No. 109 

1960 

HOW TO COLLECT AWD PROCESS 

SMALL POLYHEDRAL VIRUSES OF INSECTS 

The past few years have seen increased interest in 

and use of microbial agents for the control of destructive 

forest insects. One of the most successful applications of 

this control method has been the use of the polyhedral virus 

disease of the European pine sawfly, Neodiprion ser ti f er 

(Geoff .) . Control of this insect by its specific pathogen 

has been fully as satisfactory as chemical control, and less 

expens ive . 

The main purpose of this note is to show how the concentrated polyhedral suspension used in control operations 

is obtained. The steps described here apply to insects containing polyhedral bodies of 5 microns or smaller. Most 

sawfly polyhedroses (Neodiprion s e r t i f e r , N. lecontei, N. 

pratti, etc.) fall in this size range. 

HOW TO OBTAIN THE POLYHEDRA 

The actual pathogen, the virus rod, is embedded in a 

protective shell, the polyhedron. Hugh quantities of these 

are found in the insect immediately after death. Two general 

procedures are used to obtain the disease agent. 

Field collection. European pine sawfly larvae, as 

well as most other sawflies that have died of polyhedrosis, 

turn black and dry on the twigs and needles of the host 

tree. Dying larvae have a yellowish cast, but these should 

not be included with the rest of the collected material 

until actually dead. 
Select diseased colonies and individuals from infested trees. It is advisable not to include healthy insects in 

the collection. These healthy individuals cause difficulties 

in determining the concentrat ion of the final polyhedral 

suspension because of the fatty and resinous material in 

them. 

Field collection may be quicker than laboratory rearing if sufficient diseased colonies can be found. However, 

care must be exercised in removing not only the healthy 

larvae but also bits of bark, twigs, needles, and other 

materials that add to the difficulty of cleaning the final 

suspension. 

Laborat~r.~ rearing. 

This method, while more timeconsuming, is more efficient than field collecting when the 

disease is not prevalent or when field populations are 

sparse. In addition, the constant observation made possible 

by laboratory rearing permits placing dead larvae in the 

collecting vessel at the most advantageous time for efficient cleaning. 

Several colonies (the exact number depending upon the 

amount of polyhedral suspension desired) are brought into 

the laboratory. Each colony is placed in a separate rearing 

container and is provided with host foliage thoroughly 

sprayed with the disease agent. After the larvae have consumed this foliage, they are then fed untreated foliage 

until death ensues, usually 12 to 20 days later, depending 

on the vigor of the insects and the rearing conditions. 

Crowding and poor sanitation in the rearing containers usually aid in the onset of the disease. If the insects are 

collected when in instars 11-IV, the disease will strike 

when the larvae are nearly full-grown. This insures the 

maximum return of polyhedra because more polyhedra per larva 

are obtained from larger individuals. 

In some circumstances it may be desirable to combine 

the two methods by spraying a few infested trees with the 

disease agent and then collecting diseased larvae. Beating 

the tree with a padded stick will enable the cdllector to 

obtain the sick and dead larvae quickly. $ 

STORAGE OF DISEASED CADAVERS 

There are two ways in which the collected cadavers 

may be kept prior to preparation of the final suspension. 

Both methods have their advantages and disadvantages. 

Wet storage. The diseased larvae are collected in 

glass containers (pint or quart) half full of water. They b 
may be kept this way for a few months in the refrigerator at 

4°C. When this method is used, the polyhedra do not tend to 

clump and stick together, and a more homogeneous suspension 

can be prepared. However, wet storage tends to cause deterioration of the polyhedra more quickly than does dry storage. The action of putrefactive bacteria on the carcasses 

may cause objectionable odors, but this can be overcome by 

processing the larvae quickly. For long- term storage, wet 

storage is not recommended. 

Dry storage. The diseased larvae are collected in 

dry glass containers. For long- term storage of polyhedra$ 

material, this method is recommended. The virus material 

apparently loses very little virulence when stored in this 

manner for sever a1 years. Object ionable odors are avoided, 

and the necessity for a refrigerator will be eliminated because dry material can be stored at room temperature. However, preparation of the final suspension from the dried 

carcasses presents a more difficult problem because, as the 

insect dries, the polyhedra form large clumps, which must be 

specially treated to separate them. 

In short, if the diseased material is collected for 

use the next year in the field, wet storage is acceptable; 

but if storage of the diseased material will be longer than 

a few months before the final suspension is prepared; dry 

storage is preferred. 

HOW TO PREPARE THE POLYKEDRAL SUSPENSION 

To prepare a polyhedral suspension from the diseased 

larvae, the following equipment is required: (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, and glass 

containers. It is necessary that the operating characteristics--particularly the range of speeds--of the centrifuge 

are known. Any one of several types of hemacytometers,, or 

corpuscle-counting chambers, can be used; instruction booklets for the various types are invariably included with the 

4 particular device purchased. 

From wet storage. First, mix or blend the cadavers 

thoroughly. Then filter out coarse debris by passing the 

material through a fine-mesh plastic screen. Wash the material remaining on the screen several times with a stream of 

water, and then squeeze it dry to obtain the maximum amount 

of polyhedral bodies in the filtrate. 

