United States

Department of

Agriculture

Forest Service

Southern Forest

Experiment Station

l&search --- v-9w-w,- Note ----

SO-284

September, 1982

Reduced Brood Production of Southern Pine Beetles

by Diflubenzuron

J. W. Van Sambeek

SUMMARY

Treating the female southern pine beetle (Dendroctonus

frontalis Zimmerman) with the insect growth regulator diflubenzuron will decrease the hatch of eggs deposited in the

first 2 dm of egg gallery. Treatment of males had no effect.

Surface applications of 1 to 10 pg of diflubenzuron per

female beetle and exposure to artificial diets with 100 to

1000 pg/g of diflubenzuron inhibited initial egg hatch, resulting in a larvae-free segment of gallery. The duration of the

inhibiting effects depended on the level of initial exposure.

Neither fecundity nor the length of gallery excavated were

affected. Application of 0.3 percent diflubenzuron to host

logs did not appear to have any inhibiting effects on eggs or

emerging brood.

Additional keywords: DimilirP, egg hatch, ovicide, insect growth regulator, bark beetles.

INTRODUCTION

Synthetic insect growth regulators offer potential alternatives to non-specific pesticides in the control of bark beetles

without the hazards often associated with these pesticides.

Diflubenzuron’, a synthetic insect growth regulator, has the

potential for affecting adult beetle populations by interfering

with the hatch of eggs deposited by treated females (Grosscurt 1977, McGregor and Kramer 1976, McLaughlin 1977,

Moore and Taft 1975, Novak and Sehnall978). This effect

is usually short-lived after exposure is terminated (Grosscurt 1977, Novak and Sehnal1978, Schroeder et al. 1976).

‘Other names include DimiW, TH 6040, and 1-(4-chlorophenyl)-3-(2,6-difluorobenzyl)-urea. This paper reports the results of research only. Mention

of a pesticide, or use of a trade or proprietary name does not constitute a

recommendation for use by the US. Department of Agriculture nor does it

imply registration under FIFRA as amended.

The proposed method for application of diflubenzuron to

control bark beetles is to treat the bark surface of potential

host trees and, subsequently, .to reduce emerging adult

brood populations. Because only a small portion of the life

cycle of bark beetles is spent outside the inner bark microenvironment,. applications of diflubenzuron to bark surfaces must deliver an effective dose to the adult beetles as

they crawl over and penetrate the bark. Application of diflubenzuron to host logs before beetle attack. has been

shown to reduce the number of larvae produced per gallery

for the spruce beetle, Dendroctonus rufipennis Kirby

(Sahota and lbaraki 1980).

This report summarizes the effects of diflubenzuron

administered to adult southern pine beetles (Dendroctonus

frontalis Zimmerman) directly by surface application to the

beetles and indirectly by absorption from artificial diets or

host logs.

MATERIALS AND METHODS

All southern pine beetles used in the experiments were

reared from naturally infested bolts of loblolly pine (Pinus

taeda L.) which had been placed in 50 liter rearing cans in

the laboratory at 21” to 24°C. To minimize uncontrolled

matings, adult beetles were collected at 2 to 3 hour intervals

as recommended by Covington (1970). Beetles were immediately segregated by sex and stored for up to 3 days at

4°C to accumulate an adequate supply of virgin females.

Uninfested pine bolts used for surface treatment and for

inoculating with treated beetles were cut from loblolly pines

growing near the edge of abandoned beetle infestations on

the Kisatchie National Forest in Central Louisiana. After

treatment, all bolts were placed in screened cages in the

laboratory to prevent attacks by stray beetles.

Southern Forest Experiment Statlonm-102lO U.S. Postal Services Bldg., 701 Loyola Avenue, New Orleans, La. 70113

Forest Service, U.S. Department of Agriculture.

