Food consumption by cabbage loopers infected with nuclear polyhedrosis virus

JOURNAL

OF INVERTEBRlTE

PATHOLOGY

Food Infected

21,

191-197

Consumption with

by Cabbage

Nuclear JAMES

Department Agricultural

(1973)

Polyhedrosis D.

of Zoology-Entomology, Experiment Station, Received

Nollember

Loopers Virus

HARPER

Auburn Unie~ersity, Aubuw, Alrtbama 368830 1, 1971

The effects of age, temperature, and dose on artificial medium consumption by healthy and nuclear polyhedrosis virus-infect,ed cabbage looper larvae were measured using gravimetric methods. Instar in which lethal infection occurred was more closely related to subsequent food consumption than was larval age in days. Larval cabbage loopers, lethally infected in either the first or second instar, consumed 2% or less of their potential consumption. Larvae infected in the third instar consumed ca. 5% of their subsequent potential. In the fourth instar, this amount increased to ca. 10%. If infection occurred in the fifth instar, no significant amount of feeding was prevented. Increasing the virus dosage significantly decreased consumption and length of feeding period over the range of dosages tested. The relationship between consumption patterns of diseased and healthy insects remained constant over a 2035°C temperature range.

lem of time lag and subsequent feeding A frequent criticism of insect viruses as damage and stress the necessity for treating insects at an early stage. However, precise microbial insecticides is that a time lag occurs between ingestion of the pathogen and qualitative and quantitative measures of food consumption by virus diseased insects death of the insect, during which the host have not been reported. Working with aninsect continues to feed and cause damage other type of pathogenic process, Rahman to its food source. Glass (1958) and Cham(1970) demonstrated that parasitism could berlain and Dutky (1958), working with increase or decrease the quantity of food virus diseases of the red-banded leaf roller, consumed by Pieris Tapae, depending on the Argyrotaenia velutinana, and the tobacco pnrasitc studied. budworm, Heliothis virescem, respectively, The work reported here was designed to found that crop damage could not be preprovide information on the effects of three vented despite up to 100% virus-caused variables-age, temperature, and dosagemorality in the pest populations, because on consumption of a nutrient medium by the larvae did not die until they had virus-infected insects. almost completed development. Tanada and Reiner (1962) demonstrated that damage METHODS AND MATERIALS to corn could be prevented if corn earworm larvae, HeEiothis zen, were infected in the Consumption of medium by cabbage first or second instars, but not if infected loopers, Trichoplusia ni, was measured in later instars. Most general discussionson using gravimctric methods (Waldbauer, the use of microorganisms for insect control 1968). Solidified nutrient medium (Lee and (e.g., Bucher, 1958; Cameron, 1968; Smith, Bass, 1969) was cut into pieces large 1967; Thompson, 1963) mention the prob- enough to provide excess food for one larva 191 Copyright All rights

@I 1973 by Academic Press, Inc. of reproduction in any form reserved.

192

HARPER

for a 24-hr consumption period. Each piece was weighed and placed in a 60 X 15 mm disposable Petri dish with one test larva. Dry weight of the medium used each daJ was determined by oven drying fresh, preweighed samples at 100% for 24 hr, cooling the samples to room temperature in a dessicating jar, and reweighing. The ratio of oven dry weight (ODW) t’o fresh weight was determined and used as a constant multiplier to convert the fresh weight fed to an ODW-fed basis. Five control ratios were obtained daily and averaged for this constant. Constants were determined daily to account for batch differences and for gradual within-batch drying of the medium in storage. After 24-hr exposure to the medium, all remaining medium and excrement were removed and replaced with fresh, weighed pieces of medium. This process was continued for each test larva until it either died or pupated. Unconsumed medium from each plate was placed in a pretared, glass vial. Its dry weight was then determined using the previously described procedure. Daily dry weight consumption of medium was determined by subtracting the unconsumed dry weight from the dry weight fed. Effect of age. Seventy 5-day-old larvae were randomly selected from a laboratory culture. Twenty were randomly selected from this group and placed on medium in l-02 plastic cups. Ten of the larvae were inoculated with NPV by dispensing 0.1 ml of a 6 X IO6 polyhedra/ml suspension uniformly over the medium surface. This dosage was selected on the basis of prcliminary tests which demonstrated that it was greater than an LD,, for all ages of T. ni. The medium in the other ten cups was not treated, and larvae feeding on the untreated medium served as controls for all other phases of the entire experiment. For all inoculations, larvae were allowed to feed on the treated medium for 2 hr and then transferred to Petri dishes containing preweighed, virus-free medium. Daily consumption was then determined as previ-

