Bioassay of the nucleopolyhedrosis and granulosis viruses of Trichoplusia ni

JOURNAL

OF INVERTEBRATE

PATHOLOGY

10,

327-334

( 1968)

Bioassay of the Nucleopolyhedrosis Granulosis Viruses of Trichoplusia

and ni 1y’

J.D. PASCHKE,R.E. LOWER AND R.L.

GIESE

Department

of Entomology,

Purdue University,

Received

April

Lafayette,

Indiana

47907

10, 1967

Bioassays of nucleopolyhedrosis and granulosis viruses of Trichoplusia ni were conducted utilizing different viral preparations during a 3-year period. All data were analyzed using Berkson’s minimum logit chi-square (~2) method for estimation by the development of a computer proof the MA. The method was facilitated gram for the IBM 7094-1401 system.

INTRODUCTION

The simultaneous infection of an insect with a nucleopolyhedrosis and a granulosis virus is known to occur in relatively few species of insects. Paillot (1936) first described such a condition in the cutworm Agrotis segetum. A similar infection was noted by Tanada (1953) in larvae of Pi&s Tapae and he subsequently demonstrated (1956) that larvae of the armyworm, Pseuduletiu unipuncta, could be simultaneously infected by nucleopolyhedrosis and granulosis viruses. Steinhaus (1957) reported a double polyhedrosis-granulosis infection in the bronzed cutworm, Nephelodes emmedon&z, sent to him from Missouri; Steinhaus and Marsh (1962) found a similar natural double infection in specimens of the fall armyworm, Laphygma frugiperdu, collected in Louisiana in 1960. A double infection of larvae of the spruce budworm, 1 This investigation was supported in part by Research Grant No. 4565 of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, U. S. Public Health Service. 2 Purdue University Agricultural Experiment Station Journal Paper No. 3058. 3 Present Address: Insects Affecting Man and Animals Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Gainesville, Florida. 327

Choristoneura fumiferana, was described in detail by Bird (1959), and Paschke and Hamm (1962) reported the double infection of larvae of the cabbage looper, Trichoplusia ni. More recently, Schvetsova and Ts’ai ( 1962) reported on a simultaneous polyhedrosis-granulosis infection in Agrotis segetuna in laboratory tests. Tanada ( 1959 ) considered the effects of double and single inocula on the quantitative responses by a test population of P. unipuncta. He suggested a synergistic effect with the addition of heat-inactivated granulosis to the polyhedrosis inoculum. However, Bird ( 1959) found no evidence that one virus had a synergistic action on the development of the second virus in his study of double infection in the spruce budworm. He concluded that an advantage must be given the less virulent granuIosis virus either in time or in infectious units to produce both infections in the same insect. The conclusions drawn on the basis of the data derived from such tests and based on inadequate quantification are open to question since one viral agent by itself or in combination with the other may or may not have an undetermined advantage. Prior to undertaking an investigation of the double infection of T. ni we believed that it was first necessary to establish a routine.

328

PASCHKE,

LOWE

bioassay procedure in order to determine accurately the activity of each etiological agent. Previous studies on double infections were based on crude quantification of each viral agent. This paper describes the methods developed in our laboratory and currently employed in investigations concerned with the pathology of virus diseases. METHODS

Quantification

AND

MATERIALS

AND

GJESE

umetric flask while the remaining sample was air-dried until a stable dry weight was achieved. The concentrated aqueous sample was then serially diluted on the basis of dry weight to give the desired doses. Subsequent to the methods of quantihcation described above we have employed lyophilization in the preparation of virus inocula. After purification and concentration by differential centrifugation, each of the respective suspensions was dispensed ( 1.0 ml) to ampules and freeze-dried. The ampules were held at room temperature until they were needed for testing. The polyhedra were resuspended in sterile water, counted, and serially diluted as mentioned previously. Capsules were weighed directly on a Cahn Electrobalance (Model M-10, Cahn Instrument Co., Paramount, California) to establish a suspension containing 1.0 mg capsules/ml sterile water. This suspension was serially diluted to provide the required doses and these were held at 4°C.

