Journal of Chemical Ecology, Vol. 15, No. 1, 1989
OLFACTORY ORIENTATION RESPONSES BY WALKING FEMALE Ips paraconfusus BARK BEETLES I. Chemotaxis Assay
R. PATRICK
AKERS l and DAVID
L. WOOD
Department of Entomological Sciences University of California Berkeley, California (Received May 12, 1987; accepted October 19, 1987) Abstract--Gas-liquid chromatography of the air within the arena developed for this assay showed that a concentration gradient was established within 12 min of applying the pheromone (ipsenol, ipsdienol, cis-verbenol), and that this gradient was nearly constant for 20-95 rain after application. The concentration fell rapidly and approximately exponentially between the source and the center of the arena. Turning rate and the number of beetles that reached the source increased, and heading with respect to the source decreased, in the presence of pheromone. Responses of beetles that did and did not reach the source were significantly different, but within each group there were no significant differences among dosages. Turning rate and heading varied little with distance from the source, while walking rate decreased as distance from the release point of the beetles increased. We hypothesize that dosage exerts its major effect on source location by altering the probability that a beetle will enter into orientation behavior and that beetles orienting to sources have similar behaviors even when orienting to a wide range of dosages. Key Words--lps paraconfi~sus, Coleoptera, Scolytidae, bark beetles, taxis, chemotaxis, orientation, olfaction, pheromones.
INTRODUCTION These experiments were undertaken while developing assays for a study of quality d i s c r i m i n a t i o n
among
the components
of a multicomponent
pheromone
Present address: Worcester Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, Massachusetts 01545.
0098-0331/89/0100-0003506.00/0 © 1989PlenumPublishingCorporation
4
AKERS AND WOOD
(Akers and Wood, 1989). An assay had to provide clear, differential responses to olfactory stimuli, which could serve as a behavioral basis for defining different odor qualities. The extent to which an assay met this requirement was tested by exposing beetles to a dosage series of a standard pheromone blend. Ips paraconfusus females readily found pheromone sources 16-18 cm away in still air, and we became curious as to the mechanisms involved. A complete knowledge of the behavioral mechanism was not necessary to use the assay as an indicator of olfactory significance, but the more complete the behavioral description, the better we can visualize possible neural mechanisms. This, in turn, should assist in interpreting neurophysiological recordings and in deriving models from them (Kein 1974ab, 1975). Several other studies of orientation in still air suggested that the paths of insects become more linear as they near a source of odor (Klingler, 1958; Bell and Tobin, 1981; Fraenkel and Gunn, 1961, Chap. 18). Since higher dosages should produce steeper concentration gradients (Crank, 1956), the inference might be drawn from these previous studies that as dosage increased all the insects in a population would shift their overall directional vectors (Bell and Tobin, 1982) towards the source and then approach it in a more linear or direct manner. The proportion of beetles that reached the source would increase as the intersections of their paths with the arena wall became more narrowly distributed around the source. Alternatively, the group that reaches the source might be comprised of beetles that enter into orientation behavior while the group that does not reach the source might be comprised of beetles that do not enter into orientation. Dosage would then change the proportion of orienting beetles, while the behavior of orienting beetles in different dosages might be similar. In our first analyses the beetles appeared to head more directly to the source as dosage increased, which seemed to support the first hypothesis. However, when the second hypothesis occurred to us, a more extensive analysis indicated that it was probably the more correct of the two. The initiation of orientation therefore appears to have a strong all-or-none component. A second paper (Akers, 1989) examines the mechanisms by which an orienting beetle may reach the source.
