United States

‘@#j$ Argriculture

;@?) Department of

f-n~7 @ ? Forest Service

w Southern Fbrest

Experiment Station

soFebruary, 1984

Temporal Cold Storage of Eggs of the Poplar Tent Maker, Clostera r’nclusa,

Prior to Use in Rearing the Egg Parasite, Ooencyrfus ennomophagus

A. T. Drooz and J. D. Solomon

SUMMARY METHODS

After being retained in cold storage at - 10°C over a

24-month period, some Clostera inclusa (Hubner) eggs

were still able to be successfully parasitized by Ooencyrtus ennomophagus Yoshimoto, an egg parasite. An

equation was developed for predicting parasite yield over

time from cold-stored eggs. Predicted parasitism was 25

percent or better for up to 8 months of storage, then

dropped to below 16 percent for eggs stored 12 months,

8 percent after 18 months, declining to less than 1 percent after 24 months of storage.

Additional keywords: Parasite rearing, biological

control, Populus deltoides, Salix nigra.

Host eggs were obtained from a nursery colony of C.

inclusa reared on foliage of cottonwood, Populus deltoides Bartr., and black willow, Salix nigra Marsh. After

pupation and emergence the moths were allowed to

mate in screen cages and then transferred to brown

paper bags for oviposition. The egg masses deposited

on the sides of the bags were collected promptly and

processed for storage tests within 24 hours of deposition,

because the parasite normally develops only in unembryonated eggs. The egg masses were placed in double

plastic bags, the air was removed from each bag, and the

necks of the bags were twisted and tied. The bags of

eggs were placed in a freezer at - 10°C for storage tests.

INTRODUCTION

Egg parasites offer considerable promise for biological

control of forest defoliators, but technology must be

developed to fully utilize this control technique (Fedde, et

I. 1976). Drooz and Solomon (1980) reported that the

egg parasite, Ooencyrtus ennomophagus Yoshimoto

(Hymenoptera: Encyrtidae), a natural parasite of elm

spanworm, Ennomos subsignarius (Hubncr) eggs, could

develop successfully in eggs of the poplar tent maker,

Clostera inclusa (Hubner). Their studies demonstrated

that the encyrtid parasite developed at least as well on C.

inclusa eggs preserved at - 10°C for one month as on

fresh ones, which can therefore be stored pending availability of parasites, and that eggs of hosts such as C.

inclusa are killed by low temperature treatment, thereby

making them suitable for transport without fear of introducing the species outside the host’s native habitat. We

now report on studies made to determine how long C.

inclusa eggs can be stored and still be suitable for rearing

0. ennomophagus.

Samples of preserved eggs were removed from storage at monthly intervals of 1 to 24 months for exposure to

parasites. The 0. ennomophagus parasites were from a

laboratory colony maintained at the Forestry Sciences

Laboratory, Research Triangle Park, NC, at 24°C and 76

percent humidity.

Egg masses of C. inclusa, containing 29 to 342 eggs

(table 1) were put in cages with six adult 0. ennomophagus parasites less than one month of age and held at

24°C and 76 percent RH. The percent of adult parasites

that emerged from the C. inclusa eggs (percent = number

parasites emerged t number C. inclusa eggs exposed)

was used to gauge rearing success.

RESULTS

The preserved C. inclusa eggs remain in the liquid

state at the storage temperature of - lO”C, which is not

cold enough to freeze them, and the parasites began

ovipositing into the eggs taken directly from the freezer.

The parasite developed from egg to adult in 21 to 24

days. The resulting adult parasites were relatively longlived-most lived for 2 to 3 months. Both development

Southern Forest Experiment StationIl-10210 U.S. Postal Services Bldg., 701 Loyola Avenue, New Orleans, La. 70113

Forest Service, U.S. Department of Agriculture.

Serving Alabama, Arkansas, Louisiana, Mississippi, E. Oklahoma, Tennessee, E. Texas, Puerto Rico, U.S. Virgin Islands
oI1”“““““““““llJ 0 2 4 6 8 10 12 14 1 6 18 20 22 24

TIME STORED (MONTHS)

Figure 1 .-Observed and predicted emergence of Ooencyrtus ennomophagus from eggs of Clostera inclusa cold-stored at

- 10 “C up to 24 months prior to exposure to the parasite.

time and longevity compared favorably with those reared

from fresh eggs.

