rosaceae that do contain very little or nodulcitol. In this case it was similarly concluded that the dulcitol sensitivity might be an evolutionary relict from the ancestral host association with Celastraceae.
Although feeding in both Y. cagnagellus and Y. padellus was stimulated by benzaldehyde, Y. padellus (associated with a benzaldehyde-containing hosts) responded over a much larger concentration range than Y. cagnagellus (a species that is using a benzaldehyde-free host). It should be noted that care must be taken when interpreting these results. It is not clear what the natural concentrations of host plant odours that are experienced by the larvae, since a steep gradient in the boundary layer exists, and concentrations near the leaf surface can be much higher than those obtained from headspace samples (Schoonhoven et al. 2005). However, since the sensory responses show good dose–response relations, and the behaviour yields a clear optimum, we feel that the used concentrations are covering the natural range. It will be interesting to investigate the actual genetics underlying the observed sensitivity differences between the species.

yponomeuta cagnagellus
lepidoptera
adaptive speciation
sensory physiology
plant odours


Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.
Introduction
Fusarium head blight (FHB; scab), principally caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein.) Petch), is a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) throughout the world (Sutton 1982; McMullen et al. 1997). Between 1993 and 2001, in United States, an estimated US$ 8 billion loss was incurred from FHB (Nganje et al. 2004). The disease is favored by warm conditions with frequent rainfall and high humidity during flowering. Yield reductions result from reduction in the kernel number and the presence of dry, shriveled kernels, often referred to as ‘tombstone kernels’. Additionally, reductions in grain quality are caused by Fusarium trichothecene mycotoxins such as deoxynivalenol (DON; Bai and Shaner 1994; Sutton, 1982; Tuite et al. 1990).
The most economical and efficient way to protect wheat from FHB is to develop genetically-resistant varieties. Wheat breeding programs are utilizing resistance to initial infection (Type I) and resistance to spread of the disease (Type II) as the primary forms of resistance (Rudd et al. 2001). However, these forms of resistance are partial (Kolb et al. 2001), and the level of genetic resistance provided is generally insufficient to withstand a FHB epidemic. Therefore, novel sources of resistance are required, and genetic engineering is one approach to develop novel resistance in wheat.
Several classes of genes have the potential to provide genetically-engineered resistance to FHB in wheat. One group of genes, referred to as pathogenesis-related (PR) or defense response genes, encode proteins such as β-1,3-glucanases, chitinases, thaumatin-like proteins (tlps) and thionins whose expression often increase as part of the plant host defense response to pathogen attack (Linthorst 1991). Many defense response genes were shown to be induced in wheat (Pritsch et al. 2000, 2001; Li et al. 2001; Kang and Buchenauer 2002; Kong et al. 2005; Han et al. 2005; Zhou et al. 2005; Bernardo et al. 2006) and barley (Boddu et al. 2006) spikes during F. graminearum infection. In particular, PR1, PR-2 (β-1,3-glucanase), PR-3 (chitinase), PR-4, and PR-5 (tlp-1) transcripts accumulated in wheat spikes during F. graminearum infection (Pritsch et al. 2000, 2001). In addition, polyphenol oxidase activities were detected in resistant wheat genotypes (Mohammadi and Kazemi 2002). Furthermore, Kang and Buchenauer (2003) showed accumulation of thionin proteins in F. culmorum-infected wheat tissues. These findings demonstrated that wheat and barley mount an induced defense response to Fusarium infection that involves many defense response genes.
Overexpression of defense response genes in transgenic plants has provided enhanced resistance to a variety of fungal pathogens (Muehlbauer and Bushnell 2003). For example, transgenic wheat lines carrying a barley-seed class II chitinase exhibited enhanced resistance to powdery mildew (Bliffeld et al. 1999; Oldach et al. 2001). Varying amounts of resistance towards powdery mildew were observed in transgenic wheat lines carrying a barley chitinase or a barley β-1,3-glucanase (Bieri et al. 2003). With respect to FHB, a transgenic wheat line carrying a rice tlp and a line carrying a combination of a wheat β-1,3-glucanase and chitinase exhibited delayed symptoms of FHB in greenhouse trials (Chen et al. 1999; Anand et al. 2003). However, neither transgenic wheat line exhibited any resistance to FHB under field conditions (Anand et al. 2003). In addition, transgenic Arabidopsis carrying an overexpressed Arabidopsis thionin showed increased resistance to F. oxysporum (Epple et al. 1997). Recently, transgenic wheat expressing the Arabidopsis NPR1 gene, a gene that regulates defense responses, was shown to exhibit a high level of resistance to FHB in greenhouse evaluations (Makandar et al. 2006).