METHODS AND MATERIALS
Compounds. The attractant pheromone of L paraconfusus is a blend of ipsenol (Ip), ipsdienol (Id), and cis-verbenol (cV) (Silverstein et al., 1966; Wood et al., 1967, 1968). The compounds were obtained from Booregard Industries, Ltd., Sarpsborg, Norway. Gas-liquid chromatography (GLC) showed the following purities: ipsenol 95 %, ipsdienol 98 %, and cis-verbenol > 99 %. None of the compounds had any contamination of the other compounds. A small
BARK BEETLE CHEMOTAXIS
5
sample of the ipsenol was purified by GLC to > 9 9 % purity. This sample was tested in the chemotaxis assay and was found to have full activity. The Chemotaxis Assay. The design of the assay followed discussions in Fraenkel and Gunn (1961, pp. 271-284) and was intended to create a concentration gradient across an arena. The top and bottom of the arena were each a pane of double-strength glass, 40.6 cm square and seamed along the edges. The top had a 1-cm hole in the center, cut with a diamond-dust core drill (Equamat Distribution, Santa Clara, California). The wall of an arena was a ring of polyvinyl chloride plastic, 1 cm high, 40 cm OD, 39 cm ID, with silicon rubber gaskets permanently bonded to the edges of the wall. To assemble an arena, a wall was centered between an upper and lower pane of glass, and the edges were bound with heavy rubber bands. The hole in the upper pane was sealed with a stopper except when access to the interior was needed. A treatment was placed on a 22-mm-square cover slip. Once the solvent evaporated within a fume hood, the cover slip was slipped under the wall of an assembled arena, and placed in a standardized location just inside the wall. The spatial and temporal variation of the pheromone within the arena were estimated by taking samples of air directly from an arena with a gas-tight syringe and injecting them onto a gas-liquid chromatograph (GLC) fitted with flame ionization detectors. Peak areas were converted to quantity by comparing them to peaks produced by known amounts of linalool. The amount of compound remaining behind in the syringe after injection was not estimated. Therefore, only the amount recovered and not the actual concentration in the air is reported. Linalool, an isomer of ipsenol, was used in these studies to conserve pheromonal compounds. The molecular weight, structure, and adjusted boiling points of ipsenol, ipsdienol, and linalool are very similar (Table 1). In addition, linalool and ipsenol are indistinguishable on many GLC columns (Young et al., 1973). One milligram of linalool applied to an arena gave a concentration in the air similar to 1 mg of ipsdienol, ipsenol, or cis-verbenol. Accordingly, linalool appeared to be a reasonable substitute for the pheromonal compounds. The first experiment evaluated the behavior of the concentration gradient within the arena through time. One milligram of linalool was applied to the dispenser, and 1-ml samples of air were taken periodically (Figure 1) over the dispenser and at the center of the arena. A difference in concentration appeared almost immediately between the center and the dispenser. The concentration difference was nearly constant between 20 min and 95 min after charging the arena. Diffusion periods of 30 or 60 rain were thereafter used in all experiments, and all beetles in a single arena were run by 15 min after the diffusion period. The spatial variation within the arena was further characterized at the 60min diffusion time by taking 1-ml samples of air at four points along the line between the dispenser and the center of the arena (Figure 2), by fitting the
HO
HO
~
C ioHi60
CIoH,60
2-methyl-6-methylene2,7-octadien-4-ol~'
2-pinen-4-ol"
Ipsdienol
cis- V erbenol
H
/~L~I
I
Structure
C it)H170
Formula
2-methyl-6-methylene7-°cten-4-°l~'
Chemical name
Ipsenol
Compound
OH
II
152
152
153
MW
NA
202 a
190-193"
Boiling point
36
31
52
Source
4.6
3.6
6.2
Center
Taxis assay
7.8
9.5
4.3
Kinesis assay
Concentration in air 'j (ng/ml air)
TABLE 1. PHYSICAL PROPERTIES OF PHEROMONAL COMPOUNDS AND THEIR BEHAVIOR IN ASSAYS
o o
> z
> m
G%
3,7-dimethyl- 1,6octadien-3-ol"
C joH~sO
.L
[,
I[ 154
198-200 '~
bSilverstein et al. (1966). ' Moil et al. (1979). Corrected to standard temperature and pressure using corrections of Hass and Newton (1980). d Mori (1974), Corrected to standard temperature and pressure using corrections of Hass and Newton (1980). "CRC Handbook of Chemistry and Physics (1980).
"Dosage was 1 mg/arena. Response is mean of three arenas.
Linalool
leo. 46 5.8
4.7
rt t-t---t
Above Dispenser
1 0 0 .w
E lO
Q.
[ At c e n t e r ~0
>
0 0
6~ E 0.1
0
30
60
90
Min. after Application FIG. 1. The concentration of linalool in the air above the pheromone dispenser and at the center of the arena, near the release point of the beetles. One milligram was applied approx. 1 min before the first sample was taken at time 0. Distance between the center and the dispenser was approx. 17 cm. Error bars show standard deviations.
"~
50
E Q_ / C
30
=45.4e"0'17d
C= concentration
d) d=distanc¢from source
CD > 0 lO
6~
E
b
"s
~o
1"5
Distance from Source (cm.) FIG. 2. The concentration of linalool in the air at various distances between the dispenser and the center of the arena. One milligram was applied to the dispenser 60 min before taking the samples. Error bars show standard deviations.