Serving Alabama, Arkansas, Louisiana, Mississippi, E. Oklahoma, Tennessee, E. Texas, Puerto Rico, U.S. Virgin islands

1
Surface Treatment of Adult Beetles

Technical grade diflubenzuron (99 percent active ingredient) was dissolved -in AR grade acetone to prepare test

solutions containing 0.0, 0.01, 0.1, 1.0, and 10 kg diflubenzuron/pI. Two doses of 0.5 t.~l of test solution or

acetone only were applied 1 minute apart to each beetle’s

ventral abdomen using a 25 ~1 Hamilton syringe mounted in

a Hamilton 50-stop repeating dispenser. For each test solution the following three treatment combinations were used:

a) both sexes treated; b) females treated, males untreated;

and c) males treated, females untreated. An additional

check with both males and females untreated was also

included, resulting in a total of 16 treatment combinations.

centrations of 0.4, 1.5, and 3.0 grams of active ingredient

per liter and sprayed on uninfested pine bolts to the drip

point (approximately 5 ml/dm*). Unsprayed bolts and bolts

sprayed only with water served as check treatments. The

treated and check bolts were placed inside screened cages

and newly emerged beetles of both sexes were introduced

daily over a two week period. Approximately 5 beetles per

dm* of bark surface were placed in each cage. Thirty-five

days after initial treatment, half of the bolts were placed in 50

liter rearing cans to collect emerging brood and the other

half was examined for attacks, gallery construction, and egg

hatch.

Sixteen beetle pairs for each treatment combination were

inoculated into holes drilled into uninfested bolts cut from a

single loblolly pine. Holes (2 to 3 mm diameter, 1 dm apart)

were drilled diagonally through the outer and inner bark.

The female was inserted first and was immediately followed

by the male. The entrance hole was covered with a piece of

outer bark and taped.

Treatment With Artificial Diets

To determine the effects of diflubenzuron on gallery construction and egg hatch, bolts were shaved to expose the

individual galleries. Each gallery was measured to determine overall gallery length, the distance to the first larval

mine, and the distance to the last recently hatched larvae.

For each 5 cm segment of gallery beginning from the entrance hole, the number of egg niches, larval mines, and

eggs were counted. Percentage of egg hatch was determined for the 1st 2nd, and 3rd dm of gallery up to the

portion of gallery containing only recently hatched larvae or

freshly laid eggs.

In a preliminary experiment, the addition of the dye Calco

Oil Red N-1700 to the diet resulted in red eggs being laid by

treated females, showing that ingested fat soluble compounds like diflubenzuron can be deposited in the eggs of

treated females. This experiment was done to check if

adults could absorb diflubenzuron as they crawled over or

ingested treated materials.

Brood adults emerging from surface-treated bolts were

collected every other day until emergence was complete.

Brood emergence per dm of bark surface, time of peak

brood emergence, and brood longevity were determined for

each bolt. Beetle longevity was the mean number of days

until 50 percent of the starved brood kept in petri dishes at

23°C had died. /

For all treatments, data was analyzed by one- or two-way

An artificial diet (modified from Bridges 1979) was prepared to provide 0, 10, 100, and 1000 kg diflubenzuron per

gram of a.moist diet. Twenty newly emerged beetles of the

same sex were kept in petri dishes containing 6 grams of the

moist diet for 7 days. Beetles were carefully removed from

the media and only those that could walk were used. Dietfed females were paired with untreated males and diet-fed

males were paired with untreated virgin females. Untreated,

newly emerged males and females served as an additional

check treatment. Beetle pairs (12 to 24 pairs per treatment

combination) were inoculated into uninfested bolts as in the

previous experiment. The experiment was replicated 4

times using bolts from 12 loblolly pines.

analysis of variance and tested for significant differences (a

= 0.05 and 0.01) among treatment means and Duncan’s

New Multiple Range Test and used to separate significantly

different treatment means (CX = 0.05). The arcsin transformation was applied to all percentages before analysis.

RESULTS

Surface Treatment of Adult Beetleg

Surface Treatment of Bolts

This experiment was designed to show if application of

diflubenzuron to bark surfaces before beetle attack could

deliver to adults a dosage that would be sufficient to inhibit

egg hatch or affect subsequent brood emergence and

longevity.

