chemical communication in heliothine moths
TRANSCRIPT
J Comp Physiol A (1995) 177:527-534 �9 Springer-Verlag 1995
B. G. Berg �9 J. H. Tumlinson �9 H. Mustaparta
Chemical communication in heliothine moths IV. Receptor neuron responses to pheromone compounds and formate analogues in the male tobacco budworm moth Heliothis virescens
Accepted: 22 April 1995
Abstract Single receptor cell recordings from the sen- silla trichodea type 1 of the Heliothis virescens male antennae have revealed: 1) Three distinct types of receptor neurons, two pre- viously identified types tuned to the principal pheromone components, (Z)-ll-hexadecenal (Zll- 16:AL) (79% of neurons recorded) and (Z)-9-tet- radecenal (Z9-14:AL) (12%), and a third type (9%) that is tuned to (Z)-11-hexadecenyl acetate (Z11-16: AC). The acetate suppresses male attraction to conspecific females. 2) The Zl l -16:AL receptor neurons respond with about equal frequency to the formate analogue, (Z)-9- tetradecenyl formate (Z9-14:FO) at comparable concen- trations. The Z9-14:AL receptor neurons, however, show only weak responses to the corresponding formate (Z)-7-dodecenyl formate (Z7-12:FO). These results cor- relate well with results from field studies, showing that Z9-14: FO can substitute behaviourally for Z11-16: AL, whereas Z7-12: FO cannot substitute for Z9-14: AL. 3) The Zl l -16:AC receptor neurons also respond to Z7-12:FO at higher concentrations. Therefore, Z7- 12:FO would not be able to simulate the effect of Z9-14:AL, even at high concentration, because Z7- 12:FO would mediate suppression of pheromone at- traction through the Z11-16:AC neurons. 4) A difference in the interaction between the pheromone aldehydes and their membrane receptors is shown by the different effects of the two formate ana- logues. Furthermore, shortening and lengthening of the two moieties of the Zl l -16 :AL chain does not reduce the stimulatory effect to the same extent.
B. G. Berg �9 H. Mustaparta ( ~ ) Department of Zoology, University of Trondheim AVH, N-7055 Dragvoll, Norway
J. H. Tumlinson Insect Attractants, Behavior, and Basic Biology Research Laboratory, Agricultural Research Service, USDA, Gainsville, FL 32604, USA
Abbreviations Zll-16:AL (Z)-ll-hexadecenal �9 Z9-14:AL (Z)-9-tetradecenal �9 Z9-16:AL (Z)-9-hexadecenal �9 ZT-16:AL (Z)-7-hexadecenal �9 16:AL hexadecanal �9 14:AL tetradecanal �9 Zl l -16:AC (Z)-ll-hexadecenyl acetate �9 Z9-16:AC (Z)-9-hexadecenyl acetate �9 ZT-16:AC (Z)-7-hexadecenyl acetate �9 16:AC hexadecanyl acetate �9 Zll-16: OH (Z)-I 1-hexadecenol �9 Z9-16: OH (Z)-9-hexadecenol �9 zg- 14: FO, (Z)-9 -tetradecenyl formate �9 Z7-12: FO (Z)-7-dodecenyl formate �9 Zll-18:AL (Z)-I 1-octadecenal �9 Z13-18:AL (Z)-13-octadecenal �9 H. virescens Heliothis virescens �9 H. zea Helicoverpa z e a
Introduction
The tobacco budworm moth Heliothis virescens is a polyphagous species, the larvae of which feed on cotton, tobacco and corn. Together with Helicoverpa zea and Pectinophora gossypiella, it forms a complex of economically important lepidopteran pest insects on cotton in the American continent. The pheromones of these species have been identified, and their effects in mating disruption have been thoroughly studied. In H. virescens the pheromone is a 6 component blend, consisting of (Z)-ll-hexadecenal (Zll-16:AL), (Z)-9- tetradecenal (Z9-14:AL), (Z)-9-hexadecenal (Z9- 16: AL), (Z)-7-hexadecenal (Z7-16: AL), hexadecanal (16: AL) and tetradecanal (14: AL) (Roelofs et al. 1974; Tumlinson et al. 1975; Klun et al. 1980; Teal et al. 1986). However, only two of the compounds, the principal pheromone components Zl l -16:AL and Z9-14:AL, are necessary and sufficient to elicit attraction of males (Vetter and Baker 1983). The natural ratio which has optimal effect, is about 16:1 (Pope et al. 1982). Since aldehydes are relatively unstable compounds it has
