U.S. Department of Agriculture

Forest Service Research Note SE-302 February 198 1

BIOASSAY OF PINE BARK EXTRACTS AS BITING

STIMULANTS FOR THE SOUTHERN PINE BEETLE’

H. A. Thomas, J. A. Richmond, and E. L. Bradley’

ABSTRACT.-A bioassay was developed to compare pine outer-bark constituents extracted chemically from five species of southern pines. Extracts from inner and outer bark were also compared. Extracts

from the outer bark of shortleaf pine elicited the greatest number of biting responses which significantly

exceeded the controls. Beetles responded more often to extracts from the outer bark than to those from

the inner bark.

Keywords: Dcndrocronusfrontalis. Coleoptera: Scolytidae, solvent extractives, Pinus spp.

Chemical substances are known to influence

host finding, aggregating, and mating of southern

pine beetles (SPB), Dendroctonus frontalis

Zimmermann (Coster and Vite’ 1972; Renwick and

Vite’ 1968, 1969; Vite’ and Renwick 1971), and in

phytophagous insects, host substances are known to

influence host selection (Thorsteinson 1960). How

host substances influence host selection by SPB is

not fully understood.

Scolytids initiate attack by first biting into the

host or constructing galleries, releasing volatiles

which induce subsequent mass attack. Bark constituents may be the key to continued attack or

abandonment of a particular tree. Some host trees

in heavily infested areas may survive by overcoming beetles through resinosis. by chance escape,

or by their lack of chemical stimuli which induce

pioneer beetles to attack. Furthermore, Coster and

‘The work reported here was funded in whole or in part by a

program sponsored by the U.S. Department of Agriculture and

entitled “The Expanded Southern Pine Beetle Research and

Applications Program.” The findings, opinions, and recommendations are those of the authors and not necessarily those of

the U.S. Department of Agriculture.

bioassay used for Scolytus multistriatus (Marsham)

by Norris and Baker (1967) and for Hylobiuspales

(Herbst) by Thomas (1969). A bioassay unit consisted of the bottom half of a disposable plastic petri

dish, a moistened 9-cm-diameter disk of nonwoven

polyester fiber3 used as a floor, a 2-mm-thick elder

pith wafer treated with pine bark extract or solvent,

2Thomas is Principal Entomologist and Richmond is Ento- ‘We thank Dr. Zoltan Motd of the International Paper Co..

mologist, Forestry Sciences Laboratory, Research Triangle Corporate Research Development Division,Tuxedo Park. N.Y..

Park, N.C.; Bradley is Biologist, Forestry Sciences Laboratory, for supplying these disks, whose file is designated No. 022476-S

Athens, Ga. by that company.

Vite’ (1972) found that pheromone production by

the female SPB is related to feeding activity, and

Hughes (1973) has shown that behaviorally

important substances are produced by the beetles

from the terpene cy-pinene, which could be obtained during feeding.

The present study was undertaken to determine

(1) whether substances in pine bark induce biting

responses of the SPB, (2) whether these substances

occur in the outer or the inner bark or differ among

pine species, and (3) what general region in the bole

contains active substances.

METHODS AND MATERIALS

We modified the elder (Sambucus sp.) pith

Southccxtern Forest Experiment Staliorl- Asheuille, North Carolina
and a 3-mm-thick Plexiglas@‘disk cut to fit exactly

inside the petri dish (fig. l).4 With the Plexiglas

insert supported by the wafer, the beetles could walk

about in the dish but could not climb. This arrangement limited the beetles’ activity to the same plane

as the wafers and encouraged them to feed. Notches

were cut in the perimeter of the pith wafers to

simulate bark crevices. Test solutions were applied

to the wafers with a microsyringe at rates calculated to provide extracted substance in an amount

equal to that found in.the bark (dry-weight basis).

After treatment, wafers were air-dried for 14 to 16

hours.

C

Figure I.-Exploded view of bioassay unit: A. 3-cm-thick Plexiglas insert; B, elder pith wafer; C. ‘)-cm-diameter polyester

nonwoven fiber disk; D. bottom half of plastic petri dish.

To initiate a bioassay, the polyester disks and

treated elder pith wafers were put in place. beetles

were added at the outside edge of the petri dishes,

and the Plexiglas disks or regular covers were

added.

Petri dishes in a complete bioassay were

assigned to random positions on a table and received 1100 to 1400 lux from Daylight@fluorescent

lamps. The testing room was kept at 24°C (T2”)and

85 to 95 percent relative humidity and was without

windows.

Bark was collected at breast height from two to

five trees of five pine species: loblolly (Pinus rueda

L.), shortleaf (P. echinata Mill.), Virginia (P.