As part of our effort to increase variation for genetic resistance to FHB and to understand the relationship between defense response gene expression and FHB resistance, we produced wheat plants carrying a wheat α-1-purothionin, a barley tlp-1, or a barley β-1,3-glucanase transgene. We evaluated these plants against FHB under greenhouse and field conditions. Our results show that the overexpression of α-1-purothionin, tlp-1, or β-1,3-glucanase in wheat results in enhanced resistance to FHB.
Materials and methods
Plant materials
The spring wheat cultivars ‘Wheaton’, ‘Roblin’, ‘Alsen’, ‘2375’, ‘Sumai 3’, and ‘Bobwhite’ were used as checks for FHB responses. Wheaton and Roblin are hard red spring wheat cultivars that are highly susceptible to FHB; Bobwhite is a cultivar from CIMMYT that is susceptible to FHB; 2375 is moderately susceptible to FHB; Alsen is moderately resistant to FHB with resistance derived from Sumai 3; and Sumai 3 is a Chinese cultivar known for resistance to spread of disease in the spike (Type II resistance; Bai and Shaner 1996). The cultivar Bobwhite was used as parental material for transformations.
Plant transformation plasmids
pAHC25
The pAHC25 plasmid (Fig. 1; Christensen et al. 1992), containing the uidA and bar genes under the control of the maize ubiquitin promoter, was kindly donated by Dr. Peter Quail of the Plant Gene Expression Center, University of California at Berkeley. The uidA gene encodes β-glucuronidase and the bar gene encodes the enzyme phosphinothricin acetyltransferase which confers resistance to the phosphinothricin-containing herbicides.
Fig. 1Plasmids used for wheat transformation. Plasmids containing the wheat α-1-purothionin (pKM1), barley tlp-1 transgene (pAHCBarPR5), and barley β-1,3-glucanase (pUBKBarGluc-3) were co-bombarded with pAHC25 to develop transgenic wheat plants. The ubiquitin 1 promoter and intron is from the maize ubiquitin gene, and the T nos termination sequence is from the nopaline synthase gene from Agrobacterium tumefaciens. The uidA gene encodes β-glucuronidase and is from Escherichia coli and the bar gene encodes the enzyme phosphinothricin acetyltransferase and is from Streptomyces hygroscopicus
pKM1
A plasmid containing a 460 bp wheat α-1-purothionin gene (GenBank accession number X70665.1) under the control of the maize ubiquitin promoter was kindly provided by Dr. Ann Blechl (USDA-ARS, Albany, CA). The α-1-purothionin gene was cloned into the BamHI/BglII site (replacement of the bar gene) of pUBK BglII−. The pUBK BglII− vector, kindly provided by Drs. Ann Blechl, Pat Okubara, and Kent McCue (USDA-ARS, Albany, CA), is a derivative of the pAHC20 vector (Christensen et al. 1992). pUBK BglII− contains the ubiquitin promoter, with the BglII site removed, driving the bar gene.
pAHCBarPR5
Barley tlp-1 cDNA (GenBank accession number AM403331) was removed from the parent plasmid (kindly provided by Dr. David Collinge, Department of Plant Biology, Royal Veterinary and Agricultural University, Denmark) by XhoI digestion, blunt-end repaired and cloned into the blunt-end repaired BamHI site of pAHC17. The expression cassette in the pAHC17 vector (Christensen et al. 1992) contains the maize ubiquitin promoter and the NOS terminator element. The pAHC17 vector was kindly provided by Dr. Peter Quail of the Plant Gene Expression Center, University of California at Berkeley.
pUBKBarGluc-3
The 1234 bp barley class-II β-1,3-glucanase cDNA (GenBank accession number M62907.1; Leah et al. 1991) was removed from the parent plasmid (kindly provided by Dr. John Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark) by EcoRI digestion, blunt-end repaired and ligated into the blunt-end repaired BglII/BamHI site (replacement of bar gene) of the expression vector pUBK BglII−.