528 B. G. Berg et al.: Receptor neuron responses to pheromone mimics
been of interest to find out whether the more stable formates can substitute for aldehyde pheromones in H. virescens and H. zea. The structural similarities be- tween corresponding aldehydes and formates are strik- ing. Priesner et al. (1975) showed by EAG-recordings that the formate, (Z)-9-tetradecenyl formate (Z9- 14:FO), had at least as strong a stimulatory effect as the major pheromone component Zl l -16:AL on the antennal receptor neurons of H. zea. Furthermore, it was shown in field tests that Z9-14: FO effectively dis- rupted the pheromone communication in H. virescens and H. zea, which suggested that this compound can adapt the receptor neurons receiving the information from Zl l -16:AL (Mitchell et al. 1975, 1976). In addi- tion, it was found that Z9-14:FO and the second prin- cipal pheromone component, Z9-14:AL, in a mixture of 16: 1, was as attractive to H. virescens males as the natural or synthetic pheromone blend (Mitchell et al. 1978). As a next logical step, it was tested whether (Z)-7-dodecenyl formate (Z7-12:FO) could substitute for the other pheromone aldehyde, Z9-14: AL (cf. Tum- linson 1979). The results showed, however, that this formate failed to mimic Z9-14:AL in H. virescens (Mitchell et al. 1978). A mixture of Z11-16:AL and Z7-12:FO in the ratio 16:1 did not attract males of H. virescens, nor did Z7-12:FO evaporated around H. virescens females reduce the attraction of H. vires- cens males. This suggested that Z7-12:FO might nei- ther activate nor adapt the Z9-14:AL receptor neurons adequately.
Electrophysiological studies of receptor neurons on the male antenna of H. virescens have revealed 3 types of neurons receiving information from pheromone compounds produced by heliothine moths (Almaas and Mustaparta 1990). One of these neuron types was tuned to (i.e. specialized for, by responding significantly best to) the principal pheromone component Z l l - 16:AL, another to Z9-14:AL, and the third type of neuron responded best to (Z)-ll-hexadecenol (Z11-16:OH), however, with a lower sensitivity. In the present investigation the effects of Z9-14: FO and Z7- 12:FO, as well as of other chemical analogues, have been tested on single receptor neurons in H. virescens. The results demonstrate that the third type of receptor neuron is tuned to Zll-16:AC. Furthermore, they ex- plain why Z7-12:FO may not substitute for Z9-14:AL in the pheromone blend, whereas the Z9-14:FO can substitute for Zll-16:AL. The results also indicate a difference in the interaction between the two al- dehydes and their respective receptors.
were placed in a container in an incubator on a phase-shifted LD 14 h: 10 h photoperiod at 23~ Each day, moths that had emerged were placed in a separate container marked with the date and allowed to feed on honey-water. Most animals were studied 2 4 days after emergence.
Electrophysiological recordings
The insects were placed inside a Plexiglas jacket with the head and antennae exposed as previously described by Almaas and Mus- taparta (1990). The antennae were fastened by tungsten hooks to a soft wax platform. Recordings from single receptor neurons were made, using tungsten microelectrodes (tip diameter less than 0.3 p.m). The indifferent electrode was inserted into the haemolymph at the base of the antenna, whereas the recording electrode tip was inserted into the base of the olfactory sensillum until the extracellu- lar impulse activity was displayed. The preamplifier had an input resistance of 500 MfL capacitance < 10 pF, 1. order highpass filter, 2. order lowpass filter and 50 Hz notchfilter. The neural activity was monitored by a loudspeaker and displayed by an oscilloscope. Recordings were stored by a Hewlett Packard 3964A instrumenta- tion tape recorder and recorded by a modified Siemens-Elma Min- gophon 3 for further analysis.