BARK BEETLE CHEMOTAXIS
9
syringe with a long needle. The dispenser contained 1 mg of linaloot. All samples from a single arena were taken within a 5-min period. Five arenas were sampled. The theory of diffusion indicates that, if a material diffuses freely in two dimensions without any constraints, its concentration should fall off exponentially with the square of the distance from the source (Crank, 1956). In this experiment, the relationship was estimated as falling off exponentially with the distance, not its square. When both forms of relationships were fitted to the data using the nonlinear regression (NLIN) procedure available in the Statistical Analysis System (Goodnight and Sail, 1982), the relationship based on the distance produced the lower sums of squares. Observation and Recording Procedures. Preliminary experiments in the open laboratory indicated that the beetles were not orienting at random within untreated arenas (Akers, 1985). Accordingly, an observation chamber was developed that isolated the insects from these possibly confounding cues (Figure 3). The beetles oriented at random within the chamber when no odor cues were available. All experiments were performed using the chamber in the darkened laboratory. The movements of a beetle were recorded by marking its position by hand at l-sec intervals, with reference to an electronic metronome. The beetle's path was usually traced at the same time with the free hand. If an animal stopped walking, recording of its position ceased until it began moving again. However, the beetles are highly thigmotactic, and most did not stop moving until they contacted the wall of the arena or the pheromone source, at which time tracking ceased. Tracks were later entered into a computer via a digitizing table. The observation procedure was able to separate trails differing in average linear speeds by 0.02-0.04 cm/sec and in turning rates by about 2-5°/sec (Akers, 1985). Handling of Experimental Animals. Naturally infested ponderosa pine was collected as logging debris, mostly from the vicinity of Blodgett Research Forest near Georgetown, Eldorado County, California. Infested logs were held at 5°C until needed. At such time, the logs were moved to a rearing chamber in a greenhouse, where the emerging beetles were attracted into a refrigerator at 2-5 ° C (Browne, 1972). Beetles were stored for periods up to three weeks before use, with no signs of adverse effects (Borden, 1967). One day before they were to be used, the beetles were tested for their ability to perform in the assay. They were placed on a flat piece of glass and were accepted for assays if they had both their antennae and could walk more than 5-6 cm upon the glass. The sex of the selected beetles was then determined. If the beetles were kept uncrowded after emergence from the log, they did not damage one another, and a high proportion of the females were usable. Selected females were stored in individual glass tubes. They were removed from the cold 5-20 min before use. Recovery from the cold took a minute or
10
AKERS AND WOOD G3cn',.
k,s.s
15W Warm White Fluorescent Tube
White Plexiglas Diffusion Screen 13¢m.
,, Glass Arena Support
18
T
Black Curtains (cut away)
IOO
Lens (f=50 cm.)
ioo Surface
FIG. 3. The observation chamber used in all experiments to isolate the beetles from extraneous cues for orientation.
two at room temperature, which was 22 +_ 1 °C. They were kept cool until a few minutes before use, since their struggles within the storage tubes seemed to decrease their vigor after an hour or so. A beetle was brought out o f the cold directly onto the observation platform o f the observation chamber, where it could recover in the same light and temperature conditions under which it would be tested. A beetle was used only once. W h e n a beetle was to be released, the tape was removed from the storage tube and the beetle was slid gently but immediately into an L-shaped, glass release tube inserted through the hole in the arena. The outlet o f the tube was oriented at approx. 90 ° with respect to the source.
BARK BEETLE C H E M O T A X I S
11
Dosage Series. The experimental design was a dosage series of a 1 : 1 : 1 blend o f the three pheromonal compounds, as used in earlier behavioral tests (Byers and W o o d , 1981). Dosages differed by powers of 10, had a range of 10-4-1 mg o f each compound, and were dissolved in 0.1 ml o f pentane. The series was run with diffusion periods o f both 30 and 60 min. Solutions were stored at - 6 0 ° C or over Dry Ice until a few minutes before use. In order to minimize contamination between treatments, each treatment had its own arena. An arena was rinsed after each use in hot (approx. 70°C) water, since the pheromone compounds are moderately soluble in water. The water flowed at a rate that provided an exchange of water approximately every 4 min. The rinse lasted about an hour. The parts o f an arena were then aired for two to five days before the next use. A preliminary experiment indicated that the beetles did not exhibit any trail-following behavior or otherwise interact significantly with one another when more than one beetle was released within an arena (Akers, 1985). In this experiment, the relationships between the tracks o f two beetles that had run together within a single arena were compared to the relationships between the tracks of a pair o f beetles that had run in separate arenas. The average distance that the second beetle o f a pair maintained with respect to the trail o f the first beetle was measured, as were the walking and turning rates o f the second beetle. There were no significant differences in these measurements between the beetles that had the opportunity to follow a trail and those that did not. Observations made on different beetles moving within a single arena could therefore be considered as independent for statistical purposes, and running more than one beetle within a single arena greatly reduced the time required per replicate. Five beetles were usually run successively in each arena, with an interval o f approx. 2 - 3 min between runs. A randomized block design was used as a precaution, in case the supply of beetles was lost during an experiment, but the effects o f blocks were slight and are not reported. The experimenter did not know the identity of the treatments, either during the experiment or during the manipulation o f the data. Analysis of Orientation Data. The kinetic responses measured were orthokinesis, or linear rate o f motion, and klinokinesis, or rate of turning. The estimate of the rate o f motion was the mean distance between each point on a beetle's track. Two related measures were used to summarize the turning rate. Both depended on measuring the turn angle at each point on a track. The turn angle at a point was defined as the angle between the direction from the previous to the current point on the track and the direction from the current to the next point. Zero degrees was defined as straight ahead from the current point on the track. F o r the net turning rate, the " h a n d e d n e s s " o f a turn was taken into consideration, with left-hand turns defined as positive. In the gross turning rate, the absolute value o f each turn was used. The estimates of the rates were the mean turn angles over all the points on the track. A heading was also determined
12
AKERS AND WOOD
at each point on an insect's track by finding the angle between the direction towards the source and the direction from the present point to the next point on the track. Zero degrees was defined as straight towards the source. Both a mean net and a mean gross heading were defined. The statistical analyses available for circular variables are more complex and much more limited than those available for linear variables (Batschalet, 1965, 1981). However, the gross angles used here are defined on only half the circle and thus are actually angular distances. As such they may be treated as linear variables (Batschalet, 1981, p. 231). On the other hand, the net angles are defined on the full circle and thus are true circular variables. The mean angle and its angular (=standard) deviation may be estimated from the mean vector (Batschalet, 1981, Chap. 1). This provides an unbiased estimate of the mean, but a fairly biased estimate of the angular deviation (Batschalet, 1981, p. 46). However, once the mean net angle of a track is obtained, the major point of interest in comparing means of beetles and treatments is the magnitude of the mean, which is again an angular distance. The summaries made then were: walking rate and its variation within a track, net turning rate, gross turning rate and its variation within a track, mean net heading, and mean gross heading and its variation within a track.