ously described. The remaining 50 larvae selected from the laboratory culture were placed in Petri dishes with unweighed medium which was changed daily to control the effect of handling on medium consumption by any treated group. Each day for 6 days, 10 larvae were randomly removed from the untreated group and were placed on treated medium for 2 hr. They were then placed on weighed medium unt’il they died 3r pupated. Mean daily food consumption of larvae treated initially at ages 5 through 11 days was obt.ained and compared to consumption of the control group. The low volume of food consumed by healthy larvae during the first 5 days of their development necessitated a modification of the above described method. Five larvae were placed on each of five blocks of medium and allowed to feed for 5 clays without a food change. The remaining food was then dried and total group consumption was determined in the usual way. Only qualitative observat,ions were made of consumption by larvae infected at O-4 days after hatching. Effect of temperatwe. Twenty g-day-old fifth instar larvae were tested for temperature effects at each of four temperatures20, 25, 30, and 35%. Larvae were treated with the same dose and technique as in the age test, with 10 receiving virus and 10 being held as controls. All 20 larvae in each treat’ment were then placed in incubators maintained at the above constant temperatures. Food was changed daily and consumption determined as previously described. Effect of dosage. Three dosages of approximately 16.7, 167, and 1667 polyhedra/mm” of medium surface were used to determine effect of dosage on food consumption. Inoculation was accomplished by dispensing 0.1 ml of Tris buffer (0.1 M, pH 7.1) containing 105, 10F, or lo7 polyhcdra/ml onto the surface of medium in I-oz plastic cups. One 6-day-old, early fourth instar larva was allowed to feed in each cup for 24 hr, and 10 larvae were

FOOD

CONSUMPTION

BY

NW-INFECTED

treated with each dosage. The dosages were selected on the basis of previous work (Harper, unpubl.) which demonstrated t’hat a dosage of lo5 polyhedra/cup was close to an LD,, value, while 10” and lo6 caused >99% mortality. After treat’ment, the Iarvae were placed in individual Petri dishes with fresh, weighed food. Daily consumption was measured as in the previous tests until the larvae had either died or pupated. RESULTS

AND

Dwx~ss~o~

Consumption by healthy lnruae. The control larvae of the age test provided a qualitative and quantitative description of food consumption over time (Fig. 1). The consumption pattern, as previously stated, could not be measured for the first three instars using the methods described, so the average first &day consumption is shown as a gradually increasing line covering an area representing the 8 mg (ODW) of diet consumed during this period. Based on consumption data for older larvae, consumption increased between molts and decreased at molting periods as a result of cessation of feeding for a number of hours before and after the molt. The major feeding period, in terms of volume, began on day 7 and culminated approximately 1 day prior to pupation. Consumption by larvae during

CABBAGE

193

LOOPERS

the first three inxtars was ea. 2.0% of their total or potential consumption of 407 mg. The curve in Fig. 1 is a composite of the feeding activity of 10 larvae. While the points shown represent nctual consumption, the curve is only an approximate qualitative representation of larval feeding patterns. Control groups from the other tests had almost identical consumption patterns. Effect of age at time of infection on food consumption. After infection, larvae usually fed normally during the first 24 hr postinfection (Table 1). Consumption during the second 24 hr in most cases was significantly lower than that of the control larvae, and daily consumption then continued to decline until death. This relationship was seen in larvae inoculated at ages 5,6,9, and 10 days. Larvae inoculated at 7 days posteclosion ate less than healthy larvae during the first 24 hr postinoculation but ate a similar amount during the second 24 hr (day 9). From that time on, their consumption dropped very rapidly while consumption by the healthy larvae increased rapidly. Larvae inoculated at 8 clays posteclosion ate significantly less medium than the healthy larvae during the first 24 hr postinoculation as well as during each of the 2 following days prior to death. Many larvae at age 7 molted into the fourth instar shortly be-