Test compounds
The synthetic compounds used for determining the specificities of olfactory receptor neurons were obtained from various commercial sources, or synthesized by standard procedures in the USDA, ARS Laboratory, Gainsville, FL. They were purified to a purity greater than 99% by HPLC on silica, reverse phase, or AgNO 3 treated silica columns and analyzed for purity and confirmation of structure by capillary gas chromatography and mass spectroscopy (Heath and Tumlinson 1984). The following twelve compounds, which are pheromone constituents of heliothine moths were included: Z11- 16:AL, Z9-14:AL, Z9-16:AL, Z7-16:AL, 16:AL, 14:AL, (Z)-l l - hexadecenyl acetate (Z11-16: AC), (Z)-9-hexadecenyl acetate (Z9- 16:AC), (Z)-7-hexadecenyl acetate (Z7-16:AC), hexadecanyl acetate (16:AC), (Z)-I 1-hexadecenol (ZI 1-16:OH) and (Z)-9-hexadecenol (Z9-16:OH). In addition to these compounds naturally present in the insects, the following four chemical analogues were tested: Z9- 14:FO, Z7-12:FO, (Z)-I 1-octadecenal (Zll-18:AL) and (Z)-13-oc- tadecenal (Z 13 - 18 : AL) (Fig 1).
The compounds were diluted (decade steps) in n-hexane (HPLC- grade), from 50 mg/ml to 0.5 ng/ml. The principal pheromone com- ponents Z l l -16 :AL and Z9-14:AL were diluted to 0.0005 ng/ml. Two series of all the compounds were made independently. From each dilution, 200 pl was applied on a piece of filter paper under a weak stream of pure nitrogen which helped evaporate the solvent. Each filter paper was placed in a test cartridge (10 cm long, 4 mm i.d.). In addition, separate cartridges, each containing 0.01 ~tg of a compound, were prepared for all substances. These were used to initially screen each receptor neuron for sensitivity to the com- pounds. The neuron was then tested using the duplicated test series of the most effective compounds. These tests were always applied from low to high concentrations. Substances eliciting no response during the initial screening were also tested at higher concentrations. Empty cartridges and cartridges with pure hexane evaporated onto a filter paper were used as control stimuli.
Materials and methods
Insects
Male pupae of Heliothis virescens (Lepidoptera: Noctuidae), were provided by Dr. Max Angst, Ciba-Geigy, Basel, Switzerland. They
Stimulation
The stimuli were applied with the use of a Syringe-olfactometer (Kafka 1970). Cartridges containing the odors were fastened to the outlet of a clean 20ml syringe (Almaas and Mustaparta 1990).
B. G. Berg et al.: Receptor neuron responses to pheromone mimics
Fig. 1 Structure of the three compounds (bold) to which the receptor neurons are tuned in Heliothis virescens males. Below the two principal pheromones Zll-16:AL and Z9-14:AL, the structure of the corresponding formates and three other chemical analogues are shown
Zll-16:AL
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Z9-14:AL
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~ O ~ C H 3 0
About 7 ml of the air (and therefore more than the volume of the stimulus cartridge) was blown through the cartridge during 560 ms. The inter-stimulus interval was 1 min for low concentrations and 4 - 1 0 m i n for high concentrations, depending on the response strength. During the intervals purified air was blown over the antenna.
Results
The results are derived from recordings of 140 neurons in 22 H. virescens males, of which 108 neurons re- sponded specifically to pheromone compounds of he- liothine species. These neurons could be classified in three groups. The majority (85 neurons) were tuned to one principal component of the H. virescens pherom- one blend, Z11-16:AL. Thirteen neurons were tuned to the second principal component Z9-14:AL, and 10 neurons were tuned to Z11-16:AC which is not pro- duced by H. virescens females.
The response patterns were typically phasic-tonic for all neurons, with an initial high firing frequency which decayed to a tonic level. The response lasted until the stimulation ended or outlasted the stimulation period, depending on the stimulus intensity (Fig 2). Some vari- ations of the expressed phasic component were also seen within each group of neurons. In particular the Zl l -16:AC neurons often showed a less pronounced initial phasic response than the other neurons.