‘Mention of commercial products in this paper is solely to

identify materials used. and does not constitute endorsement h?

the US. IIepartmenr of Agriculture.

virginiano Mill.), slash (P. elk&i Engelm.), and

pitch (P. rigidu Mill.). Collections were made within

40 km of our North Carolina laboratory except for

the pitch pine, which was collected approximately

400 km west. Sample trees were 20.3 to 35.5 cm in

d.b.h. and 9.1 to 22.8 m in height. Loblolly, shortleaf, and Virginia pines are locally indigenous and

susceptible to successful attack and colonization by

the SPB. Slash and pitch pines are occasionally

attacked but grow chiefly outside the range of the

SPB. These latter two species were included to provide an indication of the sensitivity of the bioassay.

Bark samples were freeze-dried and ground to

60 mesh. Subsamples from each species were pooled

and Soxhlet extracted simultaneously for 24 hours

with six solvents: water, 95 percent ethanol, 70 percent ethanol, benzene, hexane, and diethyl ether.

These solvents were selected to provide a complete

range of polarity. The amount of material extracted from each source by each solvent was calculated.

Beetles were collected as they emerged in the

laboratory from naturally infested pine bark collected within 121 km of the laboratory. Most

infested bark came from shortleaf pine, but 10 to 20

percent came from loblolly pine. Beetles were collected within 6 hours after emergence and sexed by

the method of Osgood and Clark (1963). We

attempted to carry out each bioassay series with

beetles which emerged from bark collected in one

local area, usually from trees within a single stand.

The objective of the first series of bioassays was

to investigate the solvent/ species relationship; i.e.,

the question of which solvent extracts of the outer

bark of each pine species elicited biting activity. The

six solvents and the five species gave 30 treatments.

Since each treatment was paired with a control(solvent alone, no extract), there were 30 controls. The

extracts obtained with the six solvents from a single

pine species were bioassayed together three

times: once with male beetles, once with females,

and once with unsexed beetles. Within a single bioassay, each of the six treatments was replicated five

times; i.e., individual petri dish units containing six

beetles each were paired with five units with beetles

in which the pith wafers were treated with solvent

alone. A diagram of the typical treatment array is

given in figure 2.

The number of beetles at the wafer in each petri

dish was counted at 30-minute intervals for 3 hours.

The observations made at the end of the second

hour were selected for ANOVA, because they were

at the approximate midpoint of the test. Thirty
_Y,,,

SOLVENT (So,,, . 9 l 6)

0 0

0 0

l-l 0 0

0 0

-1

---.

‘-.

--A

-_

---,

4 5 6

-_

3

4

5

6

Figure 2.-Experimental design of biting bioassay for southern pine beetles.

individual tests of significance were performed to

compare the biting response for each treatment

combination (solvent containing extract) with its

own control (solvent only).

In the next series of bioassays, beetle biting

responses to methanolic extracts of phloem (inner

bark) were compared with responses to methanolic

extracts of outer bark (periderm) from shortleafand

loblolly pines. two preferred hosts of the SPB. Bark

was collected at breast height and at the base of the

live crown in both species. Five trees of each species

were sampled. Samples from the same species and

height were pooled, freeze-dried, ground, and extracted in a Soxhlet apparatus for 24 hours with

methanol. In preliminary studies, methanol had

been found to remove a wide range of substances

from bark. Comparisons of the methanol extracts

were replicated daily for 8 days in a balanced,

complete-block design with separate bioassays for

male, female, and unsexed beetles. Again, the

number of beetles at the wafer in each petri dish was

counted at 30-minute intervals for 3 hours. A mean

biting response was calculated by pooling the data

from similar replications, finding the mean and

standard deviation of the number of beetles at the

extract and control wafers at the end of the 3-hour

period, and finally obtaining the difference between the mean biting response to the various

extracts and corresponding controls (solvent only).

Note that this arrangement used fewer control units

than did the previously described series.

3
RESULTS AND DISCUSSION

The results of the bioassay series to compare the

responses with the species X solvent combinations

are shown in table 1.

There appeared to be no relationship between

biting activity and species of pine from which the

samples were obtained. Female biting activity on

extracts prepared with 95 and 70 percent ethanol

showed more significant ‘F’ values than on extracts

prepared with other solvents. When the solvent

means were subject to Duncan’s multiple range test,

however, no indication could be found of grouping

along any point in the range of polarity represented.

Again, when methanol extracts were prepared

from periderm and phloem of shortleaf and lob1011~ pine, there were considerable variations in

mean biting response among three bioassays (table

2). In this series of tests, only the shortleaf pine

periderm extract consistently elicited a response

greater than the control. This extract also caused an

equal or greater response than that of extracts from

shortleaf pine phloem. The biting response was

highly variable among the extracts from the other

sources.