FIG. 1. Qualitative and quantitative pattern of food consumption (artificial normal laboratory reared Trichoplz~sicl ni larvae. Points present the average-per-larva actually consumed during each day. The solid line connecting these points is only approximation of the actual feeding pattern.

medium) by amounts a qualitative

194

HARPER

TABLE 1 AVERAGE DAILY CONSUMPTION OF NUTRIMT Trichoplusia ni LSRVAE INOCULATED WITH Age in days when inoculated 5 fi 7 8 Y 10 11 Healthy control

MEDII!M (rng ODW/day) NPV AT DIFFERENT AGES

BY

Days after eclosion 1-5 (8.Oy' (8 0) (8 0) (8 0) (8 0) (8.0) (8 0) 80

6 3 8

7

(2.1) (2.1) (2 1) (2.1) (2 1)

8 9'" li 4 (24 4) (24 4) (24 4) (24.4) (24 4)

21

24.4

(2 1)

8

9

10

11

y** 0 2" 16.2

Dd D -0.6** 4.3** 52 8** 120 2 (132 2)

D D 54.8** 100 8’ 124.7

12.9 29.5 63

-0.2 12.7** 44

D 3.1 39

122 2

138 6

11.8

Pd

00

-l.l"C 2 t3** 10 7** (22 2) (22 2) (22 2) (22 2)

(1: :; (17 4) (17 4)

0 2x* 0 6" 12 2** 6.1** 59.7 (57.3) (57 3)

22 2

17 4

57 3

-0

12

13

14

15

17

TOtA'

D 1.5

D

22.9 L 30.9a 74.Ih 75. Ob 274.5c 397.7d 394.4d

00

00

404 Od

16

~1Values followed by * are significantly different from the values given for the control larvae on the same day at the 0.05 level (** = 0.01 level) according to Dunn&t’s procedure. *Values in parentheses are assumed average amounts of medium consumed by the NPV-treated larvae based on amounts consumed by the control larw?. c Negative values result from normal variation in the accuracy of the gravimetric method used for determining consumption. d All larvae dead (D) or pupated (P). c Mean total consumption values followed by the same letter are not significantly different at the 0.0; level according to Duncan’s New Multiple Range Test. Totals presented are sums of actual and expected daily consumption values.

fore or after inoculation. The combined process of infection and molting may possibly have caused greater than normal feeding reduction during day 8. A similar interference may have been responsible for the low initial consumption by larvae inoculated on day 8. Average consumption by control larvae declined from day 7 through 9, indicating that molting was affecting consumption by healthy larvae as well during this period. Larvae inoculated at ages 10 and 11 days consumed medium at essentially the same rate and in the same amounts as did healthy larvae. The only difference in this group was an inability to pupate and a prolongation of larval life by as much as 3 days. During this period, consumption was minimal. Total food consumption by larvae infected at different ages is given in Table 1 and was determined by adding the actual amounts consumed after inoculation to the amounts presumably consumed prior to inoculation. The latter figure is based on the assumption that all healthy larvae followed a similar qualitative and quantitative feeding pattern. From these data, it is seen that larvae are capable of eating more

food after infection as age at infection increases. The relationship is not, however, linear, but steplike, especially between clays 6 and 7,8 and 9, and 9 and 10. Under the experimental conditions imposed by these tests, these steps in consumption rates corresponded well with definite larval developmental stages. Thus, the O-4 age group were first and second instar larvae, ages 5-6 were third instar larvae, 7- and &day ages were fourth, g-day-old larvae were a mixture of fourth and fifth instars, and lo-day larvae were all in the fifth instar. The steps represent the points at which molting occurred. This distribution of instars at given ages agrees very well with findings of Smilowitz and Smith (1970). Their larvae developed only slightly faster at a 30°C rearing temperat~ure. To determine how much subsequent feeding was prevented by infection at a given age or developmental stage, the amount of food consumed after treatment n-as compared to consumption of healthy larvae during the same period. If infected at age 5 or 6 days, nearly 95% of subsequent consumption was prevented as compared to ea. 90% reduction by 7- and 8-clay-old larvae, ca. 55% reduction by g-day-old larvae, and