Effect of the formates and other analogues on receptor neurons tuned to Z11-16:AL
The specificities of 26 Z11-16:AL neurons were deter- mined according to the dose-response curves of the test compounds. As shown in Fig. 2, the neurons responded with increased firing rate in a dose-related manner to Z11-16:AL. The sensitivities of these neurons varied,
with thresholds from 0.01-10 ng. The responses to the chemical analogue Z9-14:FO were about the same as to the key compound Zl l -16 :AL when testing the same amount of compounds on the filter paper (Fig. 2). The very sensitive neurons also responded to Z9- 14:AL and Z l l -16 :OH at high concentrations. The other test compounds, including the formate Z7- 12: FO, had no or minimal effect on these neurons. The dose-response relationships of a single Zl l -16 :AL neuron is shown in Fig. 3A, demonstrating overlapping of the dose-response curves for Z l l -16 :AL and Z9- 14:FO, with only a slight shift to the right of the formate curve at low concentrations. The much lower effect of Z9-14:AL and Z l l -16 :OH is shown by the shift of the dose-response curves 2-3 log units to the right. The average dose-response curves for Z 11-16:AL and Z9-14:FO of 16 neurons are shown in Fig. 3B, also demonstrating the overlap of the two curves.
Nine neurons tuned to Z l l -16 :AL were tested for sensitivity to the non-biological analogues Z13-18 :AL and Z11-18:AL, as well as Z9-14:AL. All three ana- logues showed a dose-response shift to the right of about 2-3 log units (Fig. 4). At doses 1, 10, 100 and 1000 ng, Z13-18:AL elicited slightly stronger responses than Z11-18:AL and Z9-14:AL (significantly different at 1, 100 and 1000 ng, P < 0.05, Wilcoxon Matched- Pairs Signed-Ranks Test).
Effect of the formates and other analogues on receptor neurons tuned to Z9-14:AL
Dose-response relationships were determined for nine of the 13 neurons tuned to Z9-14:AL. Activities from the other four were recorded simultaneously with activ- ity from neurons tuned to Zll-16:AC. Because of the relatively small spike amplitudes of the four Z9-14:AL neurons (interfering with the spikes of the Z11-16:AC
530
Fig. 2 Responses of a single neuron tuned to the major pheromone component Z11- 16:AL. Increased response firing rates are shown for increased doses of Z l l -16 :AL (0.0001, 1, 100 and 10000 ng). The responses to 100 and 10000 ng of Z9-14:FO is shown, demonstrating about the same response strength as to the aldehyde at the corresponding doses. The stimulatory effect of Z9-14:AL at the highest concentration (10000 ng) is shown, demonstrating a moderate response. Response to control stimulation was nil. The stimulation period (560 ms) is indicated by a horizontal bar below
B. G. Berg et al.: Receptor neuron responses to pheromone mimics
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component Z l l -16 :AL. The 120 curves of Z l l - 1 6 : A L and of
Z9-14:FO are almost E 110 completely overlapping, whereas o
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independently prepared test ~ 70
series. B Average dose-response curves for some of the Z l l - -o 16:AL neurons (n = 16), ~ 60
demonstrating the close overlap 3 50 of the Z l l - 1 6 : A L and Z9- 14:FO curves ~ 40
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B. G. Berg et al.: Receptor neuron responses to pheromone mimics 531
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Fig. 4 Dose-response curves for Z l l - 1 6 : A L and three chemical analogues, recorded from some receptor neurons (n = 9) tuned to Z l l -16 :AL. The curves of all three analogues are shifted 2-3 log units to the right of the key compound curve. A slightly higher effect of Z13-18:AL is shown at low concentrations (i.e. 1-1000 ng). Con- sidering the different volatilities of these compounds, the dose- response curves (of equal numbers of molecules) for Z13-18:AL should be shifted slightly to the left for the Z l l - 1 8 : A L curve, whereas the curve for Z9-14:AL should be shifted to the right. Standard deviations are omitted for clarity
findings that the information from the two principal pheromone components Z 11-16:AL and Z9-14:AL are received by specialist types of receptor neurons. These pheromone-specific receptor neurons mediate the pheromone information to the brain via a labelled-line mechanism (Almaas and Mustaparta 1990). The third group of neurons, tuned to Zll-16:AC, which re- sponded second best to Z l l -16 :OH and Z9-16:OH, is probably the same type of neurons previously classified as Z l l -16 :OH neurons by Almaas and Mustaparta (1990). In the previous investigation these neurons were not tested for Zll-16:AC, and it was pointed out that they might in fact be tuned to another compound, since they displayed a relatively low sensitivity to Z l l - 16:OH. The presence of the Zl l -16:AC neurons sug- gested that the acetate might mediate an important message for the H. virescens males. The behavioural effect of this compound is now being studied, and preliminary results suggest that Z11-16:AC suppresses pheromone attraction of males (NJ Vickers and TC Baker, personal communication). Since the acetate is produced by sympatric heliothine species (Teal et al. 1981; Klun et al. 1982) as well as species of related genera (Underhill et al. 1977), it is quite possible that it acts as an interspecific signal, interrupting the male pheromone attraction. It is also possible that the acet- ate acts intraspecifically, since it is produced by the H. virescens males (Teal and Tumlinson 1989). It is interesting that the acetate neurons responded second best to Z l l -16 :OH (Fig. 6) which has been found to reduce pheromone attraction at relatively high concen- trations (Vetter and Baker 1983; Shaver et al. 1989).