To prepare the original polyhedral preparation, infected fifth-instar larvae were allowed to decompose in deionized water and the resulting suspension was cleared of debris by filtration. Subsequently, the filtrate was purified and concentrated by differential centrifugation and repeated washings in sterile triple-distilled water. The final sediment was resuspended in 100 ml of sterile distilled water and divided into two aliquots, one of which was held at 4°C in a sealed volumetric flask. The Bioassay Procedures remaining portion was used to determine The population of test insects used in the number of polyhedra present by visual counts on a Petroff-Hauser bacterial cell this study has been in continuous culture counter. Ten counts of both chambers of in our insectary for more than 3 years. The the counter were averaged to determine an methods used in rearing larvae, as well as estimate of the number of polyhedra per ml. procedures used for holding individual test animals, have been described previously Serial dilutions of 1: 100 in sterile distilled (Paschke, 1964). water were kept at 4°C in sealed vaccine The doses of virus inoculum and sterile bottles until they were used in bioassay water controls were administered to fourthprocedures. Because of the small size of the inclusion instar larvae by force feeding ,2.0 p liters bodies of the granulosis virus it is difficult of each test suspension. A calibrated Isco to make a direct count of the capsules and model M microapplicator (Instrument Spesuch counts are subject to large errors. In cialities Company, Lincoln, Nebraska) was this investigation the dry weight of in- used to calibrate a series of 0.25ml glass clusion bodies has been employed as a syringes . Glass capillary tubes, which had quantitative measure of viral suspensions in been finely drawn and fire-polished were 1959) an effort to reduce experimental variation as coupled to the syringes (Martignoni, much as possible. to dispense the required 2.0 p liter doses. Purified suspensions of capsules were split By injecting the suspensions while observinto aliquots and with the exception of one, ing the larvae under a stereo dissecting midispensed into tared weighing bottles. The croscope it was possible to ascertain that aqueous aliquot was held at 4°C in a vol- they had received the whole 2.0 p liters

BIOASSAY

and that it was not regurgitated. Although this method is more tedious than others, it was felt that the accuracy attained would produce more replicable results. All insects that died during larval and prepupal stages were autopsied to ascertain the cause of death. Failure to emerge as an adult was used as a final criterion of mortality.

329

OFVIRUSES

gram which plotted the lethal doses and their fiducial limits for each assay and which permitted graphic examination of the results. An example of the complete printout of a single assay is given in Tables 1 and 2 and the data plot in Fig. 1. Only test animals dying of virus disease, as determined by autopsy, were included in the analysis of the mortality data.

Statistical Analysis Graded doses were utilized in each of the bioassays and were based on preliminary tests which indicated the desired dose range. Initially, 10 larvae were treated per dose and the procedure was replicated on five successive days. The experimental designs for subsequent replicated tests were dependent upon the time of testing and the availability of larvae. All data were analyzed by the minimum logit chi-square (x2) method of Berkson ( 1953). Initially, the data were analyzed as described by Dahm et al. (1961). Computation using a desk calcuIator proved to be extremely time consuming, and because of the numerous computations required in the method, subject to human errors. It was concluded that a computer program wouId be a soIution to both problems. A FORTRAN IV program (LOCSAN) was developed to analyze data on an IBM 7094-1401 system. In this program, mortalities are corrected by Abbott’s formula ( 1925), unless the mortality is 0% or 100% (or less than the control), in which case, Berkson’s empirical rule is imposed (Berkson, 1953) where zero mortality is corrected by the expression MN, x 100 and total mortality is corrected by the expression (2N-1/2N) w h en N is the number of animals per dose. The program determines lethal doses, their fiducial limits, and tests for linearity and homogeneity of response among the test animals (chi-square test, P = 0.05). A subroutine was incorporated into the pro-

RESULTS

AND

DIS~~SSIOK

Bioassay The median lethal dose (~~50) for polyhedrosis and slopes (b) of regression, based on repeated bioassays of two virus preparations are given in Table 3. Based on the results of each of the bioassays, the ~~~~ vahres vary from approximately 2000 to 7000 polyhedra per test animal. It will be noted that the fiducial limits for each assay are quite wide but this was not unexpected. These are not atypical when comparison is made with other data derived in a similar manner. In addition, it will be noted that the slopes of regression are quite low. This is typical of the response of insect larvae inoculated with viruses, the slopes usually being approximately unity or slightly less. Table 4 presents the results of bioassays on three different granulosis virus preparations. As expected, each preparation gave different responses by the test population. It can be seen that the aqueous suspensions of Preparation I, stored at 4”C, did not attenuate during the 2-year period. This contradicts previously suggested, but not well documented evidence, that granulosis viruses as aqueous suspensions, have a rather short shelf-life. As in the case with polyhedrosis viruses, the fiducial limits of the estimated LD~~'S are quite wide; slopes are again typically low, both a characteristic of biological assays of microorganisms. If one compares the values of g (Tables 3, 4) it can be seen that when more test subjects are utilized for each of the graded

336

PASCHKE,

9.OOE

LOWE

AND

GIESE

Clt

0

X

0

I I I I I 0

x

0

:

I I 7.32E

u

I I 1 I I I

s 0

I I I 5.64E

c

0

x

0

l x

x

0

0

cl* I I 1 I I I I I

3.96E

x

Ol+

0

x

ox

0

ox

0,:

0

x

0

0 0

1

2.28E

0

x

0

x

oa

x

0.