RESULTS
A logistic regression showed that dosage had a significant effect on the number of beetles that reached the source (Figure 4) (30-min diffusion period: a = 1.42, B = 0.58; 60-min diffusion period: a = 2.22, B = 0.86. All parameters are significantly different from 0 at P < 0.001). Increasing dosage decreased the net and gross headings (Table 2, all beetles; Figures 5F, G; 6F, G), implying that beetles headed more directly towards sources of higher dosage. Increasing dosage also increased the gross turning rate (Figures 5C, 6C). It decreased the walking rate in the 60-rain diffusion period (Table 2: dosage, all beetles; Figure 6A), but the effect was not significant in the 30-min diffusion period (Table 2: dosage). There was no significant effect on the magnitude of the net turning rates. Since turning rate increased with dosage while walking rate either decreased or remained constant, turning radius therefore decreased with increasing dosage, meaning that the animals made tighter turns in the higher dosages. Such behavior was difficult to reconcile with the notion that the tracks of the beetles were more linear in the higher dosages. Further, a comparison of trails of beetles that did (Figure 7: AI, Bi) and did not (Figure 7: A2, B2) reach the source
BARK BEETLE CHEMOTAXIS
13
(24~'12
80
e) .......""
60 ct,_, (3) (:1.
/
40
24)
......~12s)
( 25) .'
/(o j~1241
20
/ ( 2 4 )J.[' (24),~ uJ *i'l z -4
I -3
m -2
| -1
| 0
Log mg. Each of Ip, ld, and cV FIG. 4. The effect of pheromone dosage on the proportion of beetles that reached the pheromone source. Solid line represents experiment with 60-min diffusion period and dotted line represents experiment with 30-rain diffusion period. Numbers in parentheses are sample sizes for respective treatments, cV = cis-verbenol, Id = ipsdienol, Ip = ipsenol, z --- 0 dosage level.
suggested that the behavior of the beetles differed fairly strongly between the two groups. Samples of trails of beetles that reached the source looked similar, even though the beetles were exposed to different dosages (Figure 7: A~, B 0. Therefore, the behavior of beetles that did and did not reach the source was compared. The mean gross headings were significantly different between the two groups (Table 2: rs vs. nrs; Figures 5G, 6G). Beetles that did not reach the source had mean gross headings that did not differ among dosages (Table 2: dosage, nrs; Figures 5G, 6G). Beetles that reached the source also had mean gross headings that did not differ among dosages (Table 2: dosage, rs; Figures 5G, 6G), even though these beetles might have been expected to approach higher dosages more directly. Similar results were apparent from other behavioral responses. Walking
14
AKERS AND WOOD
TABLE 2. ANALYSES OF VARIANCE FOR EFFECTS OF DOSAGE AND WHETHER BEETLES LOCATED THE SOURCE ON BEHAVIORAL RESPONSES
Behavioral response Walking rate
Comparison between Dosages, all beetles rs vs nrsI' Dosages, rs Dosages, nrs
Standard deviations of walking rates
Dosages, all beetles rs vs. nrs Dosages, rs Dosages. nrs
Gross turning rate
Dosages, all beetles rs vs. nrs Dosages, rs Dosages, nrs
Standard deviations of gross turning rates
Dosages, all beetles rs vs. nrs Dosages, rs Dosages, nrs
Magnitudes of net turning rates
Dosages, all beetles rs vs. nrs Dosages, rs Dosages, nrs
Diffusion period (min)
Number of beetles
Fob~
P value"
30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60
149 146 125 122 70 73 55 49 149 146 125 122 70 73 55 49 149 146 125 122 70 73 55 49 149 146 125 122 70 73 55 49 149 146 125 122 70 73 55 49
1.16 3.54 26.20 19.42 0.16 0.36 1.26 1.78 0.55 3.03 19.97 4.82 1.48 1.70 1.39 1.38 5.00 t 3.82 104.8 71.8 1.57 5.76 0.52 0.60 2.72 10.36 114.40 59.73 0.75 3.95 0.55 0.56 1.08 0.29 0.81 0.71 0.87 0.16 1.50 0.62
0.330 0.005
OLFACTORY ORIENTATION RESPONSES BY WALKING FEMALE Ips paraconfusus BARK BEETLES I. Chemotaxis Assay
R. PATRICK
AKERS l and DAVID
L. WOOD
Department of Entomological Sciences University of California Berkeley, California (Received May 12, 1987; accepted October 19, 1987) Abstract--Gas-liquid chromatography of the air within the arena developed for this assay showed that a concentration gradient was established within 12 min of applying the pheromone (ipsenol, ipsdienol, cis-verbenol), and that this gradient was nearly constant for 20-95 rain after application. The concentration fell rapidly and approximately exponentially between the source and the center of the arena. Turning rate and the number of beetles that reached the source increased, and heading with respect to the source decreased, in the presence of pheromone. Responses of beetles that did and did not reach the source were significantly different, but within each group there were no significant differences among dosages. Turning rate and heading varied little with distance from the source, while walking rate decreased as distance from the release point of the beetles increased. We hypothesize that dosage exerts its major effect on source location by altering the probability that a beetle will enter into orientation behavior and that beetles orienting to sources have similar behaviors even when orienting to a wide range of dosages. Key Words--lps paraconfi~sus, Coleoptera, Scolytidae, bark beetles, taxis, chemotaxis, orientation, olfaction, pheromones.