532 B.G. Berg et al.: Receptor neuron responses to pheromone mimics
Fig. 5 Dose-response curves 1 40 from a single receptor neuron tuned to Z9-14:AL (A), and 130
average dose-response curves for 6 neurons (B). A Dose-response 120 curves for Z9-14:AL, Z9-16:AL ~ 110 and Z7-12:FO obtained for o a very sensitive receptor neuron. ~ 100 The Z9-16:AL and Z7-12:FO curves are shifted 2 3 and ~ 90 3-5 log units to the right, .~ respectively. B Average dose- ~ 80 response curves (n = 6) for Z9- ~ 70 14:AL and Z7-12:FO, demonstrating the much lower "~ effect of the formate. Duplicated ~ 60
o~ curves indicate separately "~ 50 prepared test series
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Fig. 6 Average dose-response curves from six neurons tuned to Z 11-16: AC. The biological analogues Z9 - 16: OH and Z 11-16: OH had a relatively high stimulatory effect on these neurons, showing dose-response curves shifted only 1 log unit to the right of the acetate curve. A further shift to the right of about 1 log unit is seen for the dose-response curves of Z7-12:FO. Standard deviations are omitted for clarity
Also in other species it has been shown that neurons tuned to interspecific signals respond relatively strong- ly to more than one biological compound (Lucas and Renou 1989). It is, therefore, possible that some insects possess one type of receptor neuron that mediates in- terruption from several interspecific signals. The signifi- cance of the Z l l -16 :AC neurons is further demon- strated in the two following papers of this volume. It is
shown that the Z 11-16: AC neurons project to a specific part of the macro-glomerular complex of the antennal lobe in H. virescens males (Hansson et al. 1995, this volume). Furthermore, some projection neurons in the antennal lobe of the males responded specifically to the Z11-16:AC (Christensen et al. 1995, this volume). This suggests that the information from the interspecific signal is received by specific receptor neurons and fur- ther transmitted to and processed in separate pathways throughout the antennal lobe in H. virescens, like in H. zea.
The similar effect of the same dosage of Zl l -16:AL and Z9-14: FO on the receptor neurons tuned to Z11- 16:AL (Figs. 1 and 2) is in accordance with results from field studies, showing that the formate can completely substitute for the aldehyde both as regards pheromone attraction and disruption (Mitchell et al. 1975, 1976, 1978). Similar results from electrophysiological invest- igations have been reported for the Zl l -16:AL recep- tor neurons in the sympatric species H. zea (Grant et al. 1989), which is also in accordance with field tests (Mitchell and McLaughlin 1982). Grant et al. made calculations, correcting for the different volatilities of the formate and the aldehyde, indicating a shift of the formate dose-response curve less than one log unit to the right of the aldehyde curve. This suggests that more formate than aldehyde molecules are needed in order to induce the same response strength of the Zl l -16:AL receptor neuron, both in H. zea and in H. virescens.
