No. 130 

1962 

HOW TO COLIXCT AND PROCESS 

LARGE POLYHEDRAL VIRUSES OF INSECTS 

Polyhedral viruses have proved highly effective and 

very practical for control of certain pine sawflies; and a 

method of collecting and processing the small polyhedra (5 

microns or less) characteristic of sawflies has been described. 1 There is experimental evidence that the virus 

diseases of many Lepidopterous insects can be used similarly 

for direct control. The polyhedra of most caterpillars, such 

as the gypsy moth and forest tent caterpillar, are larger 

(6 to 15 microns) and the method of processing them for control use is somewhat simpler. The preparation of polyhedral 

material from the gypsy moth, Porthetria dispar (L), will be 

used as an example. 

HOW TO OBTAIN THE POLYHEDRA 

The polyhedra containing the infectious agent, the 

virus rod, are found in tremendous quantities in the caterpillar immediately after death. Care is necessary when collecting diseased caterpillars, since the integument of the 

dead larva is very fragile and easily punctured. Much of 

the polyhedral material will be lost if the insect is ruptured. Two general methods may be used to obtain the polyhedra. 

'~ewis, F. R. How to collect and process small polyhedral viruses of insects. Northeast. Forest Expt. Sta. Forest Res. Xote 109. 8 FP. 1960. 
Field collection. --In not iceably infested areas, the 

polyhedral disease may be present in an ample number of 

caterpillars so that field collection is easy and rapid. 

Gypsy moth, tent caterpillar, and other Lepidopterous 

larvae die of polyhedrosis in a characteristic posture, 

sometimes described as "wilting" . The dead insect hangs 

from the twigs, leaves, or other parts of a host tree by a 

proleg in an inverted V position. 

The diseased larvae are collected in a dry pint or 

quart glass jar. Each larva is loosened and removed from 

the hanging position with tweezers. The collecting jar is 

held immediately beneath the insect to catch any liquid 

material (containing huge quantities of polyhedra) in case 

the larva inadvertantly is ruptured. It is possible for two 

people to collect 16 to 20 pints of diseased material per 

day in this way. A pint of virus-killed gypsy moth caterpillars in the V-VI instar will yield up to 10 grams of purified polyhedra powder, depending upon the condition of 

collected material. 

Laboratory rearing.--When the host insect density and 

disease incidence are not sufficient for making field collection of diseased specimens efficiently, it is possible to 

rear artificially infected larvae in the laboratory. With 

the gypsy moth, 50 larvae are placed in a cage and fed suitable foliage thoroughly sprayed with the disease agent. A 

single feeding of contaminated foliage usually is sufficient 

to initiate the disease. Unsprayed foliage is used to feed 

the larvae until the disease kills them. Approximately 4 

gram of purified polyhedral powder may be obtained per cage 

lot . 

Young larvae, usually in instars 11-111, are fed the 

polyhedra because death does not occur till 12 to 20 days 

after infection, depending on rearing conditions (80-90° F. 

and 90 percent relative humidity are optimum for polyhedrosis). By the time mortality occurs, the larvae will be 

large enough to produce a good yield of polyhedra, but not 

old enough to pupate and emerge as adults. The dead larvae 

are collected as soon as they die, in the same manner as 

described for field collecting. 

STORAGE OF DISEASED CADAVERS 

The polyhedra-f illed cadavers are usually kept in 

closed glass jars under refrigeration (40° C.) until the 
final clean, dry powder is prepared. The larvae can be kept 

in this manner for several months without loss of viral 

virulence. It is advisable, however, to prepare the purified 

polyhedra powder the same year the diseased material is 

collected. 

HOW TO PREPARE THE PURIFIED POLYHEDRA POWDER 

Preparation of the purified polyhedra powder from the 

diseased caterpillars requires the following equipment: (1) 

a good compound microscope, (2) an electric centrifuge capable of speeds to 5,000 rpm, (3) a Waring-type blender, (4) a 

hemacytometer, and (5) pipettes, large beakers, large watch 

glasses, and miscellaneous glass containers. It is necessary that the operating characteristics--particularly the 

range of speeds--of the centrifuge be known. Any one of 

several types of hemacytome ters or particle -count ing instruments can be used. 

The process of purifying the polyhedra is essentially 

one of freeing the polyhedra from the tissues of the cadaver, removing the insect debris by filtering, concentrating the polyhedra by centrifugation and gravity, and finally decanting and evaporating the supernatant (liquid) from 

the polyhedra. 