INTRODUCTION These experiments were undertaken while developing assays for a study of quality d i s c r i m i n a t i o n
among
the components
of a multicomponent
pheromone
Present address: Worcester Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, Massachusetts 01545.
0098-0331/89/0100-0003506.00/0 © 1989PlenumPublishingCorporation
4
AKERS AND WOOD
(Akers and Wood, 1989). An assay had to provide clear, differential responses to olfactory stimuli, which could serve as a behavioral basis for defining different odor qualities. The extent to which an assay met this requirement was tested by exposing beetles to a dosage series of a standard pheromone blend. Ips paraconfusus females readily found pheromone sources 16-18 cm away in still air, and we became curious as to the mechanisms involved. A complete knowledge of the behavioral mechanism was not necessary to use the assay as an indicator of olfactory significance, but the more complete the behavioral description, the better we can visualize possible neural mechanisms. This, in turn, should assist in interpreting neurophysiological recordings and in deriving models from them (Kein 1974ab, 1975). Several other studies of orientation in still air suggested that the paths of insects become more linear as they near a source of odor (Klingler, 1958; Bell and Tobin, 1981; Fraenkel and Gunn, 1961, Chap. 18). Since higher dosages should produce steeper concentration gradients (Crank, 1956), the inference might be drawn from these previous studies that as dosage increased all the insects in a population would shift their overall directional vectors (Bell and Tobin, 1982) towards the source and then approach it in a more linear or direct manner. The proportion of beetles that reached the source would increase as the intersections of their paths with the arena wall became more narrowly distributed around the source. Alternatively, the group that reaches the source might be comprised of beetles that enter into orientation behavior while the group that does not reach the source might be comprised of beetles that do not enter into orientation. Dosage would then change the proportion of orienting beetles, while the behavior of orienting beetles in different dosages might be similar. In our first analyses the beetles appeared to head more directly to the source as dosage increased, which seemed to support the first hypothesis. However, when the second hypothesis occurred to us, a more extensive analysis indicated that it was probably the more correct of the two. The initiation of orientation therefore appears to have a strong all-or-none component. A second paper (Akers, 1989) examines the mechanisms by which an orienting beetle may reach the source.