Table 1 .-‘F’ values solvent X species

Solvent I

Loblolly Pitch Shortleaf Slash Virginia

Water

95% ethanol

70% ethanol

Benzene

Hexane

Diethyl ether

FEMALES ONLY

0.16 2.50 7.20* 0.29 0.0

2.02 .33 2.25 4.90* .08

7.72* 5.76* 3.20* .07 12.72*

.09 1 .oo 1.20 0 .80

I .91 .57 .29 I .52 .23

.93 2.67 2.67 3.20 0

Water

95% ethanol

70% ethanol

Benzene

Hexane

Diethyl ether

MALES ONLY

.03 I .38 .05 .55 0

4.92* .20 8.06* .64 .91

.02 .71 2.93* 3.66* I.1 I

1.67 10.29* 1 .oo .24 6.45*

1.02 .35 1.06 .60 .I3

1.66 6.40* .18 .57 .44

*Significantly different from control (P = 0.10)

Table 2.-Biting response of southern pine beetles to methanolic extractives of

loblolly and shortleaf pine phloem and periderm during three bioassays

Biting response per bioassay (x Ilr SD)”

Sources

1 1 1 111

Loblolly phloem 7.97 -i- 2.40 7.10 k 2.93 5.66 “- 2.66

Shortleaf phloem 6.12 2 3.39 5.06 ” 2.15 4.08 “- 2.25

Loblolly periderm 10.50 2 2.90 3.50 2 3.08 5.85 _; 2.88

Shortleaf periderm 9.54 -I- 5.57 5.04 -I- 3.21 7.75 -!- 3.31

Control (solvent only) 7.33 rir 3.26 4.37 -” 3.00 5.13 2 2.03

“Means were calculated from the number of beetles rrspondlng durung a j-hour period. HIO~~~S;I\S fortxch wurcc c<>n\~\icJ 011

beetles from the same population (area) and tested In groups 01 30 male nnd 30 t’emalr hertIe\
Our findings indicate that methanol extracts Unpublished research at this laboratory has shown

from the periderm of shortleaf pine contain sub- the importance of volatile substances in initiating

stances that induce more biting initiation by the feeding by the SPB. Those results show that virtualSPB than do those from the phloem of shortleaf, the ly all of the substances affecting this behavior are

periderm and phloem of loblolly, or the periderm of removed during ovendrying but not during freezeVirginia, pitch, and slash pines. There was no drying. Nevertheless, it is probable that some

significant difference in the biting response to important volatiles (or at least critical amounts of

samples collected at breast height or at the base of them) are lost even during freeze-drying. Other

the live crown. Although the biting response of male work on pine bark composition and its effect on

and female beetles to all sources was similar, tests initiation of beetle tunneling (White, this Laborawith unsorted beetles often produced relatively low tory, unpublished) suggests that compositionvaries

responses or none. Data from these tests were elim- significantly between trees. Because bark samples

inated because of the possibility of interaction be- from several trees were pooled in this study, this

tween the sexes. variation may have been obscured.

The results show that positive biting responses

can be obtained in the laboratory to substances

extracted from the bark of hosts of the SPB, but

these responses are relatively weak and variable.

Our work suggests that other factors are more

important than biting stimuli in governing host

selection by the SPB.

LITERATURE CITED

Coster. J. E.. and J. P. Vith

1972. Effects of feeding and mating on pheromone release in the southern pine beetle. Ann. Entomol.

Sot. Am. 65263-266.

Hughes. P. R.

1973. Effect of a-pinene on transvjerbenol synthesis in

Dendroctonus ponderosoe. Naturwissenschaften

60:261-262.

Norris. D. M.. and J. E. Baker

1967. Feeding responses of the beetle Sco!t,rus to chemical stimuli in the bark L;lmus. J. Insect Physiol.

13:955-962.

Osgood. E. A.. Jr.. and E. W. Clark

1963. Methods of sexing and sex ratios of the southern

pine beetle. Dendrocronus ,fronrah.s Zimm. Can.

Entomol. 95: 1106-l 109.

Renwick. J. A. A.. and J. P. Vith

1968. Isolation of the population aggregating pheromone

of the southern pine beetle. Contrib. Boyce 7 hompson Inst. 24:6568.

Renwick. J. A. A., and J. P. Vite’

1969. Bark beetle attractants: Mechanisms of colonization by Dendrocronus ,fronta/is. Nature (Land.)

22411222-1223.

Thomas. H. A.

1969. Quantitative bioassay used to study inhibitory

effects of two organotins in pales weevil feeding.

USDA For. Serv., Res. Note SE-I 18.3 p. Southeast.

For. Exp. Stn.. Asheville, N.C.

Thorsteinson. A. J.

1960. Host selection in phytophagous insects. Annu. Rev.

Entomol. 5: 193-218.

Vim’, J. P., and J. A. A. Renwick

197 I. Inhibition. of Dendroctonus ,/~ontalis response

to frontalin by isomers of brevicomin. Naturwissenschaften 58:418.

5


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