0

01:

I I 1 I

0 l

I I

1 I &.OOE

-7.70E FIG.

program

1.

0

x

0

. oo*------------.--+---------------+--------------00

-6.49E

l - -

00

Data plot of computed lethal doses subroutine in IBM 7094-1401 program

-5.28E

00

and 95% ( LOGAN).

doses g becomes negligible (see Finney, 1962, for discussion of g). Since the value of g increases as a result of heterogeneity of response by the test animals, it becomes clearly evident that the use of more insects per dose reduces heterogeneity to a minimum. This is apparently more critical in the case of granulosis virus, probably as a result of unavoidable settling of capsules from the suspension during the period of force feeding. It is also evident that a bioassay can be conducted utilizing fewer

-4.07E

fiducial

limits

-

- -

-

- -

--

00

----

-+

-2.WE

computed

--

---

00

from

incorporated

replications but only by increasing the number of larvae per dose listed. The use of the computer in analysis of these data greatly shortens the time spent in statistical consideration of each or all of the assays. Use of desk calculator for analysis requires a minimum of 3-4 hrs computation time. Computer analysis reduces this to a fraction of a minute and increases reliability in the results and as a result more time can be spent in consideration of biological phenomena.

EXAMPLE

-4.69097 -4.69897

0.2OOOE-64 0.2000E-34

---em_

-5.69897 -5.6909’7

0.2000E-05 0.200clt-05

50. 59.

50. 50.

59. 50.

32. 35.

13. 12.

8. 3.

64~00 70.00

26.00 24.0@

16.00 6.00

TABLE

1

C.57536 0.0473@

-1.04597 -1.15268

-1.65823 -2.75154

-2.75154 -2.44235

LOG1 T 2.82000 3.6tlOCO

SUHNNL SL’WNWLZ SLL LEAR eC195L x2

= = = = = =

-40.69962 117.12363 07.96061 -0.71654 0.91529 8.68219

SUMS = 18.74OC10-106.79869 --_--------------------------------------C.23046 11.52OCO -54.13214 0.21OOG 10.50CGC -49.33919 _----------------------------------------22.02000-103.47132 SUMS = -___--------------------------------------

15.52482

6.62819 8.89663

-20.57465

-10.06222 -10.51244

-10.90262

suns = 9.54OLO -63.90017 _-----------------------------------------9.62060 -54.82409 C.19240 9.12OtO -51.97461 0.1824@ ______-------_-____----------------------_

-16.74717

-50.04330

-11.14329 -7.75933

-7.75933 -8.98784

NYL

-21.71109 -28.33221

NYX

INCI.UDING

-45.01708 -18.89109

-----------------__----------------------c. 13440 6.72000 C.05640 2.82000 -------------------------------------------

C. ?564C C.07360

NW

-----------------------------------------stus = 6.5OODO

w

e

PROGRAM (LOGAN) AND WEIGHTING FACTORS

56.8OCOO 299.86769 67.54917 5.98274 = 1.17364 = 0.01737 -----------------------------------------------------------

= = =

64.00030 70.0000C

26.CCOOO 24.OOQO”u

16.OCOO3 6.00003

6.OCC3J 8.coooc

PP

FROM IBM 7094-1401 LOG OF DOSE, LOGITS,

----------------------------------------------~-------------------------------------------------SUMNhX = -324.22149 SUMNW SLMNWXi = 1’306.25200 SUHNWXL sxx = 57.55525 SXL XRAK = A= -5.70812 t!CI95U = 1.43199 8 VARX5J = 0.01748323 VARE ---------------------------------------------

-I

-6.69897 -6.69897

0.2003f-G6 0.2000E-06

6.CO 8.00

P

OF PRINTED OUTPUT INTERNALLY COMPUTED

PROGRAM FEL1612 - LOCSAN BY A. L. GIESE FOREST EhTOMCLOGY LAbORATORY DEPARTMENT OF EMTOPOLilGY PURDUE UNlVERSIrY --------------------------CONTROL PERCEVT MORTALITY = G. NUMBER OF DOSES = 4 YUMBER OF REPLICATES = 2 -------WV-----------,----- ----TABLE 1. LOGIT STATISIICS. -------__---------------------------------------------------------------------------------------------------------------_-_------------------------------------------------------------------------------------N 2 X RES -------------------------------------------------------------------------------------------------------0.2DOOE-07 -7.69091 53. 3. 0.2000E-07 -7.69897 51). 4.

