b?$!i!F$ ResearchNo&

1978

/iivtimm Praparation For lmunodiffusion

In Southern Pine B88Ue Predation Studies

M. C. MILLER, W. ADRIAN CHAPPELL

WILLIAM C. GAMBLE, AND J. ROBERT BRIDGES

SUMMARY

An anti-adult southern pine beetle serum was

produced by subcutaneous injection of rabbits

with southern pine beetle (SPB) adult antigen.

Initial tests demonstrated the ability of the

anti-adult SPB serum to detect adult SPB antigen in the body of the adult predator,

Thanasimus dubius (F.). Cross reactivity was

found between the anti-adult serum and extracts

of immature stages of Dendroctonus frontalis

Zimmerman, adult 0. terebrans (Olivier), lps

grandicollis (Eichhoff), and I. calligraphus (Germar).

Adult southern pine beetle protein can be

detected in a whole predator extract at least 28

hours after feeding.

Additional keywords: Dendroctonus frontalis,

Thanasimus dubius, Agar gel double diffusion,

precipitin test, predation.

STUDY OF PREDATORS

Through most of its life cycle the southern

pine beetle (SPB), Dendroctonus frontalis Zimmermann, is a cryptic insect. Except for the

adult stage, SPB life cycle stages are hidden

beneath the bark of the host tree. A sizeable

SPB insect-associate complex exists (Moser and

others 1971), but direct measurements of successful attack by SPB natural enemies have not

been possible. Accepted methods of estimating

insect predation are indirect (Berryman 1967).

Identification of biological roles of insect associates of the SPB and related bark beetles has been

limited to small-scale, time-consuming rearings

and to laboratory or field observations. Work

has concentrated mainly on adult stages of those

predators easy to obtain, rear, or maintain in the

laboratory (Thatcher and Pickard 1966, Berryman 1967, Amman 1970, 1972, Moore 1972,

Nagal and Fitzgerald 1975). Few adult insect

associates have been studied in a way that

provides information regarding their direct impact on the SPB population (Thatcher and

Pickard 1966).

Precipitin reactions provide a means for determining the biological roles of predatory insects

and mites by indicating their food preferences

(West 1950, Dempster 1960, Dasgupta and

Cunliffe 1970). These reactions also provide a

means for measuring the minimum number of

prey consumed (Dempster 1960, Kiritani and

Dempster 1973).

This study concerns preparation of an antiadult southern pine beetle serum, determination

of sensitivity and selectivity of the antiserum,

and development of a laboratory feeding test for

SP B predators.

MATERIALS AND METHODS

Antigen preparation

Newly emerged SPB adults were collected in

an emergence box and refrigerated collector

similar to that of Browne (1972). Beetles were

starved for 24 h, screened, and hand sorted to

remove debris. They were washed with distilled

water, dried on paper towelling, and crushed in

equal volumes of 0.85 percent NaCI in a mortar

with a pestle. To inhibit melanization, a few drops

of 0.001 M KCN were added to the resulting

mixture (Dempster 19601, which was then

Southern Forest Experiment Station/T-l0210 U.S. Postal Services Bldg., 701 Loyola Avenue, New Orleans, La. 70113

Forest Service, U.S. Department of Agriculture.

Serving Alabama, Arkansas, Louisiana, Mississippi, Eastern Oklahoma, Tennessee, Eastern Texas.
stored at 4°C in 30-ml portions for 24 h. Gross

beetle fragments were removed by filtration

through glass wool and cheesecloth. When

filtering was completed, the glass wool was

compressed to extract the liquid, which was

then centrifuged at 23,500 g for 30 min. at 4°C

and filtered sequentially through Gelman

MetricaTM” 5.0,1.2,0.8,0.45, and 0.20 urn pore

filters. Before the liquid was sterilized with the

0.20 urn filter, a sample of supernatant was

taken for protein determination (Lowry and

others 1951). The antigen contained 2.8 mg/ml

of protein. Seventy mg of protein antigen was

placed in each presterilized bottle and lyophilized before shipment to the Center for Disease

Control (CDC) for immunization of rabbits.