Surface treatment of adult beetles with a known amount

of diflubenzuron demonstrated the sensitivity of the beetle

to the test compound and formed the basis for evaluating

the effectiveness of the more indirect methods for absorbing

the compound (table 1). Diflubenzuron was not acutely toxic

at rates up to 10 Fg per beetle as judged from the number of

pairs excavating egg galleries. Neither diflubenzuron nor

acetone alone had a significant effect on the number of egg

niches per dm of gallery nor the length of gallery excavated

during a 24-day period when compared to newly emerged

untreated pairs.

Diflubenzuron (WP-25) was suspended in water at conDiflubenzuron at dosages of 1 or 10 pg per beetle caused

a significant inhibition in egg hatch in the 1 st and 2nd dm of

Van Sambeek is a research plant physiologist, formerly with the Southern Forest Experiment Station, Pineville, Louisiana 71360. Currently with the North

Central Forest Experiment Station, Carbondale. Illinois 62901. Requests for information should be sent to Pineville, Louisiana. The author wishes to thank B.C.

Weber and D.J. Polak for their suggestions and assistance in preparation of this manuscript and Thompson-Hayward Co. for supplying the diflubenzuron.

2
Table 1 .-Southern pine beetle gallery excavation and brood production 24 days after surface treatment of adults with diflubenzuron in acetones

Sex of Diflubenzuron Average Egg niches Egg hatch per dm gallery Initial

treated concentration Galleries gallery per dm of larvae-free

beetles per beetle produced length gallery+ 1st dm 2nd dm 3rd dm gallery segment

w number dm number . . . . . percent . . . . . . . . . . cm

Both 10.0 7 3.06 30.4 1.8 8.3 37.9 21.1

1.0 6 2.40 24.2 0.0 5.2 31.3 18.1

0.1 4 2.80 30.5 36.3 61.0 54.7 6.4

0.01 6 3.22 26.7 57.7 61.5 54.5 5.3

0.0 5 2.58 27.5 45.8 55.7 49.3 6.1

Females 10.0 7 2.80 26.9 11.5 36.4 41 .o 15.8

1.0 4 3.75 29.0 6.5 30.5 50.8 15.4

0.1 7 3.27 31.4 42.7 51.9 47.5 7.2

0.01 5 2.80 24.5 48.4 70.6 58.1 5.1

0.0 9 2.99 31 .o 57.3 63.7 57.7 5.1

Males 10.0 8 2.91 26.1 55.0 73.1 70.2 4.9

1.0 1 3 3.60 28.0 54.3 63.9 57.7 5.1

0.1 9 2.83 26.1 53.5 54.0 55.9 4.0

0.01 5 3.80 27.5 42.9 72.9 62.0 6.0

0.0 5 3.00 28.4 53.4 68.9 73.5 6.2

Neither

. . . . . . . 9 3.36 24.1 46.2 70.4 68.9 4.7

Source df: . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . ANOVA Fvalues..................................

Sex (S) 2 (*) 0.52 0.27 6.52** 10.82** 2.42 11.59**

Dosage (D) 4 0.25 0.55 5.69** 7.11** 0.49 12.27**

SxD 8 0.39 0.74 2.42 2.91*= 0.31 4.05**

“Treatment values are means from all galleries out of 16 inoculations for each treatment combination. Two way analysis of variance was performed and tested

for significance at a = 0.05 (*) and C Y = 0.01 (**).

+The first decimeter of gallery had approximately 25 percent fewer egg niches than the average for the entire gallery.

*A chi-square test showed no significant differences between treatments in the number of pairs excavating galleries (X’ = 7.095, 8 df).

gallery for treated females mated with either treated or

untreated males. Although few eggs hatched in the 1 st dm

of gallery at these dosages, egg hatch was normal by the

3rd or 4th dm of gallery. Crosses between untreated

females and treated males did not result in a reduction in

egg hatch, indicating the effect is not mediated through the

males.

Increasing the diflubenzuron dosage to females significantly increased the initial larvae-free gallery segment, but

treatment of males had no effect. Egg niches normally begin

within 0.2 to 0.3 dm of the entrance hole (Van Sambeek and

Kile 1981) thus it appears that diflubenzuron is quickly

absorbed by the female and is deposited in the first few

eggs that are laid, causing them not to hatch. Length of the

initial larvae-free gallery segment would appear to be an

excellent parameter for evaluating the effect of diflubenzuron on southern pine beetle reproduction.