METHODS AND MATERIALS
Compounds. The attractant pheromone of L paraconfusus is a blend of ipsenol (Ip), ipsdienol (Id), and cis-verbenol (cV) (Silverstein et al., 1966; Wood et al., 1967, 1968). The compounds were obtained from Booregard Industries, Ltd., Sarpsborg, Norway. Gas-liquid chromatography (GLC) showed the following purities: ipsenol 95 %, ipsdienol 98 %, and cis-verbenol > 99 %. None of the compounds had any contamination of the other compounds. A small
BARK BEETLE CHEMOTAXIS
5
sample of the ipsenol was purified by GLC to > 9 9 % purity. This sample was tested in the chemotaxis assay and was found to have full activity. The Chemotaxis Assay. The design of the assay followed discussions in Fraenkel and Gunn (1961, pp. 271-284) and was intended to create a concentration gradient across an arena. The top and bottom of the arena were each a pane of double-strength glass, 40.6 cm square and seamed along the edges. The top had a 1-cm hole in the center, cut with a diamond-dust core drill (Equamat Distribution, Santa Clara, California). The wall of an arena was a ring of polyvinyl chloride plastic, 1 cm high, 40 cm OD, 39 cm ID, with silicon rubber gaskets permanently bonded to the edges of the wall. To assemble an arena, a wall was centered between an upper and lower pane of glass, and the edges were bound with heavy rubber bands. The hole in the upper pane was sealed with a stopper except when access to the interior was needed. A treatment was placed on a 22-mm-square cover slip. Once the solvent evaporated within a fume hood, the cover slip was slipped under the wall of an assembled arena, and placed in a standardized location just inside the wall. The spatial and temporal variation of the pheromone within the arena were estimated by taking samples of air directly from an arena with a gas-tight syringe and injecting them onto a gas-liquid chromatograph (GLC) fitted with flame ionization detectors. Peak areas were converted to quantity by comparing them to peaks produced by known amounts of linalool. The amount of compound remaining behind in the syringe after injection was not estimated. Therefore, only the amount recovered and not the actual concentration in the air is reported. Linalool, an isomer of ipsenol, was used in these studies to conserve pheromonal compounds. The molecular weight, structure, and adjusted boiling points of ipsenol, ipsdienol, and linalool are very similar (Table 1). In addition, linalool and ipsenol are indistinguishable on many GLC columns (Young et al., 1973). One milligram of linalool applied to an arena gave a concentration in the air similar to 1 mg of ipsdienol, ipsenol, or cis-verbenol. Accordingly, linalool appeared to be a reasonable substitute for the pheromonal compounds. The first experiment evaluated the behavior of the concentration gradient within the arena through time. One milligram of linalool was applied to the dispenser, and 1-ml samples of air were taken periodically (Figure 1) over the dispenser and at the center of the arena. A difference in concentration appeared almost immediately between the center and the dispenser. The concentration difference was nearly constant between 20 min and 95 min after charging the arena. Diffusion periods of 30 or 60 rain were thereafter used in all experiments, and all beetles in a single arena were run by 15 min after the diffusion period. The spatial variation within the arena was further characterized at the 60min diffusion time by taking 1-ml samples of air at four points along the line between the dispenser and the center of the arena (Figure 2), by fitting the
HO
HO
~
C ioHi60
CIoH,60
2-methyl-6-methylene2,7-octadien-4-ol~'
2-pinen-4-ol"
Ipsdienol
cis- V erbenol
H
/~L~I
I
Structure
C it)H170
Formula
2-methyl-6-methylene7-°cten-4-°l~'
Chemical name
Ipsenol
Compound
OH
II
152
152
153
MW
NA
202 a
190-193"
Boiling point
36
31
52
Source
4.6
3.6
6.2
Center
Taxis assay
7.8
9.5
4.3
Kinesis assay
Concentration in air 'j (ng/ml air)
TABLE 1. PHYSICAL PROPERTIES OF PHEROMONAL COMPOUNDS AND THEIR BEHAVIOR IN ASSAYS
o o
> z
> m
G%
3,7-dimethyl- 1,6octadien-3-ol"
C joH~sO
.L
[,
I[ 154
198-200 '~
bSilverstein et al. (1966). ' Moil et al. (1979). Corrected to standard temperature and pressure using corrections of Hass and Newton (1980). d Mori (1974), Corrected to standard temperature and pressure using corrections of Hass and Newton (1980). "CRC Handbook of Chemistry and Physics (1980).
"Dosage was 1 mg/arena. Response is mean of three arenas.
Linalool
leo. 46 5.8
4.7
rt t-t---t
Above Dispenser
1 0 0 .w
E lO
Q.
[ At c e n t e r ~0
>
0 0
6~ E 0.1
0
30
60
90
Min. after Application FIG. 1. The concentration of linalool in the air above the pheromone dispenser and at the center of the arena, near the release point of the beetles. One milligram was applied approx. 1 min before the first sample was taken at time 0. Distance between the center and the dispenser was approx. 17 cm. Error bars show standard deviations.
"~
50
E Q_ / C
30
=45.4e"0'17d
C= concentration
d) d=distanc¢from source
CD > 0 lO
6~
E
b
"s
~o
1"5
Distance from Source (cm.) FIG. 2. The concentration of linalool in the air at various distances between the dispenser and the center of the arena. One milligram was applied to the dispenser 60 min before taking the samples. Error bars show standard deviations.