Immunization of Rabbits

At the CDC the adult SPB antigen in each

bottle was reconstituted with 5 ml of sterile

distilled water to produce a stock antigen, which

was further diluted with water to prepare an

antigen containing 1 mg of SPB protein per kg

of animal weight. Freund’s incomplete adjuvant

was added in equal volumes to the aqueous SPB

antigen and emulsified as described by White

and others, (1975). Three New Zealand white

rabbits, ranging in weight from 3.8 to 5.0 kg,

were immunized by footpad-subcutaneous injection (Chappell and others 1976) with enough

adult SPB antigen to constitute 1 mg/kg of gross

body weight (GBW). The dose of adjuvantantigen was divided between the 2 front footpads. Ten ml of preimmunization blood was

collected from the central artery of an ear of

each rabbit. Dn the 32nd day after the initial

injection, all rabbits were given booster footpad

injections of 1 mg/kg GBW SPB antigen without

adjuvant.

On days 11, 18, 25, 32, 39 and 46 after the

initial injection of antigen, samples of blood

were collected for antisera titer determinations.

Sera were stored at -2o’C.

We determined antiserum titers by reacting

twofold dilutions (l/10, l/20, 1/40,...1/5120)

of SPB antigen stock with undiluted anti-adult

SPB serum by agar gel double diffusion, using

the glass slide method described by Chamberlain

and Sudia (1967). The adult SPB antigen was

diluted with 0.85 percent NaCI. The greatest

dilution of adult SPB antigen producing adistinct

precipitin line with undiluted anti-adult SPB

serum was considered the titer of the antiserum.

Laboratory Testing

Individual adult lps grandicollis (Eichhoff,

adult 1. avulsus (Eichhoff), adult platypodid, and

SPB larva, pupa, teneral adult /. calligraphus,

(Germar), Dendroctonus terebrans (Olivier),

adult, and a day-old adult were crushed in 0.25 ml

of 0.85 percent NaCI. The resulting suspensions

were tested by agar gel double diffusion with

undiluted anti-adult SPB serum.

Thanasimus dubius IF.) adults were collected

in a bucket trap (Moser and Browne in press)

baited with Frontalurem and hung in an active

SPB infestation. T. dubius were stored separately in gelatin capsules until they arrived at

the laboratory, where each was held in a 6.0 cm

plastic Petri dish and presented with 1 or more

adult SPB. Those seizing and feeding on the SPB

adults were manipulated into gelatin capsules

where feeding was completed. Those not feeding

were held either in plastic Petri dishes or in

gelatin capsules for 18-24 hours and were

assumed to be starved.

Ten starved and 10 laboratory-fed T. dubius

adults were crushed individually on a small piece

of filter paper and given code numbers. Each

smear was suspended overnight in 0.5 ml of 0.85

percent NaCl at 4°C. Coded aliquots of the

undiluted predator suspensions were tested by

agar gel double diffusion with undiluted rabbit

anti-adult SPB serum.

Nineteen .adult T. dubius were fed various

numbers of SPB adults; 8 fed on 1 SP8 each, 8

fed on 2 SPB each, and 3 were left overnight to

feed at will. Specimens of T. dubius were killed

and crushed on filter paper after feeding was

complete. All were held over P205 at room

temperature (Dempster 1960) until extracted in

0.25 ml of 0.85 percent NaCl at 4°C. An

additional 20 T. dubius were starved to death at

room temperature, then individually crushed

and extracted in 0.25 ml of 0.85 percent NaCl at

4°C. These uncoded, undiluted, predator suspensions were tested in separate groups by agar

gel doubl#e diffusion with undiluted anti-adult

SPB serum.

Because 15 ul of predator extract might not

contain e’nough antigen to produce a positive

test, we used a concentrated predator extract to

determine duration of SPB protein in the

predator.

T. dub&s adults, collected from baited bucket

traps, were held with an excess of SPB adults for

24-48 hours in gallon plastic containers to assure

removal of any other prey material from their

M. C. Miller and J. Robert Bridges are Research Entomologists, Southern Forest Experiment Station, Forest Service - USDA,

Alexandria, Louisiana. W. Adrian Chappell and William C. Gamble are Chief and Supervisory Microbiologist, respectively, Viral and

Rickettsial Products Branch, Biological Products Division, Center for Disease Control, Public Health Service, U.S. Department of

Health, Education, and Welfare, Atlanta, Georgia.

2
digestive systems. Each T. dubius adult was

placed in a 6.0 cm plastic petri dish and

presented with 1 or more adult SPB. Those

seizing and feeding on the adult SPB were

manipulated into gelatin capsules. These T.

dub&s were held without feeding for 0 to 36

hours and were killed by freezing at 4-hour

intervals. They were extracted as previously

described and a 15 ul portion of each 0.25 ml

extract was tested against undiluted homologous

anti-adult SPB serum by agar gel double diffusion. The remaining 235 ul were concentrated

by lyophilization, reconstituted in 15 ul of

distilled, demineralized Hz0 and tested as previously described.