Treatment With Artificial Diets

Female beetles tunnelled extensively in the artificial diet

with or without diflubenzuron. Male beetles, however, tunnelled very little during the 7 days on the diet, but many were

still active and were capable of mating with virgin females

when inoculated into bolts. Exposure of either sex to diets

with or without diflubenzuron had no significant effect on the

percent of beetles excavating galleries (i.e. attacks), the

average gallery length after 24 days, or the number of egg

niches per dm of gallery (table 2).

The highest amount of diflubenzuron in the diet-fed

females reduced the ability of eggs to hatch (table 2).

Female beetles placed on diets containing 1000 pg diflubenzuron per gram of moist diet deposited significantly

fewer viable eggs in the 1 st and 2nd dm of egg gallery. The

length of the initial larvae-free gallery segment was significantly increased by females which fed on diets containing

either 100 or 1000 pg diflubenzuron per gram.

Surface Treatment of Bolts

Results from this experiment showed no significant differences due to diflubenzuron on length of gallery excavated,

egg niches per dm of gallery, or egg hatch in any of the first 3

dm (table 3). Even the best parameter for assessing diflubenzuron effects, i.e. length of initial larvae-free gallery,

was essentially equal to that observed in other experiments

(Van Sambeek and Kile 1981). Similarly, diflubenzuron had

no effect on the number of days to peak brood emergence

or mean longevity of starved emerging brood (table 4).

Bolts sprayed with 3 grams per liter of diflubenzuron had

the highest brood production per dm* of bark surface and

were apparently more heavily attacked initially than bolts in

the other treatments. Brood production near the ends of the

galleries and from late attacks was greatly reduced due to

heavy fungal contamination originating at the ends of the

bolts. Bolts with the earliest peak brood emergence tended

to produce the most brood beetles.

3
DISCUSSION

Diflubenzuron had no direct effect on adult southern pine

beetles, whether topically applied or ingested. However,

Richmond et al. (1978) reported that topical application of 5

pg diflubenzuron per female reduced both the number of

galleries and the number of eggs laid during the first 5 days

following treatment. Application of 10 kg per female showed

no effect in this study on either the number of galleries or

egg-niche excavation during a 24-day period. The extended

length of the study period could account for the discrepancy

in the egg-niche response,, but diflubenzuron generally

does not affect the production of eggs (Grosscurt 1977).

In these experiments, as in experiments with other coleopteran beetles, the primary effect of diflubenzuron was

egg hatch inhibition. Brood production inhibition has also

been recently demonstrated for the spruce bark beetle

(Dendroctonus rufipennis) following ingestion of diflubenzuron by adults from both artificial diets and surfacetreated bolts (Sahota and lbaraki 1980). The spruce beetle

is apparently more sensitive than the southern pine beetle

to diflubenzuron because feeding on diets containing 200

kg per gram of diet for 2 days completely inhibited egg

hatch along the entire gallery. In most Coleoptera, the effect

of an initial application of diflubenzuron is short lived, and

continued exposure is necessary for control (Grosscurt

1977, Novak and Sehnal 1978). In this study, the first eggs

laid by diflubenzuron-treated females did not hatch,

although the last eggs laid hatched normally. For the southern pine beetle, diflubenzuron can significantly reduce egg

hatch in the first and second dm of gallery, but may not for

the entire gallery. Galleries produced under laboratory conditions were usually 3 dm or more long after 24 days, but

galleries produced under field conditions average only 1 to

1.5 dm in length (Thatcher and Pickard 1964). Diflubenzuron has the potential to reduce brood production of the

southern pine beetle in the field.

Table 2.-Southern pine beetle gallety excavation and brood production following exposure to diets containing diflubenzuron’

Sex of Diflubenzuron Average Egg niches Egg hatch per dm gallery initial

treated concentration Pairs gallery per dm of larvae-free

beetles in moist diet inoculated Attacks length gallery* 1st dm 2nd dm 3rd dm gallery segment

Ids number percent d m number . . . . . . . . . . . percent . . . . . . . . .