This first filtrate must now be further cleaned of 1 debris by differential centrifugation. The debris is re- 
FLOW SHEET FOR 

CENTRIFUGATION OF SMALL POLYHEDRA, SUCH AS N. SERTIFER VIRUS 

Initial filtration after several 

washings and squeezings 

Centrifuge at 500 rpm for 

2 minutes 

Re-suspend in water and 

for 15 seconds 

Re-suspend in water and 

centrifuge at 2,000 rpm supernat ant 

for 15 seconds 
tn 

Z and disca~d if only a 

small number oy polyhedra 

are seen. Repeat previous &dimen 

L 

t Supernat ant 

step if many polyhedra * a 

are pr es en t Re-suspend in water, 

* 

Examine for polyhedra. 

mix well in blender If present in quantity * repeat previous step; 

discard if few polyhedra Centrifuge at 1,500 are present. 

rpm for 1 min. - * 

Sediment Supernat ant 

v 

Re-suspend in water and 

centrifuge at 2,000 rpm 

for 1 minute if appreciable number of polyhedra 

are present. * 

Sediment Supernat ant * 

Repeat precious step if 

many polyhedra are present. 

Discard if most polyhedra 

are removed. 

FINAL 

1 

CLEAN 

SUSPENSION 

& * 

1 
moved to make the determination of the concentration easier. 

However, it is not necessary to completely remove all the 

debris from the suspension. 

It can be seen that several microscope examinations 

of the material (using the hemacytometer) are necessary in 

the process of centrifugation. It has been our practice to 

discard any material that has a concentration of less than 

1 x lo5 polyhedra per milliliter. The polyhedral bodies of 

N. serti fer virus disease are small (1.0-1.5 mu.) but can be 

recognized as apparently spherical bodies under the high dry 

objective (43x) of the compound microscope. These bodies 

tend to appear reddish and give a bullseye effect when the 

focus is changed slightly . Actual practice in observing 

known polyhedra is the best way to become familiar with the 

appearance of these bodies. Bright field, dark field, and 

phase-contrast illumination can be used. The type of illumination will be dictated by the equipment and experience of 

the individual. 

From dry storage. 

Preparation of the suspension from 

dry material requires additional steps at the beginning of 

the process to separate the clumped polyhedra and free them 

from the dried remains of the host tissues. First grind the 

dried larvae with a mortar and pestle with a few drops of a 

wetting agent such as Santomerse and sufficient water to 

make fluid. After thoroughly pulverizing the larvae, allow 

the material to stand overnight to permit softening. Mix 

this material completely in a blender and then proceed as 

with the wet-stored material. 

DETERMINING THE CONCENTRATION 

The concentration of the cleaned polyhedral suspension is determined by use of a blood cell counting chamber 

(hemacytometer) such as the Levy counter with improved double Neubauer rulings. 

In practice, it is usually necessary to dilute the 

final suspension to 10 or even 100 times its volume with 

water before reading in the hemacytometer, because it is so 

concentrated that accurate counts of the polyhedra are difficult to make. Take several separate samples (usually 

four), and use the average to calculate the concentration of 

polyhedra in the final suspension. 

The detailed steps in determining the concentration are as follows: (1) mix the suspension well in the blender, 

(2) take a sample (1-5 ml.) and dilute with water, (3) assemble the clean hemacytometer and place a quantity of the 

diluted sample under the cover slip by means of a pipette, 
(4) allow the polyhedra to settle for 5 minutes, and (5) 

count the number of polyhedra. 

For making the count, the improved Neubauer ruling 

has a central 1-mm. square, which is subdivided into 400 

smaller squares. These 400 squares are divided into 25 

groups of 16 squares. Double rulings separate the 25 units 

from one another. Count the number of polyhedra in each of 

the four corner units of 16 small squares plus a central 

unit of 16 small squares. 1n this manner the number of 

polyhedra in 80 small squares are determined. Repeat this 

process several times and calculate the average count. Then 

insert the calculated average in the following formula to 

determine the concentration of the particular sample: 

Concentration of 

polyhedral bodies - (ave. count)(dilution)(4,000) 

per milliliter of - 80 (1,000) 

sample 

= (ave. count) (dilution) (50,000) 

The dilution is the ratio of the diluted volume to 

the original volume of the cleaned suspension. Thus, if 

9 ml. of water were added to 1 ml. of suspension, the dilution factor would be 10 and the number 10 would be inserted 

in the formula. If 99 ml. of water were added to 1 ml. of 

suspension, the dilution factor would be 100, and this would 

be entered in the formula. 

The concentration of the suspension should be determined immediately after preparation and again just before 

the preparation of the finished field spray. 

STORAGE OF THE FINAL SUSPENSION 

The suspensions of N. s e r t i f e r and other small polyhedra store well in water at 4OC. The pH of the suspension 

should be checked and the material buffered if necessary to 

keep the pH at approximately 7.0, because alkaline suspensions will deteriorate rapidly. Stored suspensions will 

keep their virulence for several years, but a gradual lessening of virulence may occur. Suspensions more than 2-3 

years old should not be used in the field. 

The insect pathology section of the Station's Forest 

Insect Laboratory at New Haven has a quantity of N. sert i fer 

polyhedral virus and will send a sample to anyone who is in- 
terested in examining the suspension. Sufficient material 

will be sent to provide a starter culture for those who wish 

to use the laboratory-rearing method to build up a stock for 

field use. 

Any additional information on the collecting and 

processing of small polyhedral suspensions will be gladly 

sent on request. 

--FRANKLIN B. LEWIS 

Entomologist, Forest Insect Laboratory 

Northeastern Forest Experiment Stat ion 

Forest Service, U.S. Dept. Agriculture 

New Haven, Conn.