AN

4 4

3 3

1 2

2

1

1 2

1 2

1 1

2 2

J

1

82 w

332

PASCHKE,

LOWE

AND

TABLE

GIFSE

2

PRINTED OUTPUT FROM IBM 7094-1401 PROGRAM OF ANALYSIS OF VARIANCE AND COMPUTED LETHAL

(LOCSAN) INCLUDING THE DOSES WITH 95% FIDUCIAL

RESULTS LIMITS

IAtiLF 2. ANOVA. --________________---------------------------------------------------__________________---------------------------------------------------SCURCE OF VARIATION OF SUM OF __--______-__-___-----------------------------------------------------

1

79.2784

2

5.6956

DCSES

3

84.9741

REPLICATES/DOSE

4

2.9066

7

87.9606

REGRESSION DEVIATIUNS

&ETYEEN RETkEEN

SQUARES

FKOH

REGRESSION

TOTAL

MEAN

SQLARE

1.X1-2

2.BCl0

5.991

0.7406

9.440

PO5

________----___-------------------------------------------------------

NO SIGNIFICANT

THE

RESPONSE ..

DEPARTURE

IIF

IRWIYS

FROM LINEARITY

ANIMALS

BETWEEN

I;

=

VALUE

REPS

0.34846

TABLE 3. LETHAL DOSES AND __-___----------------------------------------------------______--_-------------------------------------------------PERCENT --------1;. 20. 25. 3:. 35. 42. 45. 50. 55. 60. 65. 7i.

75. 80. 90.

UPPER LIMIT ----------------C.Z167E-06 0.9013E-06

O.l54bE-05 C.2532E-05 0.4347E-05 0.6393E-05 O.l006E-34 O.l5BBE-04 0.2532E-04 0.4108E-04

O.b847E-04 O.l189E-03 0.2193E-03 0.4402E-03 C.,3OlE-02

EXISTS.

WITHIN

A DOSE

IS

HOMOGENEOUS

. .

THEIR

95

LETHAL

PERCENT

COSE

O.l072E-06 0.5263E-06 0.9254E-06 011515E-05 0.2371E-05 0.3605E-05 0.538BE-05 O.T98lE-05 O.llB4E-04 0.17706-04 0.2691E-04 0.4211E-04 0.6894E-04 O.l204E-03 0.595OE-03

FICUCIAL

LOWER

LIMITS.

LIMIT

0.3946E-07 0.26BBE-06 0.5134E-06 0. BB34E-06 O.l41bE-05 0.2163E-05 0.319BE-05 0.4635E-05 C.6651E-05 0.9540E-05 O.l3BlE-04 0.2038E-04 0.3115E-04 0.5007E-04 O.l920E-03

LCHI-SQUARE

lES1,

P-.05).

BIOASSAY

OF

TAEILE

333

VIRUSES

3

SLOPES OF REGRESSION, MEDIAN LETHAL DOSES ( LD~~), THEIR FIDUCIAL LIAUTS, VALUE OF g AS DETERMINED FROM REPEATED BIOASSAYS OF TWO PHEPARATIONS NUCLEOPOLYHEDHOSIS OF Trichoplusia ni

Datt tested __.

so. of replicatious

Total No. larvae per close

Slope (h) fiiducial

Median lethal dose (polyhedra/larva) and 95% fiducial limits

and 95% limits

Lower

b

Lower

Upper

~.

Preparation 5/63 6/63 12/64 l/65

3 3 -’ L!

50 50 50 40

0.42 0.65 0.83 0.92

0.64 0.90 1.17 1.27

4/66 5/66

2 2

100 20

1.30 0.87

1.62 1.61

MILD

c’

Upper

I

0.86 1.14 1.51 1.61

5.5 2.0 7.7 2.2

Preparation

x x x x

10’ 10” 10” 10”

2.6 6.9 2.0 6.4

x 10” x 10” x 10” x 10’:

1.4 2.3 6.4 2.1

x 104 x 104 x 103 x 104

0.11949 0.07641 0.08692 0.07465

9.0 x 102 4.7 x 10”

0.03710 0.21405

II

1.93 2.36

TABLE

AND THE OF A

4.2 X 102 3.0 Xl01

6.2 X 10’ 1.7 x 102

4

SLOPES 0~ REGRESSION, MEDIAN LETHAL DOSES ( LD~,,), THEIR FIDUCIAL LIMITS, VALCE OF g AS DETERMINED FROM REPEATED BIOASSAYS OF Two PREPARATIONS GRANULOSIS OF Trichoplusia ni