RESULTS AND DISCUSSION

Titers for the rabbit-produced, anti-adult SPB

sera reached l/80 on day 18, and all 3 rabbit

antisera reached titers of l/160 on days 25 and

32. Downe and West (1954) indicate that

acceptable results in detecting prey in a predator

can be obtained with titers as low as l/100.

Although anti-SPB sera from all 3 rabbits were

used in the experiments described above, the

results with antiserum from rabbit No. 3 were

the most consistent and are presented in table 1.

The anti-adult SPB serum is not genus specific. We obtained positive reactions to extracts

of adult I. calligraphus, adult I. grandicollis,

SPB larva, pupa, teneral adult, day-old adult,

and adult 0. terebrans. Downe and West (1964)

and Boreham found that antisera made from

adult antigen of insects with paurometabolous

development are sensitive to antigen from at least

the older nymphal instars. Downe and West

(1964) hypothesize that adult hemolymph may

contain nymphal antigens and discrete adult

antigens. Our results indicate that the same may

be true of insects that exhibit holometabolous

development.

Table 1 shows the increase in sensitivity of

the anti-adult SPB serum to its homologous

antigen as time passes. With day-46 anti-adult

SPB serum, all 10 of the T. dubius adults that

fed on adult SPB produced positive precipitin

reactions. Seven of the 10 predators starved

from 18-24 hours after feeding produced negative precipitin tests. There were 3 positive tests,

#2,7, and 14. These may be the result of the

more sensitive antisera reacting to small amounts

of the SPB protein in the 15 ul samples from the

Table 1 -Pncipitin raactions to individual predator exwacb

Days antiserum collectad after initial

T. dubius Fed on immunization

number SPB 1 1 16 2 5 32 39 4 6

1 No -

2 No -

3 Yes -

4 YW +

5 Yes -

6 No -

7 No -

8 Yes -

9 YW +

10 No -

1 1 Yes -

12 No -

13 Yes -

14 No -

15 No -

16 YW +

17 No -

18 YW +

19 Yes -

2 0 No -

- -

+ +

+ +

+ +

+ +

- -

- -

t +

+ +

- -

+ +

- -

t +

+ t

- -

t t

- -

+ +

- +

- -

t

+

+

t

+

+

t

+

+

t

+

t

+

T. dubius adults that retained incompletely

digested $PB material in their gut from eating

adult SPB captured in the bucket trap with

them. Greenstone finds that some spiders stop

digesting when starved and retain detectable

prey for a week.3

Positive reactions in tests 2 and 14 first occur

with day-32 antiserum, and only with day-46

antiserum for test 7. An explanation for the

delayed positive test is that, since each visible

precipitin line is the result of more than one

antigenlantibody system (Crowle 1973), not

enough of the particular antibody required for a

visible precipitin line was produced until at least

days 32 or 46.

In another experiment; 19 T. dubius adults

that had fed on either 1 or 2 SPB adults, or had

fed overnight, gave positive precipitin tests in

every case. When extracts of the 20 T. dubius

adults that were starved to death were tested, no

precipitin tests were positive. Control agar gel

double diffusion tests with undiluted SPB antigen against homologous anti-adult SPB serum

were positive in every case.

Table 2 shows the effect of time on starvation

and the use of dilute and concentrated predator

extractions in the agar gel double diffusion test.

The number of positive tests decreases rapidly

when 15 ul of predator extract are used until

1 Usa of trade names is tolrlv for the purpow of identification and does not constitute endorsement by the U.S. Dapammt of Agriculture or U.S. Department of Health, Education and Welfare.

2 Dr. P. F. L. Boreham, Imperial CoIlego Field Station, Silwood Park, Ascot, Barks. SLEJPV,parsonal communicatfon.

3 Dr. Matthew Greenrtone, University of California, lrvins 92717, parsonal communication.

3
Table 2. - Effect of starvation and USI of partial and whole predator extracts on

the agar gel double diffusion test

Hours No. of

Post-Feeding T. dubius

0 13

4 1 4

8 1 2

12 7

16 7

20 15

2 8 10

3 6 8

% Positive Tests

15 ul Concentrated

extract Extract (235 ull

66.6 100

85.7 100

83.3 100

57.1 100

0 100

0 100

100

75

there are no positive tests for 16 and 20 hours.