Females 1000 7 0 48.2 3.04 18.5 20.9 32.8 39.9 Ei!:

100 7 1 51.1 3.03 20.2 33.5 54.3 58.2 6.6

1 0 7 7 50.0 2.87 23.5 40.5 54.0 66.0 4.7

0 7 0 40.9 3.19 22.5 46.6 53.0 57.3 2.6

Males 1000 6 3 61 .O 2.96 22.2 46.7 55.8 57.8 3.5

100 5 9 55.8 3.05 21.8 47.7 61.1 63.8 3.2

1 0 6 1 51.0 3.02 20.5 49.4 58.9 61.0 2.6

0 6 3 55.0 3.32 24.2 54.9 63.8 59.3 1.8

Neither . . . . . . . . . . 7 4 51.1 3.41 23.2 51.5 59.5 59.7 2.8

Source df: .,.,..........,.................... ANOVA Fvalues..................................

Sex (S) 1 2.79 0.04 0.37 15/W* 9.65** 1.79 15.19**

Dosage (D) 3 0.32 0.25 0.64 3.86 3.31* 2.78 5.35**

SxD 3 0.43 0.04 0.76 1.26 1.23 1.55 1.70

+Treatment values are means of four replications of entire experiment with 12 to 24 beetle pairs inoculated/treatment combination/replicate. Two way analysis

of variance was performed and tested for significance at C Y = 0.05 (*) and 01 = 0.01 (**).

*The first decimeter of gallery had approximately 25 percent fewer egg niches than the average for the entire gallery.

Table 3.-Southern pine beetle galley excavation and brood production 35 days after surface-treatment of bark with a

diflubenzuron solution to the drip point§

Diflubenzuron Attacks Average Egg niches

concentration Galleries per dm2 gallery per dm of

in water examined + bark length gallery*

g/liter number numberldm’ d m number

3.0 4 5 1.06 3.67 30.7

1.5 4 3 0.76 4.03 29.3

0.4 4 2 0.61 4.23 31.3

0.0 4 5 0.77 4.27 30.0

Egg hatch per dm gallery Initial

larva-free

1st dm 2nd dm 3rd dm gallery segment

. . . . . . . . . . percent cm

76.3 87.6 86.7 1.9

66.0 77.0 71 .o 3.2

74.7 73.3 75.3 1.9

71 .o 82.3 32.3 2.4

. . . . . . . . . . . . . . . . . . . . . . ANOVA F values . . . . . . . . . . . . . . . .

1.68 0.79 0.52 0.29 2.21 1.98 0.59

“Treatment values are means of three replications of entire experiment. One way analysis of variance was performed and tested

for significance at ( Y = 0105.

+The first decimeter of gallery had approximately 25 percent fewer egg niches than the average for the gallery.

*Only galleries without heavy fungal contamination were examined to measure gallery parameters.
Table 4.-Southern pine beetle brood emergence and longevity from

diflubenzuron surface treated bolt.@

Diflubenzuron

concentration

in water

Emerging

brood

Days to peak

emergence

Brood

longevity

g/liter numberldm2 days days

3.0 7.96 a 47.7 3.17

1.5 2.42 b 55.5 2.73

0.4 3.75 b 50.4 3.30

0.0 4.27 b 52.9 3.52

Untreated 5.32 b 48.9 2.62

. . . . . . . . . . . ANOVA F values . .

5.504* 2.277 0.667

§Treatment means values in the same column not sharing the same letter

are significantly different at a = 0.05 (‘) according to Duncan’s New

Multiple Range Test. ANOVA was done on treatment means for three

replications of entire experiment.

Exposure of males to diflubenzuron produced no significant changes in the percentage of eggs hatching or the

initial larvae-free gallery segment. Sahota and lbaraki

(1980) also found no significant changes in brood production following treatment of male spruce bark beetle males.

McLaughlin (1978) and Moore et al. (1978) found that

changes observed following treatment of males resulted

from a direct uptake by the female during mating or contact

within the microenvironment.