Wheat transformation
Spring wheat (cv. Bobwhite) was used for all transformations. Particle gun bombardment of embryos, selection, and regeneration were carried out as described by Mackintosh et al. (2006). We conducted cotransformation of pAHC25 with pKM1, pAHCBarPR5, or pUBKBarGluc-3.
RNA isolation and transcript analysis
RNA was isolated from leaf tissue using the Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturers’ instructions. RNA was subjected to RT-PCR based on the protocol accompanying the Calypso RT-PCR kit (GenSys Ltd., Farnborough, UK) using primers synthesized by Integrated DNA Technologies Inc. (Coralville, IA). The 5′ sense primer was a maize ubiquitin promoter sequence (5′-GATGCATATACATGATGGCATATGCAG-3′) and the 3′ antisense primers were oligonucleotides that corresponded to defense response gene coding sequences for α-1-purothionin (5′-GTTACAGAAATTGACACAAGCATCGCC-3′), tlp-1 (5′-GACAGAAGGTGATCTGGTAGTTATTATT-3′) and β-1,3-glucanase (5′-GATGTTCACGGCAGGGTAGT-3′ and 5′-GCCACGTCCGTCATGTAGGCGTTC-3′). A wheat actin gene (GenBank accession number AB181991) with the primer sequences 5′-GCCACACTGTTCCAATCTATGA-3′ and 5′-TGATGGAATTGTATGTCGCTTC-3’ was used as a positive control. Sizes for the amplified products from the α-1-purothionin and tlp-1 transgenes were 600 and 805, respectively. Sizes for the two primers for the β-1,3-glucanase transgene were 577 and 777 bp. Size for the wheat actin gene was 369 bp.
Greenhouse evaluation of transgenic lines against Fusarium graminearum infection
Seeds of each transgenic line were planted in the greenhouse. At anthesis, one spikelet at the central node of the main spike of each plant was inoculated with 10 μl of a macro-conidial spore suspension (100,000 conidia/ml) of Fusarium graminearum isolate Butte 86ADA-11 (Evans et al. 2000; NRRL 38661). Plants were placed in a dew chamber for 72 h following inoculation and then returned to the greenhouse. Disease severity was assessed at 20 days after inoculation by counting the number of infected spikelets and expressing the infection level as a percentage of the total number of spikelets for each spike. Bobwhite, Wheaton, and Sumai 3 were used as checks in each greenhouse test.
Field evaluation of transgenic lines against Fusarium graminearum infection
Transgenic wheat lines were examined for their reaction to FHB in the field. RT-PCR positive plants of each of the lines were selected as the source of seed for the field plantings. In addition to the test lines, Bobwhite was included as the untransformed control. Two experiments were conducted during the summers of 2004 and 2005 at the University of Minnesota Agricultural Experiment Station, Crookston, Minnesota. T6 and T7 of the transgenic lines were used for the 2004 and 2005 field tests, respectively. The field tests were each a randomized complete block design with four replications. Entries were established in two-row plots; rows were 2.4 m (8 ft) long and were spaced 0.3 m (1 ft) apart. Within rows, seed was planted at a rate of 3.3 g of seed/m. Alsen, 2375, Roblin and Wheaton were also included in the experiment as disease response checks. Additional plantings of noninoculated Wheaton were included in the field trial to determine the level of disease.
Inoculum consisted of a mixture of 12 isolates of F. graminearum. These came from naturally infected samples of grain from commercial fields of wheat and barley in Minnesota from 2002 to 2004. Plots were inoculated twice; the first time at anthesis and then 3 days later. Inoculum (1 × 105 macroconidia/ml) was applied at a rate of 33 ml/m of row using a CO2-powered backpack sprayer, at a pressure of 276 kPa and fitted with a flat-fan spray tip (TeeJet SS8003, Spraying Systems Co., Wheaton, IL).
FHB incidence and severity were evaluated visually 21 days after the initial inoculation. Incidence was recorded as the percentage of spikes with symptomatic spikelets and severity as the percentage of symptomatic spikelets in 20 spikes of primary tillers arbitrarily selected per plot.
Plots were harvested with a Wintersteiger classic combine (Wintersteiger, Ried, Austria) at maturity. The percentage of visually scabby kernels (VSK) was assessed on a hand-cleaned 50 g sample by comparison to standards with a known percentage of scabby kernels according to the procedure of Jones and Mirocha (1999).