BARK BEETLE CHEMOTAXIS
9
syringe with a long needle. The dispenser contained 1 mg of linaloot. All samples from a single arena were taken within a 5-min period. Five arenas were sampled. The theory of diffusion indicates that, if a material diffuses freely in two dimensions without any constraints, its concentration should fall off exponentially with the square of the distance from the source (Crank, 1956). In this experiment, the relationship was estimated as falling off exponentially with the distance, not its square. When both forms of relationships were fitted to the data using the nonlinear regression (NLIN) procedure available in the Statistical Analysis System (Goodnight and Sail, 1982), the relationship based on the distance produced the lower sums of squares. Observation and Recording Procedures. Preliminary experiments in the open laboratory indicated that the beetles were not orienting at random within untreated arenas (Akers, 1985). Accordingly, an observation chamber was developed that isolated the insects from these possibly confounding cues (Figure 3). The beetles oriented at random within the chamber when no odor cues were available. All experiments were performed using the chamber in the darkened laboratory. The movements of a beetle were recorded by marking its position by hand at l-sec intervals, with reference to an electronic metronome. The beetle's path was usually traced at the same time with the free hand. If an animal stopped walking, recording of its position ceased until it began moving again. However, the beetles are highly thigmotactic, and most did not stop moving until they contacted the wall of the arena or the pheromone source, at which time tracking ceased. Tracks were later entered into a computer via a digitizing table. The observation procedure was able to separate trails differing in average linear speeds by 0.02-0.04 cm/sec and in turning rates by about 2-5°/sec (Akers, 1985). Handling of Experimental Animals. Naturally infested ponderosa pine was collected as logging debris, mostly from the vicinity of Blodgett Research Forest near Georgetown, Eldorado County, California. Infested logs were held at 5°C until needed. At such time, the logs were moved to a rearing chamber in a greenhouse, where the emerging beetles were attracted into a refrigerator at 2-5 ° C (Browne, 1972). Beetles were stored for periods up to three weeks before use, with no signs of adverse effects (Borden, 1967). One day before they were to be used, the beetles were tested for their ability to perform in the assay. They were placed on a flat piece of glass and were accepted for assays if they had both their antennae and could walk more than 5-6 cm upon the glass. The sex of the selected beetles was then determined. If the beetles were kept uncrowded after emergence from the log, they did not damage one another, and a high proportion of the females were usable. Selected females were stored in individual glass tubes. They were removed from the cold 5-20 min before use. Recovery from the cold took a minute or
10
AKERS AND WOOD G3cn',.
k,s.s
15W Warm White Fluorescent Tube
White Plexiglas Diffusion Screen 13¢m.
,, Glass Arena Support
18
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Black Curtains (cut away)
IOO
Lens (f=50 cm.)
ioo Surface
FIG. 3. The observation chamber used in all experiments to isolate the beetles from extraneous cues for orientation.
two at room temperature, which was 22 +_ 1 °C. They were kept cool until a few minutes before use, since their struggles within the storage tubes seemed to decrease their vigor after an hour or so. A beetle was brought out o f the cold directly onto the observation platform o f the observation chamber, where it could recover in the same light and temperature conditions under which it would be tested. A beetle was used only once. W h e n a beetle was to be released, the tape was removed from the storage tube and the beetle was slid gently but immediately into an L-shaped, glass release tube inserted through the hole in the arena. The outlet o f the tube was oriented at approx. 90 ° with respect to the source.
BARK BEETLE C H E M O T A X I S
11
Dosage Series. The experimental design was a dosage series of a 1 : 1 : 1 blend o f the three pheromonal compounds, as used in earlier behavioral tests (Byers and W o o d , 1981). Dosages differed by powers of 10, had a range of 10-4-1 mg o f each compound, and were dissolved in 0.1 ml o f pentane. The series was run with diffusion periods o f both 30 and 60 min. Solutions were stored at - 6 0 ° C or over Dry Ice until a few minutes before use. In order to minimize contamination between treatments, each treatment had its own arena. An arena was rinsed after each use in hot (approx. 70°C) water, since the pheromone compounds are moderately soluble in water. The water flowed at a rate that provided an exchange of water approximately every 4 min. The rinse lasted about an hour. The parts o f an arena were then aired for two to five days before the next use. A preliminary experiment indicated that the beetles did not exhibit any trail-following behavior or otherwise interact significantly with one another when more than one beetle was released within an arena (Akers, 1985). In this experiment, the relationships between the tracks o f two beetles that had run together within a single arena were compared to the relationships between the tracks of a pair o f beetles that had run in separate arenas. The average distance that the second beetle o f a pair maintained with respect to the trail o f the first beetle was measured, as were the walking and turning rates o f the second beetle. There were no significant differences in these measurements between the beetles that had the opportunity to follow a trail and those that did not. Observations made on different beetles moving within a single arena could therefore be considered as independent for statistical purposes, and running more than one beetle within a single arena greatly reduced the time required per replicate. Five beetles were usually run successively in each arena, with an interval o f approx. 2 - 3 min between runs. A randomized block design was used as a precaution, in case the supply of beetles was lost during an experiment, but the effects o f blocks were slight and are not reported. The experimenter did not know the identity of the treatments, either during the experiment or during the manipulation o f the data. Analysis of Orientation Data. The kinetic responses measured were orthokinesis, or linear rate o f motion, and klinokinesis, or rate of turning. The estimate of the rate o f motion was the mean distance between each point on a beetle's track. Two related measures were used to summarize the turning rate. Both depended on measuring the turn angle at each point on a track. The turn angle at a point was defined as the angle between the direction from the previous to the current point on the track and the direction from the current to the next point. Zero degrees was defined as straight ahead from the current point on the track. F o r the net turning rate, the " h a n d e d n e s s " o f a turn was taken into consideration, with left-hand turns defined as positive. In the gross turning rate, the absolute value o f each turn was used. The estimates of the rates were the mean turn angles over all the points on the track. A heading was also determined
12
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at each point on an insect's track by finding the angle between the direction towards the source and the direction from the present point to the next point on the track. Zero degrees was defined as straight towards the source. Both a mean net and a mean gross heading were defined. The statistical analyses available for circular variables are more complex and much more limited than those available for linear variables (Batschalet, 1965, 1981). However, the gross angles used here are defined on only half the circle and thus are actually angular distances. As such they may be treated as linear variables (Batschalet, 1981, p. 231). On the other hand, the net angles are defined on the full circle and thus are true circular variables. The mean angle and its angular (=standard) deviation may be estimated from the mean vector (Batschalet, 1981, Chap. 1). This provides an unbiased estimate of the mean, but a fairly biased estimate of the angular deviation (Batschalet, 1981, p. 46). However, once the mean net angle of a track is obtained, the major point of interest in comparing means of beetles and treatments is the magnitude of the mean, which is again an angular distance. The summaries made then were: walking rate and its variation within a track, net turning rate, gross turning rate and its variation within a track, mean net heading, and mean gross heading and its variation within a track.