FOOD CONSUMPTION

BY NPV-INFECTED

essentially 0% reduction in lo-day or older larvae. To determine what an insect is capable of consuming during its entire lifespan if infected at a given age, the amounts of food consumed prior to infection must also be considered. In this case, larvae infected at age 5 or 6 clays had ca. 92% reduction in lifetime consumption as compared to ~a. 82% for larvae infected at 7 or 8 days, ea. 30% at age 9 and 0% at age 10 or greater. If infected during the first 5 days after eelosion, a 98% or greater reduction in potential food consumption resulted. Effect of temperature. The relative amounts of food consumed by virusinfected larvae as compared to healthy larvae did not change over the range of t’emperatures tested. This lack of interaction is clearly illustrated in Fig. 2. Virus-infected larvae ate more food at each temperature than did healthy larvae, but the difference was constant over the entire range of temperatures. From Fig. 2, it is also evident that total consumption by the test insects was essentially unaffected by temperature in the 20-30% range. Only at 35°C was a significantly different amount of food consumed. This sharp increase in food intake may reflect a higher metabolic rate, or it may be due to an increased need for water. At 35”C, the medium dried noticeably faster than at the lower temperatures, so that the larvae would have to eat more food to obtain the same amount of water that they DAILY

LARVAE TIXP gerature (“C)

CONSUMPTION INOCULATED

OF

20

AND

20 20 25 25 30 30 35 35 a H = healthy;

10 1.3 4.4 46.1 35.7 61.4 42.2 155.0 141.3 I = infected.

11

64.2 56.8 152.7 120.2 181.3 120.1 221.3 211.8

30

35

FIG. 2. Relative food consumptionby healthy versus NPV-infected Trichoplusin xi larvae over a 15°C temperaturerange. Amounts given represent total food consumed per larvae between age 9 days and time of deat,h or pupation. got from their food at the lower temperatures. At any given temperature, consumption by healthy and virus-infected larvae was not significantly different. However, based on data from all larvae at all temperatures, virus-infected larvae consumed significantly more food in this test than did healthy larvae. The daily consumption patterns indicate other temperature-infection-consumption relationships (Table 2). At all temperatures, infected larvae continued to live and 2 (mg ODW/day)

MEDIUM REARED

Drays Treatmerit=

25 'C

NUTRIENT

NPV

WITH

195

LOOPERS

L

TABLE AVF:R.IGE

CABBAGE

from

AT

DIFFERENT

BY Trichoplusia

ni

TRMPER.\TURES

hatch

12

13

14

15

80.8 68.3 107.3 97.4 62.5 103.7 71.1 79 5

93 9 80 8 9.1 90 1 P 80 8 P 51.2

60 2 59 4 P 14.1

4.1 54.5

16

22

1.1

17

9

52

0.5

D

0.6

n

18

-0.5

10

Totals

D

-. I .!a 14.9

-0

2 D

--.

-~-

304.5 351.8 315.2 359.1 305.2 349.1 446.6 498.7

feed for l-4 days after the healthy larvae had pupated. As in the age test, the amounts consumed during this period were very small. Insect development required 6 days from molt to pupation at 2O*C, and only 3 days at 30 and 35’C, despite the fact that the same amount of food was consumed at both 20 and 30°C. The rate of intake was thus much lower at the lower temperature. A similar relationship is seen in the consumption patterns of the infected insects. Effect of dosnge. Sixty percent of the larvae fed the lower dosage (16.7 polyhedra/mm’) died from virus infection and 40% pupated. Survivors consumed normal amounts of medium, and consumption followed a pattern similar to that in Fig. 1. All larvae fed at the two higher dosages (167 and 1667 polyhedra/mm’) died from virus infection. The relationship between dosage and consumption is presented in Fig. 3. A significant (0.01 level) inverse relationship was found over the range of dosages tested. Each ten-fold increase in dosage resulted in an average reduction of ea. 31 mg of food consumed. Larvae surviving the low dosage treatment fed an average of 7 days after treatment while the virus-killed larvae fed for only 6 days. Larvae receiving the medium and high dosages

105

10'