Following VSK analysis, the samples were ground for 2 min with a Stein Laboratory Mill (model M-2, Stein Laboratories, Atchison, KS) and analyzed for deoxynivalenol (DON) concentration using gas chromatography and mass spectrometry according to the procedures of Mirocha et al. (1998) with the following modifications. DON was extracted from 4 g of the ground wheat placed in a 50 ml centrifuge tube to which 16 ml of acetonitrile:water (84:16 v/v) was added. Samples were derivatized using 100 μl of the silylating reagent (TMSI/TMCS, 100:1), 1 ml of isooctane and 1 ml of distilled water.
Western blot analysis
Spike tissue was ground using liquid nitrogen and protein was extracted by vortexing the tissue at 4°C for 10 min in extraction buffer (50 mM NaH2PO4, pH 6.8, 100 mM PMSF). After micro-centrifugation at 4°C, full speed, for 5 min, supernatant protein measurements were conducted using Biorad reagent (Biorad) with bovine serum albumin as a standard. Extracts containing 10 μg protein were used to determine the amount of transgenic protein present in transgenic lines using Western blotting.
Samples were subjected to SDS-PAGE using 12% gels, transferred to PVDF-PLUS transfer membrane (Micron Separations Inc., Westborough, MA) and cross reacted with an affinity-purified polyclonal antibody (1:1000 dilution of supplied material). The tlp-1 and β-1,3-glucanase antibodies were provided by Quality Controlled Biochemicals Inc., Hopkinton, MA. For tlp-1, two peptides (QAYQHPNDVATHAC and CINVPAG TQAGRIWAR) were used to raise the antibody. For β-1,3-glucanase, one peptide (CGLFNPDKSPAYNIQF) was used to raise the antibody. Protein was visualized using an ECF Western Blotting Reagent Pack (rabbit) (Amersham Biosciences, Piscataway, NJ), and fluorescence detection was carried out using a Storm 840 (Molecular Dynamics, Sunnyvale, CA). Specificity of β-1,3-glucanase and tlp-1 antibodies was confirmed through cross-reacting the antibodies with the peptides on Western blots.
Southern blot analysis
DNA isolation, gel electrophoresis, gel blotting, hybridization, and washing were conducted according to de la Peña et al. (1996). Radio-labeled probes for tlp-1, β-1,3-glucanase, and α-1-purothionin were used in the hybridization reactions. The subsequent banding patterns were visualized using autoradiography.
Statistical analysis
For the greenhouse evaluation data, t-tests were used to compare each transgenic line to the parental Bobwhite controls. For the field evaluation data from 2004 and 2005, all analyses were performed with SAS® Version 9.1 (SAS Institute, Cary, NC). Analyses of variance were performed using PROC MIXED procedure. The statistical model included genotype, year and genotype-by-year interactions as fixed factors, and replication nested within year as a random factor was used as an error term for testing year effect. For each experiment, homogeneity of variances among genotypes was checked for each trait using PROC UNIVARIATE. For each trait, when variances were found to be more than four times different from each other A REPEATED/GROUP = statement of PROC MIXED was used to account for the heterogeneity of variances. Least square means and pairwise comparisons between means were obtained using LSMEANS and PDIFF options.
Results
Generation of transgenic wheat plants
The wheat cultivar Bobwhite was used as parental material for all transformation experiments. The pAHC25 plasmid and either the pKM1 (wheat α-1-purothionin), pAHCBarPR5 (barley tlp-1), or pUBKBarGluc-3 (barley β-1,3-glucanase) plasmids were used in cotransformation experiments. Figure 1 shows a schematic of each plasmid. The correct orientation within the vector and open reading frame integrity of the inserted cDNA in pKM1, pAHCBarPR-5, and pUBKBarGluc-3 was confirmed by DNA sequence analysis. pAHC25 carries the uidA gene for visual scoring of β-glucuronidase (GUS) activity and the bar gene which confers tolerance to the herbicide phosphinothricin for selection. Both the uidA and bar genes were driven by the promoter from the maize ubiquitin gene. Selection and regeneration of plants was conducted as described in Mackintosh et al. (2006).