Date tested

so. of replications

Total No. larvae per close

b

2 5 .5 5 5 2

40 100 100 100 100 50

0.53 0.88 0.80 0.57 0.60 0.49

0.97 1.23 1.04 0.83 0.81 0.87

1.40 1.57 1.29 1.06 1.02 1.24 Preparation

S/66 7/66

.3 2

100 40

0.92 0.86

1.17 1.33

1.43 1.79

hlLD

Lower

Upper Preparation

5/63 S/63 lo/63 3/65 6/65 12/65

Median lethal dose (mg of dry capsules/larva) and 95% fiducial limits

Slope ( h ) and 95% fiducial limits Lower

AND THE OF A

g

Upper

I 9.89 1.45 1.55 8.95 4.87 2.41

x x x x x x

10-g 10-r 10-r 10-r 10-r 10-s

2.53 3.06 2.70 2.10 9.22 1.08

x x X x x x

10-r lo-’ 10-r 10-c 10~7 10-T

x 10-G x 10-O

7.99 7.31

x 10-l; x 10-a

9.92 X 5.28 X 5.19 X 7.94 x 1.85 x 3.07 x

10-r 10-r 1O-7 10-e lo-” 10-r

0.19936 0.07992 0.05448 0.09047 0.06936 0.18740

1.59 x 10~5 1.52 X lo-”

0.04846 0.12182

II 4.64 3.12

a Original analysis indicated heterogeneity of response in one replication; values computed with the omission of the replication contributing excess variance; reanalysis homogeneity on the basis of two replications. b Dose range lowered by one decimal dilution, resulting in highest dose giving mately 50% response and an imprecise estimate of MLD.

as given indicated approxi-

D

rJ

334

PASCHKE,

LOWE

Data derived from the above-described bioassays were utilized in determining doses of each viral agent administered in the study of double infections (Lowe, unpublished). Heretofore, studies of double viral infections in insects have neglected an accurate assessment of the doses of each virus inoculated into test subjects. As a result, one virus may or may not have had a biological advantage and the results of such tests could be easily misinterpreted. ACKNOWLEDGMENTS

The authors wish to express their appreciation to Mrs. E. Ogborn and Miss Susan Foster for their invaluable assistance in certain phases of this work. REFERENCES

W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18, 265-267. BERKSON, J. 1953. A statistically precise and relatively simple method of estimating the bioassay with quanta1 response, based on the logistic function. J. Am. Statistical Assoc., 48, 565-599. BIRD, F. T. 1959. Polyhedrosis and granulosis virus causing single and double infections in the spruce budworm, Choristoneura fumiferana Clemens. .I. Insect PathoE., 1, 40-30. DAWM, P. A., GURLAND, J., LEE, I., AND BERLIN, J. 1961. A comparison of some house fly bioassay methods. J. Econ. Entomol., 54, 343347.

ABBOTT,

AND

GIESE

FINNEY,

318 don.

I. J. 1962. “Probit Analysis,” pp. Cambridge University

2nd ed., Press, Lon-

M. E. 1959. Preparation of glass needles for microinjection. J. Insect Pathol., 1, 294-296. PAILLOT, A. 1936. Contribution a I’etude des maladies Q ultravirus des insects. Ann. Epiphyt. et Phytogenet., 2, 341-379. PASCHKE, J. D. 1964. Disposable containers for rearing loopers. J. Insect Pathol., 6, 248-251. PASCHKE, J. D., AND HAMM, J. J. 1962. Granulosispolyhedrosis complexes in loopers. Proc. North Central Branch, Entomol. Sot. Am., 17, 148. SHVETSOVA, 0. I., AND TS’AI, H. 1962. Virus diseases in Agrotis segetum Schiff. and Hadena sordida Bkh. ( Lepidoptera, Noctuidae) under conditions of simultaneous infection with granulosis and polyhedral disease. Entomol. Rev., 41, 486489. STEINHAUS, E. A. 1957. New records of insectvirus diseases. Hilgardia, 26, 417430. STEINHAUS, E. A., AND MARSH, G. A. 1962. Report of diagnoses of diseased insects 1951-1961. Hilgardia, 33, 349490. TANADA, Y. 1953. Description and characteristics of a granulosis virus of the imported cabbageworm. Proc. Hawaiian Entomol. Sot., 15, 235-260. TANADA, Y. 1956. Some factors affecting the susceptibility of the armywonn to virus infections. J. Econ. Entomol., 49, 52-57. TANADA, Y. 1959. Synergism between two viruses of the armyworm, Pseuduletia unipunctu (Haworth) (Lepidoptera, Noctuidae). J. Insect Pathol., 1, 215-231.

MARTIGNONI,