POLYHEDRA FIG. 3. consumption

lo6

PER CUP

Relationship between by Trichoplusin ni

dose larvae.

rind

food

fed for only 5 and 4 days, respectively, fore death occurred.

be-

Food consumption by virus-infected pest insects is of practical importance in two different situations. In the first, a decision must be made as to whether a population already present at a given stage of developmcnt can be prevented from doing economical damage by use of a viral insecticide. In the second, a decision must be made as to when a developing population should be treat,ed with virus so that it does not reach an economically important level. The age tests provide quantitative data which sl~oulcl aid in such decisions. Larval cabbage loopers, lethally infected in either the first or second instars, consumed 2% or less of their potential consumption. Larvae infected in the late third instar consumed ea. 5% of t,heir subsequent potential. In the fourth instar, this increased to ca. 10% and if infection occurred in the fifth instar, no significant amount of feeding was prevented. From these data, stage of development at time of infection appears to be the most critical factor in determining subsequent food consumption. Although small differences were found in daily consumption averages within each instar, they were not as great as the differences between instars. NPV dosages within the range tested also had an effect on consumption. Higher dosages caused earlier mortality and significantly lower food consumption than lower doses. Temperature, under the conditions of the t,ests, had no selective effect on consumption rates of normal as compared t,o diseased larvae. If the data obtained from the present tests can be extrapolated to consumption of natural food, a method of predicting the value of treatment of a pest population of a given age structure should be devisable. Studies current’ly being conducted on soybean foliage consumption by cabbage loopers indicate that a similar qualitative feecl-

FOOD

CONSU~LIPTION

BY

NPV-INFECTED

ing pattern does occur on natural food (Harper, unpubl.). The dosage used in such treatments could be selected on the basis of the amount of damage which one wishes to prevent rather than on the percentage of the population which one wishes to eliminate. Results of these tests suggest that even though different dosages are capable of killing all larvae which receive them, less feeding damage would bc done by those which received higher doses. Such refinements in use could aid in our ability to utilize pathogens for maximum effectiveness. ACKNOWLEDGMENTS I thank Mrs. JoDeane Owens for her technical assistance in carrying out the various tests prcsented in this paper. REFERENCES G. E. 1958. General summary and review of utilization of disease to control insects. Proc. Int. Congr. Entomol. 10th Congr. Montreal, 1956,4, 695-701. CAMERON, J. W. MACBAIN. 1967. Suitability of pathogens for biological control. In “Insect Pathology and Microbial Control” (P. A. van der Laan, ed.), pp. 182-196. North Holland Publ., Amsterdam.

BWHER,

CABBAGE

LOOPERS

197

F. S. AND R. S. DUTKY. 1958. Tests of pathogens for the control of tobacco insects. J. Econ. Entomol., 51, 560. GLASS, E. H. 1958. Laboratory and field tests with the granulosis of the red-banded leafroller. J. Econ. Entomol., 51, 454-457. LEE, B. L. AND M. H. Bass. 1969. Rearing technique for the granulate cutworm and some effects of t,emperaturc on its life cycle. Ann. Entomol. Sot. Amer., 62, 1216. RAIIMAN, M. 1970. Effect of parasitism on food consumption of Pie& rcrpne larvae. J. Econ. Entomol., 63, 820-821. SMILOWITZ, Z. AND C. L. SMITH. 1970. Distributions and frequencies of weight of cabbage looper larvae reared on artificial diet. J. Econ. Er~tomol., 63, 1106-1107. SMITH, K. M. 1967. “Insect Virology.” Academic Press, New York. TANADA, Y. AND c. REINER. 1962. The use of pathogens in the control of the corn earworm, Heliothis zea (Boddie). J. Inuertebr. Pathol., 4, 139-154. THOMPSON, C. G. 1963. The use of diseases as a practical control for insect pests. Proc. 9th Pacific Science Congr., 1957, 9, 109-111. W.ALIIB.IUER. G. P. 1968. The consumption and utilization of food by insects. In “Advances in Insect Physiology” (J. W. I,. Beament, J. E. Treherne, V. B. Wigglesworth, eds.), Vol. 5, pp. 229-288. Academic Press, New York. CHAMBERL.~IN,