To identify transgenic wheat plants carrying the α-1-purothionin, barley tlp-1 and barley β-1,3-glucanase transgenes, we conducted RT-PCR analysis on the T0 plants. We identified 25, 25, and 31 transgenic wheat lines carrying expressed wheat α-1-purothionin, barley tlp-1, and barley β-1,3-glucanase, respectively. Table 1 shows the number of embryos bombarded for each plasmid, and the percent transformed plants carrying the expressed transgene of interest. Our efficiency for recovering transgenic wheat plants expressing the transgenes of interest ranged from 1.4 to 3%.
Table 1Production of transgenic wheat plantsTransgeneNumber of embryos bombardedNumber of plants expressing transgeneaTransformation (%)Wheat α-1-purothionin1787251.4Barley thaumatin-like protein-1825253.0Barley β-1,3-glucanase1079312.9aExpression based on RT-PCR of each transgene.
To obtain T2 lines for further characterization, we grew five T1 seeds from each T0 plant. Each T1 plant was tested by RT-PCR for expression of the appropriate transgene, and T2 seed was collected from plants expressing each transgene.
Greenhouse evaluation of transgenic plants for response to Fusarium head blight
To identify transgenic lines with enhanced resistance to FHB and to eliminate susceptible lines, we conducted two greenhouse evaluations for FHB resistance. Of the 81 transgenic wheat lines developed, 70 (18 of 25 wheat α-1-purothionin, 23 of 25 barley tlp-1 and 29 of 31 barley β-1,3-glucanase) lines produced enough T2 seed for FHB evaluations. Sixteen to 20 seeds were planted for each line and inoculated with F. graminearum. We assayed the spread of the disease following point inoculation, and analyzed the results as the percent disease severity at 20 days after inoculation. In addition, each plant in the α-1-purothionin lines was assayed for transgene expression using RT-PCR. Only those plants expressing the α-1-purothionin transgene were used to evaluate the efficacy of α-1-purothionin against FHB. The plants carrying the tlp-1 and β-1,3-glucanase transgenes were not assayed for transgene expression in the initial T2 FHB screen. For the lines carrying the tlp-1 and β-1,3-glucanase transgenes, data from all plants assayed for FHB resistance, which would have included transgene null together with transgene homozygous and hemizygous plants, were used to calculate the percent FHB severity. We compared FHB severity in the transgenic lines against the nontransformed Bobwhite parent.
Based on this initial experiment, we eliminated the most susceptible lines (>50% disease severity) and reevaluated 6, 13, and 16 T2 lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively. We also evaluated the T3 lines of the same 6 lines carrying the α-1-purothionin transgene, and 13 lines carrying the tlp-1 transgene. Again, we planted 16–20 plants per line, and evaluated the lines against FHB. In this screen, all plants were assayed for transgene expression. Only those plants expressing the transgene were used to calculate disease severity for comparison against the nontransformed Bobwhite.
From the initial disease screens on the 70 transgenic lines, we identified seven lines with enhanced FHB resistance. One line had the α-1-purothionin transgene and is referred to as CM 17, two lines carried the tlp-1 transgene and are referred to as CM21 and CM23, and four lines had barley β-1,3-glucanase transgene and are referred to as CM27, CM28, CM30, and CM33. The results for these initial two screens of the resistant transgenic lines are shown in Table 2. The resistant transgenic lines were evaluated in further FHB disease screens in the greenhouse (Table 2). For these additional greenhouse evaluations, only plants expressing the transgene, based on RT-PCR assays, were used to calculate the percent FHB severity. One line carrying the α-1-purothionin transgene (CM17) significantly reduced FHB severity in four of five screens (P < 0.05), and had an overall average reduction of 34%. The tlp-1 transgenic CM23 and CM21 lines significantly reduced FHB severity when compared to the Bobwhite control in three of four or five screens, respectively (P < 0.05). Taking the average disease severity over all screen replicates, CM21 and CM23 reduced disease severity compared to Bobwhite by 30% and 36%, respectively. The ß-1,3-glucanase transgenic CM27, CM28, CM30 and CM33 lines significantly reduced FHB severity compared to the Bobwhite control in two to three screens (P < 0.05). The average reduction in disease severity compared to Bobwhite for CM27, CM28, CM30, and CM33 was 40, 49, 47, and 38%, respectively. While all seven transgenic lines had similar levels of enhanced disease resistance, the lines with the β-1,3-glucanase transgene had slightly better disease control.