In contrast to the similar effect of the aldehyde and the formate discussed above, the Z9-14:AL neurons showed very different responses to the key compound
B. G. Berg et al.: Receptor neuron responses to pheromone mimics 533
Z9-14:AL and the corresponding formate. Here, the pheromone aldehyde was 1000 times more effective than Z7-12:FO. In principal, this is also in accordance with the field study, showing that the formate could not substitute for Z9-14:AL (Tumlinson 1979). However, the present electrophysiological results raise the ques- tion of whether Z7-12:FO could eventually mimic Z9- 14: AL by increasing the concentration of the formate. This seems not to be possible, since the Z l l -16 :AC neurons also respond to the Z7-12:FO. Thus, when increasing the concentration of the formate, the acetate neurons would respond before the Z9-14:AL neurons (Figs. 5 and 6). This suggests that the formate would mediate interruption, via the Z l l -16 :AC neurons, in- stead of attraction via the Z9-14:AL neurons.
The results from the two types of pheromone recep- tor neurons, showing different relationships with re- spect to the stimulatory effects of the key aldehydes and the corresponding formates, suggested that different parts of the two aldehyde molecules, the m- and the n-chain ( C H 3 - ( C H 2 ) m /=~, ( C H z ) n - C H O ) might domin- ate in the interaction with their membrane receptors. In one case, changing the key stimulus (Zll-16:AL) by substituting one methylene group by an oxygen in position C2, did not influence the stimulatory effect. In contrast, the same substitution of the key compound for the Z9-14:AL neurons, reduced the stimulatory effect by a factor of at least 1000. These results led to the assumption that, in the interaction with the Z1 l-16:AL receptor neuron the m-chain of the signal molecule dominates, whereas in the interaction with the Z9- 14:AL receptor the n-chain is most important. Thus, the higher effect of Z9-16:AL (with 2C too long m- chain and n-chain correct) than of ZII -16:AL (with m-chain correct and 2C too long n-chain) supports the assumption that for the interaction with the Z9-14:AL receptor, a correct n-chain is more important than a correct m-chain. However, both chains must have the correct length in order to elicit full response. In spite of this, the Z7-12:FO, having about the correct length of both the m- and the n-chain, had a very low effect. This suggests that, in addition, the methylene group of the C2 position is very important for the interaction.
In the case of the Z l l -16 :AL receptor neuron, the methylene group in the C2 position does not seem to be so critical, since Z9-14:FO had the same stimulatory effect as Z l l -16 :AL at the same amount on the filter paper. In order to compare the importance of the m- and n-chain in the interaction with the Z l l -16 :AL receptor, the three chemical analogues, Z13-18:AL, Z l l -18 :AL and Z9-14:AL, were tested on the same neurons. In this case it is important to consider the different chemical properties of the compounds, in- fluencing the number of molecules given off from the filter paper. Here Z13-18:AL and Z l l -18 :AL would evaporate slower than Z 11-16: AL, whereas Z9-14: A L would evaporate about 6x faster than Zl l -16:AL. Furthermore, Z13-18:AL would evaporate slightly
slower than Z11-18:AL. By ranking the compounds in respect to their stimulatory effects (at the same amount on the filter paper) (Fig. 4), it follows that: Z13-18:AL (m correct and n 2C too long) > Zl1-18 :AL (m 2C too long and n correct) > Z9-14:AL (m correct and n 2C too short). Moreover, this ranking would not change, but rather be strengthened by considering the different volatilities of the compounds. This suggests that correct length of both m and n is important. However, with the n-chain 2C too long and m correct, the stimulatory effect is better than when the m-chain is 2C too long and n correct, which is better than when the n-chain is 2C too short and m correct. This means that the as- sumption that the m-chain of the signal molecule is more important than the n-chain in the interaction with the Z 11-16:AL receptor is too simple. The length of the n-chain is important, but a shorter n-chain gives a lower stimulatory effect than a longer n-chain. Never- theless, the results demonstrate that there is an impor- tant difference between the two receptor types as con- cerns the interactions with their key stimulants, the Z l l -16 :AL and Z9-14:AL. In one case, the substitu- tion of the methylene group with an oxygen does not matter much, whereas in the other case the same substi- tution strongly decreases the stimulatory effect.
Acknowledgements The project was supported by grants from the Norwegian Research Council, NFR, project # 510009. We are also grateful to Dr. Max E. Angst, Ciba-Geigy, Switzerland for providing insect materials and to Dr. Thomas A. Christensen, University of Arizona for comments on the manuscript.
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