Unfortunately, the spraying of bolts with diflubenzuron

provided no evidence for an increase in length of the initial

larvae-free gallery or a reduction in the percentage of eggs

hatching. Sahota and lbaraki (1980) reported that using a 1

percent diflubenzuron spray containing the adjuvant,

Savol@, reduced the number of spruce bark beetle larvae

produced per gallery by 55 percent and that in 42 percent of

the galleries none of the eggs hatched. Grosscurt (1977)

and McLaughlin (1977) found that the diflubenzuron formulation, the type of carrier, and the amount of exposure to

the carrier-diflubenzuron solution can markedly change the

rate of absorption or effective concentration.

This study demonstrated that brood production for the

southern pine beetle can be reduced in the laboratory by

diflubenzuron. Before field experimentation is attempted,

however, the use of better carriers or adiuvants should be

explored to improve absorption of the compound by the

adult beetles as they move around in the bark microenvironment.

LITERATURE CITED

Bridges, J. Ft.

1979. An artificial diet for rearing the southern pine beetle,

Dendroctonus front&s Zimm. (Coleoptera:Scolytidae).

J. Ga. Entomol. Sot. 14:278-279.

Covington, C. C.

1970. Laboratory rearing of the southern pine beetle,

Dendroctonus frontalis Zimm., with emphasis on the

production of virgin females. M.F. Thesis. Stephen F.

Austin State University. 36 p.

Grosscurt, A. C.

1977. Mode of action of diflubenzuron as an ovicide and

some factors influencing its potency. In: Vol. 1, Proc.

1977 British Crop Protection Conference - Pests and

Diseases, p. 141-147.

McGregor, H. E. and K. J. Kramer.

1976. Activity of Dimilin@ (TH 6040) against coleoptera in

stored wheat and corn. J. Econ. Entomol. 69:479-480.

McLaughlin, R. E.

1977. Dose-responses of the boll weevil to topical formulations of TH-6040. J. Ga. Entomol. Sot. 12:369-

373.

McLaughlin, R. E.

1978. Contact transfer of diflubenzuron (Dimilina) by boll

weevils and the relation on site of application and effect

on egg hatch. Entomol. Exp. and Appl. 23:171-176.

Moore, R. F., R. A. Leopold, and H. M. Taft.

1978. Boll weevils: mechanism of transfer of diflubenzuron from male to female. J. Econ. Entomol. 71:587-

590.

Moore, R. F. and H. M. Taft.

1975. Boll weevils: chemosterilization of both sexes with

busulfan plus Thompson-Hayward TH-6040. J. Econ.

Entomol. 68:96-98.

Novak, V. and F. Sehnal.

1978. Sterilization of the pine weevil, Hylobius abietis,

with diflubenzuron. Acta entomologica bohemoslovaca

75:349-351.

Richmond, J. A., A. B. DeMilo, H. A. Thomas, and A. B.

Borkovec.

1978. Mortality and sterility of southern pine beetles

treated with chemosterilants and growth regulators. J.

Ga. Entomol. Sot. 13: 237-240.

Sahota, T. S. and A. Ibaraki.

1980. Prolonged inhibition of brood production in Dendroctonus rufipennis (Coleoptera:Scolytidae) by Dimilin. Can. Entomol. 112:85-88.

Schroeder, W. J., J. B. Beavers, R. A. Sutton, and A. G.

Selhime.

1976. Ovicidal effect of Thompson-Hayward TH 6040 in

Diaprepres abbreviatus on citrus in Fiorida. J. Econ.

Entomol. 69:780-782.

Thatcher, R. C. and L. S. Pickard.

1964. Seasonal variations in activity of the southern pine

beetle in East Texas. J. Econ. Entomol. 57:840-842.

Van Sambeek, J. W. and B. W. Kile.

1981. Egg gallery excavation and brood production by

reemerged and newly emerged females of Dendroctonus frontalis Zimm. J. Ga. Entomol. Sot. 16345-352.

*U.S. GOVERNMENT PRINTING OFFICE: 1982-569-082/39