Table 2Percent Fusarium head blight severity in greenhouse evaluations of seven wheat lines carrying wheat α-1-purothionin, barley thaumatin-like protein 1, or barley β-1,3-glucanase that were selected in initial tests and three wheat varieties used as disease checksGeneration testedaGenotypebT2T2T2T3T3T3T3T4CM1738*(9)44**(8)–c38**(10)74 (14)--28* (22)CM2139*(12)38 (4)26*** (11)89 (11)––57* (22)–CM2337*(14)55 (14)44** (18)41*(12)––––CM2738*(12)34**(12)––55* (17)–––CM2826**(10)37*(10)––41** (12)–––CM3019***(8)36*(11)––51 (4)–––CM3333 (8)48*(11)44* (7)––40* (12)––Bobwhite63 (31)71 (28)78 (28)71 (28)78 (28)64 (18)73 (33)54 (36)Wheaton70 (33)85 (45)–85 (45)–91 (21)94 (57)99 (60)Sumai 326 (22)9 (25)10 (78)9 (25)10 (78)21 (16)16 (46)7 (61)Numbers in parenthesis represent the number of plants in the screen.aIndicates the generation that was evaluated. T2 and T3 lines that were evaluated in the initial FHB disease screens are in bold. Each column, except for the second T2 screen and the first T3 screen, represent individual experiments where lines were evaluated simultaneously.bCM17 is a transgenic wheat line carrying the wheat α-1-purothionin, CM21 and CM23 are transgenic wheat lines carrying barley thaumatin-like protein 1, and CM27, CM28, CM30, and CM33 are the transgenic wheat lines carrying barley β-1,3-glucanase transgene. Bobwhite is the variety transformed and susceptible check, Wheaton is a susceptible check, and Sumai 3 is a resistant check. It is not known whether the transgenic lines were homozygous for the transgene or segregating.cIndicates that this line was not examined in this screen.*Significance at the 0.05 compared to Bobwhite.**Significance at the 0.01 compared to Bobwhite.***Significance at the 0.001 level compared to Bobwhite.
Molecular characterization of transgenic plants
To verify that one, two, and four lines transformed with α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, were transgenic, we conducted Southern blot analysis. Our Southern blots also provided the opportunity to determine if the two tlp-1 lines and four β-1,3-glucanase lines were independent events. Genomic DNA was isolated from each line, digested with the appropriate restriction enzyme, blotted, and hybridized with a radio-labeled probe from the α-1-purothionin, tlp-1, or β-1,3-glucanase transgenes (Fig. 2). Each transgenic line contained at least one unique band compared to the nontransformed Bobwhite, indicating that each of the lines were transgenic for the appropriate transgene. In addition, the banding patterns of the two tlp-1 lines and the four β-1,3-glucanase lines were distinct, indicating that the two tlp-1 lines were independent events, and the four β-1,3-glucanase lines were also independent events.
Fig. 2a–c Southern blot analysis of transgenic wheat plants. aEcoRI-digested genomic DNA from untransformed Bobwhite (BW), and pUBKBarGluc-3 transgenic CM27, CM28, CM30, and CM33 plants hybridized with a probe designed to bridge the ubiquitin promoter and the β-1,3-glucanase transgene junction. bHindIII-digested genomic DNA from untransformed Bobwhite (BW), and pAHCBarPR5 transgenic CM21 and CM23 plants and hybridized with a probe designed to bridge the ubiquitin promoter and the tlp-1 transgene junction. cXhoI-digested genomic DNA from untransformed Bobwhite (BW), and pKM1 transgenic CM17 plants hybridized with a probe designed to bridge the ubiquitin promoter and the α-1-purothionin transgene junction
To confirm transgene expression, we conducted RT-PCR and Western blot analyses. As stated earlier, RT-PCR was conducted on each plant used in the greenhouse disease evaluations, except where indicated. Figure 3 shows an example of the RT-PCR analysis of the lines carrying each transgene. We also conducted Western blot analysis on plants carrying the tlp-1, and β-1,3-glucanase transgenes. We isolated protein from spikes, blotted the protein, and cross-reacted the blots with antibodies specific for tlp-1 and β-1,3-glucanase proteins. Our results showed that the transgenic lines exhibited an increase in their appropriate transgene protein compared to the nontransgenic Bobwhite control (Fig. 4).