Using the concentrated predator extracts, we

achieved 100 percent positive tests through 28

hours after feeding. The tests were arbitrarily

terminated at 36 hours when less than 100

percent results were achieved. Probably J. dubius adults in the field would not be without

food this long (Thatcher and Pickard 1966).

Negative findings for 7 of the 10 starved T.

dubius adults in the previous experiment (table

1) are more likely the result of using a portion

of the predator extract (15 ul) rather than the

whole insect concentrate.

Use of a whole insect concentrate and a broad

spectrum antiserum produced positive results in

the controlled laboratory feeding study (table

2). Research is in progress to increase test

sensitivity and produce a species specific antiadult SP8 serum.

1967. Methods for the study of mosquitoes as

virus hosts and vectors. Methods in Virology 1:63-103.

Chappell, W. A., W. C. Gamble, H. S. Lindsey,

and E. H. George.

1976. Antisera for identifying insect blood

meals by double immunodiffusion. A~hfopod5orne virus Information Exchw.

No. 31:41-44,

Crowle, A. J.

1973. Irnmunodiffusion. 2nd ed., Academic

Press, Inc., New York. 545 pp.

Dasgupta, A., and A. C. Cunliffe

1970. Common, antigenic determinants in

extracts of house dust and Dermtophr-

~~~8g~ecies. Clin. Exp. tnimynok

* .

Dempster, J. P. *

REFERENCES CITED

Amman, G. D.

1970. Prey consumption and variations in

larval biology of Enoclerus sphegeus

(Coleoptera: Cleridae). Can. Entomol.

102:1374-1379.

Amman, G. D.

1960. A quantitative study of the predators

of the eggs and larvae of the broqm beetler,

Phytodecta olivetm Forester, using the

precipitin test. J. Anim. Ecol. 29:149-167.

Downe, A. E. R., and A. S. West.

1954. Progress in the use of the precipitin test

in entomological studies. Can. Entomol.

86:181-184.

1972. Prey consumption of Jhanasinus unduletus, a predator of the mountain pine

beetle. Environ. Entomol. 1:528-530.

Berryman, A. A.

1967. Estimation of Dendroctonus brevicomis

(Coleoptera: Scolytidae) mortality caused

by insect predators. Can. Entomol.

99: 1009-1014.

Browne, L. E.

Downe, A. 5. R., tid A. S. West.

1964. The serological specificity of

hemolymph antigens in various stages of

insect development. Pages 555-562 in C. A.

Leone, ed. Taxonomic biochemistry and

serology. Ronald Press Co., New York.

Kiritani, K., and J. P. Dempster.

1973. Different approaches to the quantitative evaluation of natural enemies. J. Appt.

Ecol. 10:323-330.

Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and

R. J. Randall.

1972. An emergence cage and refrigerated’ 1951. Protein measurement with the folin

collector for wood-boring insects and their phenol reagent. J. Biol. Chem.

associates. J. Econ. Entomol. 193:265-275.

65:1499-1501. Moore, G. E.

Chamberlain, R. W., and W. 0. Sudia. 1972. Southern pine beetle mortality in

,

4
North Carolina caused by parasites and

predators. Environ. Entomol. 1:58-65.

Moser, J. C. and L. E. Browne.

In press. A nondestructive trap for

Dendroctonus frontalis Zimmerman

(Coleoptera:Scolytidae). J. Chem. Ecol.

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

1971. Relative abundance of southern pine

beetle associates in east Texas. Ann.

Entomol. Sot. Am. 64:72-77.

Nagel, W. P., and T. D. Fitzgerald.

1975. Medetera aldrichii larval feeding behavior and prey consumption (Diptera:-

Dolichopodidae).

20:121-127.

Entomophaga

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

1966. The clerid beetle, Thanasimus dubius,

as a predator of the southern pine beetle. J.

Econ. Entomol. 59:955-957.

West, A. S.

1950. The precipitin test as an entomological

tool. Can. Entomol. 82:241-244.

White, L. A., W. C. Gamble, and W. A. Chappell.

1975. Production of influenza virus complement-fixing antibody in mouse ascitic fluid.

J. Clin. Microbial. 2:287-291.

ACKPIOWLEDGEMENT - The authors thank

Mr. Edwin George, CDC, for technical assistance

with this research.

5