Development of the parasite on cold-stored eggs is

summarized in table 1, and a prediction equation for parasite yield over time is presented in figure 1. Percent of

parasite emergence (P) was fitted to the exponential

equation P = 67e -c.t*m , where m = accumulative

months in storage (in this case any whole number from 1

to 24) and e = 2.71828 (the base of the natural logarithm),

by means of the SAS nonlinear package (SAS Institute

1979). The equation gives a residual error mean square

of 151, resulting in a residual standard deviation of 12.28.

If we compare data on the percent parasitism of fresh

eggs (25 percent in the Drooz and Solomon 1980 study)

with current results, equal or better results were obtained

with cold-stored eggs for the first 8 months. The data from

table 1 indicate that the quality of eggs was quite variable

throughout the test. Predicted parasite yield (fig. 1) fell

below 16 percent by year’s end. After 18 months, predicted emergence dropped below 8 percent, and

declined slowly thereafter. Part of the variation in parasite

yield may be related to the number of infertile host eggs

in the tests, some is related to damage due to length of

storage, and some may have been caused by handling.

The contents of infertile eggs dry up quickly at - 10°C.

After the 18th month the decline in parasite yield most

likely signaled the loss in quality of the fertile host eggs

stored at - 10°C. Better packaging, possibly with impervious coatings, while host eggs are first held in evacuated

plastic bags, might prevent dessication and forestall the

decline.

Genduso (1978) and Gennadiev and Khlistovsky

(1980) preserved eggs for parasitism for 3 and 5 years,

respectively, using liquid nitrogen (- 196°C). However,

liquid nitrogen requires special handling techniques and

is not readily available in most laboratories. The temperaTable 1 .--Development of the parasite, Ooencyrtus ennomophagus,

on eggs of Clostera inclusa heldat - 70°C up to 24 months

Clostera inclusa eggs

Months Number per No. parasites

in storage egg mass emerged

1 8 8 4 1

2 4 7 3 2

3 8 2 5 1

4 210 4 0

5 156 8 9

6 9 9 2 4

7 7 8 1 1

8 100 8

9 162 4 8

1 0 174 7 2

1 1 246 1 9

1 2 342 9 1

1 3 326 5 1

1 4 121 1 1

1 5 4 3 1 1

1 6 3 2 3

1 7 3 4 3

1 8 2 9 3

1 9 4 5 0

2 0 4 6 0

2 1 5 3 6

2 2 7 4 3

2 3 186 1

2 4 276 1

ture we used has the advantage of being easily achieved

even in the freezer unit of a household combination refrigerator/freezer. Therefore, our storage technique is readily

adaptable to most field situations.

LITERATURE CITED

Drooz, A. T.; Solomon, J. D. Rearing the egg parasite

Ooencyrtus ennomophagus (Hymenoptera: Encyrtidae) on eggs of Clostera inclusa (Lepidoptera: Notodontidae) kept below freezing. Can. Entomol. 117:

739 - 40; 1980.

Fedde, G. F.; Fedde, V. H.; Drooz, A. T. Biological control prospects of an egg parasite Telenomus alsophilae

Viereck. P. 123 - 27 in Current Topics in Forest Entomology. Selected papers from the XV Intl. Cong. of

Entomol. U.S. Dep. Agric., For. Serv., Gen. Tech. Rep.

WO-8; 1976. 174 p.

Genduso, P. Cryopreservation of eggs of Heteroptera in

liquid nitrogen for rearing of oophagous parasitoids.

ATTI Xl Italian Natn. Congr. Ent. 365 - 70; 1978.

Gennadiev, V. G.; Khlistovsky, E. D. Long-term cold storage of host eggs for reproduction of egg parasites of

pest insects. Zhur. Obschei Biol. 41: 314 - 19; 1980.

SAS Institute. SAS users guide, 1979 Edition. SAS Institute Inc., Cary, N.C.; 1979.494 p.

Drooz is principal entomologist at the Naval Stores and Timber Production Laboratory, Olustee, Florida, Southeastern Forest Experiment Station,

USDA Forest Service. J. D. Solomon is principal entomologist, Stoneville, Mississippi, Southern Forest Experiment Station, USDA Forest Service.

2 * U.S. GOVERNMENT PRINTING OFFICE: 1984-769-089:IZ