Fig. 3RT-PCR analysis of transgenic wheat lines carrying the wheat α-1-purothionin (CM17), barley tlp-1 (CM21 and CM 23), and barley β-1,3-glucanase (CM27, CM28, CM30, and CM33) transgenes. The fragment sizes for the α-1-purothionin, barley tlp-1, and barley β-1,3-glucanase amplified the expected products of 600, 805, and 577 bp, respectively. The wheat actin gene was used as a positive control and it exhibited the expected size of 369 bpFig. 4a–b Western blot analysis of transgenic wheat lines. a  Protein extracted from spikes of transgenic lines carrying barley β-1,3-glucanase (CM27, CM28, CM30, and CM33) transgene was subjected to SDS-PAGE using a 12% polyacrylamide gel. Our barley β-1,3-glucanase antibody does not distinguish the transgenic barley protein from the endogenous wheat protein. The transgene-specific protein band in line CM33 appears to exhibit a higher molecular weight. Molecular markers indicated the protein to be the expected 35.2 kDa size. b Protein extracted from spikes of lines carrying barley tlp-1 (CM21 and CM23) transgene was subjected to SDS-PAGE using a 10% polyacrylamide gel. Molecular markers indicated the protein to be the expected 17.5 kDa size
Field evaluation of transgenic plants for response to Fusarium head blight
To further examine the level of effect on FHB, we conducted field tests of these seven lines in the summers of 2004 and 2005. Seed for each of the seven lines was derived from plants expressing the transgene based on RT-PCR analysis. We scored the lines for percent FHB severity, DON concentration, and percent visually scabby kernels (VSK) and compared the lines to the parental cultivar Bobwhite (Table 3). All transgenic lines tested in the field except for CM28 showed a significant reduction in at least one FHB disease measure in comparison with Bobwhite. Four, four, and two lines exhibited significant reductions in percent FHB severity, DON concentration, and percent VSK, respectively. Although CM30 showed a significant reduction in percent FHB severity, it showed a significant increase in DON concentration. CM27 was the only transgenic line that exhibited a significant reduction in all three disease measures.
Table 3Percent Fusarium head blight (FHB) severity, deoxynivalenol (DON) concentration, and percent visual scabby kernels (VSK) in transgenic wheat plants carrying wheat α-1-purothionin, barley thaumatin-like protein l, and barley β-1,3-glucanase and check wheat varieties evaluated in the field in 2004 and 2005GenotypeaFHB severity (%)DON concentration (ppm)bVSK (%)Bobwhite65.116.329.6Alsen15.4***3.7***5.4***Wheaton81.2***26.2***51.9***Wheaton (noninoculated)64.317.832.2Roblin70.918.842.2*237546.2**8.3***11.6***CM1752.7*15.724.7CM2155.111.4***19.8*CM2357.213.4*21.0CM2746.5***9.9***17.7*CM2858.217.625.8CM3048.3***22.8**34.3CM3349.2***14.320.3aCM17 is a transgenic wheat line carrying the wheat α-1-purothionin, CM21 and CM23 are the transgenic wheat lines carrying barley thaumatin-like protein 1, and CM27, CM28, CM30, and CM33 are the transgenic wheat lines carrying barley β-1,3-glucanase transgene. T6 and T7 were used for the 2004 and 2005 field screens, respectively. Bobwhite is the variety transformed and susceptible check, Wheaton and Roblin are the susceptible checks, 2375 is a moderately resistant check, and Alsen is a resistant check.bParts per million deoxynivalenol concentration.*Significance at the 0.05 level compared to Bobwhite.**Significance at the 0.01 level compared to Bobwhite.***Significance at the 0.001 level compared to Bobwhite.