RESULTS
A logistic regression showed that dosage had a significant effect on the number of beetles that reached the source (Figure 4) (30-min diffusion period: a = 1.42, B = 0.58; 60-min diffusion period: a = 2.22, B = 0.86. All parameters are significantly different from 0 at P < 0.001). Increasing dosage decreased the net and gross headings (Table 2, all beetles; Figures 5F, G; 6F, G), implying that beetles headed more directly towards sources of higher dosage. Increasing dosage also increased the gross turning rate (Figures 5C, 6C). It decreased the walking rate in the 60-rain diffusion period (Table 2: dosage, all beetles; Figure 6A), but the effect was not significant in the 30-min diffusion period (Table 2: dosage). There was no significant effect on the magnitude of the net turning rates. Since turning rate increased with dosage while walking rate either decreased or remained constant, turning radius therefore decreased with increasing dosage, meaning that the animals made tighter turns in the higher dosages. Such behavior was difficult to reconcile with the notion that the tracks of the beetles were more linear in the higher dosages. Further, a comparison of trails of beetles that did (Figure 7: AI, Bi) and did not (Figure 7: A2, B2) reach the source
BARK BEETLE CHEMOTAXIS
13
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Log mg. Each of Ip, ld, and cV FIG. 4. The effect of pheromone dosage on the proportion of beetles that reached the pheromone source. Solid line represents experiment with 60-min diffusion period and dotted line represents experiment with 30-rain diffusion period. Numbers in parentheses are sample sizes for respective treatments, cV = cis-verbenol, Id = ipsdienol, Ip = ipsenol, z --- 0 dosage level.
suggested that the behavior of the beetles differed fairly strongly between the two groups. Samples of trails of beetles that reached the source looked similar, even though the beetles were exposed to different dosages (Figure 7: A~, B 0. Therefore, the behavior of beetles that did and did not reach the source was compared. The mean gross headings were significantly different between the two groups (Table 2: rs vs. nrs; Figures 5G, 6G). Beetles that did not reach the source had mean gross headings that did not differ among dosages (Table 2: dosage, nrs; Figures 5G, 6G). Beetles that reached the source also had mean gross headings that did not differ among dosages (Table 2: dosage, rs; Figures 5G, 6G), even though these beetles might have been expected to approach higher dosages more directly. Similar results were apparent from other behavioral responses. Walking
14
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TABLE 2. ANALYSES OF VARIANCE FOR EFFECTS OF DOSAGE AND WHETHER BEETLES LOCATED THE SOURCE ON BEHAVIORAL RESPONSES
Behavioral response Walking rate
Comparison between Dosages, all beetles rs vs nrsI' Dosages, rs Dosages, nrs
Standard deviations of walking rates
Dosages, all beetles rs vs. nrs Dosages, rs Dosages. nrs
Gross turning rate
Dosages, all beetles rs vs. nrs Dosages, rs Dosages, nrs
Standard deviations of gross turning rates
Dosages, all beetles rs vs. nrs Dosages, rs Dosages, nrs
Magnitudes of net turning rates
Dosages, all beetles rs vs. nrs Dosages, rs Dosages, nrs
Diffusion period (min)
Number of beetles
Fob~
P value"
30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60
149 146 125 122 70 73 55 49 149 146 125 122 70 73 55 49 149 146 125 122 70 73 55 49 149 146 125 122 70 73 55 49 149 146 125 122 70 73 55 49
1.16 3.54 26.20 19.42 0.16 0.36 1.26 1.78 0.55 3.03 19.97 4.82 1.48 1.70 1.39 1.38 5.00 t 3.82 104.8 71.8 1.57 5.76 0.52 0.60 2.72 10.36 114.40 59.73 0.75 3.95 0.55 0.56 1.08 0.29 0.81 0.71 0.87 0.16 1.50 0.62
0.330 0.005