To start, place the stored diseased material in a 

Waring-type blender. Do not fill the blender more than half 

full. Rinse the storage jar carefully with a small amount 

of tap water and add to the blender. The rinsing must be 

done because many of the polyhedra will have settled to the 

bottom.of the jar. Blend this material for 2 to 3 minutes 

and then follow the process outlined on the flow sheet (p.4). 

The critical steps in the process are the decanting 

and evaporation. Careful decanting of the water from the 

settled polyhedra will remove a large amount of contaminating small debris and bacterial material. During evaporat ion, additional brown contaminating material can be removed 

by very careful aspiration with a fine-tipped pipette. Since 

the large polyhedral bodies settle quickly to the bottom of 

the beaker or watch glass as a white layer, the brown, 

smaller contaminants can be seen on the surface of the polyhedral layer and removed. 
now SHEET FOR PREPARATION 

OF PURIFIED POLYHEDRA POWDER 

Blend diseased larvae 

v 

Filter twice through 40-gage plastic screen. Squeeze 

material on the screen dry and discard. * 

Centrifuge at 

0 

5,000 rpm for 5 minutes. 

Sediment Supernatant (discard) * 0 

Thoroughly resuspend in small amount of water. 

-C 

Filter through a double layer of 40-gage plastic screen 

several times, alternating between two beakers. Squeeze 

screen dry and discard material on screen. 

w 

Place beaker(s) in cold (4O C.) and let p~~lyhedra settle 

for 2 weeks. * 

Carefully siphon off supernatant and discard. (CAUTION 

--sediment very easily resuspends.) 

-C 

Resuspend polyhedra layer on bottom with a very small 

amount of water. 

-C 

Place suspension in large water glass and evaporate 

water off with the aid of a light bulb placed directly 

over the water glass. * 

Scrape dry film of polyhedra off water glass. Grind 

in mortar and place in stoppered glass containers. 
a STORAGE OF THE PURIFIED POWDER 

The purified powder is weighed and placed in tightly 

capped glass containers. The powder can be kept at room 

temperature, but by preference it is kept under refrigeration (40° F.). The powder will keep its potency for several 

years. One gram of purified gypsy moth polyhedra powder 

will contain approximately 6 x 1012 polyhedra. 

I USING THE PURIFIED POLYHEDRAL POWDER 

Determine the number of polyhedra per gram of the 

purified powder by taking several 0.1-gram samples and place 

each sample in 100 ml. of water. Blend each thoroughly and 

count the number of polyhedra per ml. by using a hemacytometer or similar counting instrument.2 Multiply the number 

of polyhedra per ml. (as counted in the hemacytometer) by 

100 to get the total number of polyhedra in each 0.1-gram 

sample. Average these sample totals and multiply by 10 for 

the estimate in terms of grams. 

Weigh out the grams of powder required to treat the 

infested acreage at the specified dosage. For example, 

gypsy moth polyhedra powder with a low count of polyhedra, 

say 25 x 109 polyhedra per gram, is to be applied to 50 

acres at a concentration of 50 x 109 polyhedra per acre in 1 

gallon of water. Two grams of this powder will be required 

per gallon, and a total of 100 grams of purified powder will 

be needed to spray the entire plot to give the required 

dosage. 

For mixing, 100 grams of the powder would be weighed 

out, placed in 400-500 ml. of water, blended thoroughly to 

make a homogenous suspension, and diluted with water and 

sticker to make 50 gallons of the finished spray. The 

blended suspension of powder can be prepared several days in 

advance or can be left in the finished spray should application be delayed. No loss of potency will occur in this short 

space of time. 

The insect pathology section of the Northeastern 

Station's Forest Insect Laboratory at New Haven has on hand 

a small supply of virus polyhedra powders of the gypsy moth, 

instructions for use general ly accompany the instrument. 
forest tent caterpillar, and eastern tent caterpillar, and 

will send a sample to anyone interested in examining the 

material or building up stock. 

Further information on collecting, processing, determining concentrations, or other matters relating to the 

larger polyhedral virus diseases will be gladly sent upon 

request. 

--W. D. ROLLINSON, Biological Aide 

F. B. LEWIS, Entomologist 

Forest Insect Laboratory 

Northeastern Forest Experiment Station 

Forest Service, U.S. Dept. Agriculture 

New Haven, Conn.