Discussion
Large-scale wheat breeding efforts have not resulted in the development of highly resistant varieties to FHB. This is due to the fact that resistance in wheat is partial and quantitative. That is, multiple loci in wheat explain just a portion of the variation for FHB resistance (e.g., Kolb et al. 2001). Single genes conferring a high degree of resistance to FHB have not been found despite extensive searches of wheat germplasm resources (Leonard and Bushnell 2003). One characteristic of the wheat response to F. graminearum infection is the induction of defense response genes such as β-1,3-glucanase, tlp-1, and thionin genes (Chen et al. 1999; Pritsch et al. 2000, 2001; Li et al. 2001; Kang and Buchenauer 2002; Han et al. 2005; Zhou et al. 2005; Bernardo et al. 2006). These genes are thought to provide basal resistance during infection because they encode proteins with differing modes of action against fungal pathogens. Thionins and tlps damage fungal cell membranes by making them permeable (Bohlmann et al. 1988; Yun et al. 1998), whereas β-1,3-glucanases degrade cell wall polysaccharide linkages (Leah et al. 1991). In this study, we produced transgenic wheat lines overexpressing either α-1-purothionin, a tlp-1, or a β-1,3-glucanase to test their efficacy against FHB.
Numerous studies reveal that over-expression of defense response genes in transgenic plants results in enhanced resistance to various fungal pathogens (reviewed in Muehlbauer and Bushnell 2003). In general, these studies show that partial resistance can be achieved from over-expressing defense response genes in plants. In this study, we defined enhanced resistance as exhibiting a reduction in any of the three disease parameters. To date, there are no reports of commercially practical levels of fungal resistance derived from over-expressing defense response genes.
From our initial 70 transgenic lines, there were seven lines carrying either α-1-purothionin, tlp-1, or β-1,3-glucanase transgenes that resulted in enhanced FHB resistance in the greenhouse. Enhanced resistance was not detected in each of these seven lines in every greenhouse screen conducted (Table 2). These results are likely due to the high variability inherent in FHB disease screens. However, over multiple disease screens, the transgenic lines provided a level of resistance above that present in the nontransgenic control cultivar Bobwhite. In particular, we identified CM27, a line carrying a β-1,3-glucanase transgene that exhibited low FHB severity, low DON concentration, and low percent VSK in the field. Interestingly, in the field screens we observed lines, such as CM30, with significantly lower FHB severity and a high DON level. As seen in the greenhouse screens, these results are likely due to the variation in FHB disease screens. Variation in FHB readings from field grown plants can be difficult to control (Campbell and Lipps 1998).
Consistent with our results, Chen et al. (1999) and Anand et al. (2003) showed that overexpression of tlp-1 and a combination of β-1,3-glucanase and chitinase transgenes in wheat resulted in enhanced FHB disease in the greenhouse. Interestingly, these authors only detected enhanced resistance during early stages of disease progression. They interpreted the action of these transgenes as delaying the development of FHB. Unfortunately, field disease screens of their lines lacked resistance (Anand et al. 2003). For their field study, these authors used inoculated corn kernels, which provided a continuous source of inoculum, whereas in our field study, we sprayed fungal spores on the spikes twice. Thus, there is a distinct difference in the inoculation methodology between the two studies, which could lead to different disease reactions. Developmental differences such as the timing of flowering have resulted in different disease reactions to FHB. When inoculated grain is used for the inoculum, early heading plants can exhibit greater susceptibility than late heading plants as they are exposed to the inoculum for a longer period of time. In our study, we controlled the timing of inoculation through spraying the spikes, and we did not observe any obvious developmental differences in our transgenic lines. Thus, our results demonstrate that enhanced resistance to FHB can be obtained through overexpressing defense response genes.
To date, there are no wheat germplasm sources that exhibit immunity to FHB. The best available lines, such as Sumai 3 and Alsen, exhibit resistance to initial infection and spread of the disease but this resistance is partial and plants may become severely diseased when conditions are highly favorable for disease development. The transgenic lines described in this study may provide a potential wheat germplasm source for enhanced FHB resistance. Although the level of transgene resistance is not high enough to alone provide useful protection to FHB, our transgenic lines may extend and enhance FHB resistance germplasm when combined with other resistance sources. To increase the level of resistance, we crossed our β-1,3-glucanase and tlp-1 transgenic lines and combined the transgenes into a common background because developing lines with multiple transgenes in tobacco increased resistance to a fungal pathogen (Jach et al. 1995). We have also initiated crosses of our transgenic lines with the moderately resistant genotype, Alsen. Alsen contains the chromosome 3BS QTL for FHB resistance (Waldron et al. 1999). Our goal is to develop populations containing the 3BS QTL in combination with each of the three transgenes. Our expectation is that these combinations may result in enhanced resistance to FHB over the levels present in Alsen.

transgenic wheat
wheat
fusarium head blight
fusarium graminearum
triticum aestivum