Ent. exp. & appl. 13 (1970): 37--42. North-Holland Publishing Co., Amsterdam

A R A P I D L A B O R A T O R Y T E S T F O R D E T E R M I N I N G D E A T H

I N S O M E A R M O R E D S CA LE SPECIES

( C O C C O I D E A : D I A S P I D I D A E ) *

BY

I. ISHAAYA and E. SWIRSKI Division of Entomology, The Volcani Institute o~ Agricultural Research, Bet Dagan, Israel

A rapid laboratory test for determining death in the Florida red scale (Chrysomphalus aonidum (L.)) and the California red scale (AonidieIla aurantii Mask.) based on enzymatic activity is described. This test is based on the ability of the live scale enzymes to react with iodine, yielding iodine products which in turn are unable to give the blue color reaction with starch. The reaction seems to be due to the decrease in the oxidation number of the iodine from I ~ -~ by the scale reductive enzymes.

The longevity of the females after detachment from the host or after treatment with insecticides commonly used in the field, was determined by this test.

The California and the Florida red scales are members of the armored scale group and known to attack citrus and various other plants. The California red scale still remains one of the most serious threats to citrus crops in various coun- tries. The armored scales have a good protective covering, which is a result of a wax secretion from the body of the insect; it consists of one or two protective shields: a dorsal one - - thick and hard - - placed above the body, and in some cases also a ventral one - - thin and delicate - - placed between the body and the surface of the plant on which the insect is feeding. The Florida red scale has only the dorsal cover, while the California red scale has both the dorsal and the ventral cove r s .

One of the problems faced by biologists and toxicologists is determination of the longevity of the scale with or without treatment. Removal of the protective covering, which is necessary for determining the vitality of the scale, actually causes death. When control measures are used, it is usual to wait 2---6 weeks and even more after treatment in order to evaluate the percent mortality (Chandler & Porter, 1925; Melander, 1925; Ebeling, 1936; Klein, 1939; Ben-Amotz, 1939; Cressman & Broadbent, 1944; Bachmann, 1953; Riehl et al., 1959; Cressman & Gilmore, 1964; Kehat & Swirski, 1964; Wood, 1964; Griinberg, 1967). With a decrease in temperature the waiting period is prolonged accordingly, but it can be shortened to a few days after heat treatment (Avidov, 1957). After this waiting period the dead scales are usually dry and the presence of fluid after the body is

* Contribution from The Volcani Institute of Agricultural Research, Bet Dagan, Israel. 1969 Series; No. 1581-E.

38 I. ISHAAYA AND E. SWIRSKI

punctured signifies a live scale. In some cases a change in body color which may occur several days after death, also signifies a dead scale. By these methods it is not possible to determine the longevity and date of death of the scale, or to evaluate the insecticidal activity shortly after treatment.

In this paper we report a rapid biochemical test, based on enzymatic activity, to determine the death of these scales.

M A T E R I A L S AND M E T H O D S

Iodine test preparation. Twenty ml of 1 % bacto-agar (Difco) and 2 % starch (BDH) were boiled and poured into a petri dish 5 cm in diameter. After 15-min cooling at 0 ~ C a block of starch-agar was produced. A solution of 0.05 M iodine in ethanol was poured on the starch-agar block and left for 90 sec. The iodine solution was then removed and a dark blue film on the starch-agar block was formed. The petfi dish was placed for some minutes near a ventilator until the ethanol on the starch-agar block had evaporated. The block was then ready for the test.

Iodine test. After removing the insect's armor the body was punctured with a sharp needle and put on the blue film of the starch-agar block. (Care must be taken to prevent the puncturing of the blue film of the agar block with the needle.) When the body of the insect was connected strongly to the protective shields, as with the California red scale female, the body was punctured while still in the shields and then placed on the blue agar,starch block. After a few minutes a discolored spot beneath and around the live scale was formed; its radius increased gradually and reached its maximum after 60 min. Disappearance of the blue color seems to be due to a decrease in the oxidation number of the iodine by the live scale (see Results); dead scales failed to give this reaction.

Rearing method. The scales were reared on lemon fruit according to the method of Tashiro (1966). The infested fruits were kept at room temperature.

Chemicals. The following compounds for scale treatment were used: 1) PNR 1, narrow range oil (80o/0). Pazchem Ltd., Tel Aviv; 2) methidathion ( = GS 13005 = Supracide) ( 4 0 % e.s.), 0,0 - dimethyl s - 5 - methoxy - 1,3,4 - thiadiazol -2 -

(3H) - onyl - (3) methyl dithiophosphate. J. R. Geigy S.A., Basel.

R E S U L T S AND DISCUSSION

In order to examine the reliability of the iodine test, the following experiments were set up:

Exp. 1: Live female scales were removed from the host and placed in a closed petri dish at 25 ~ and 9 8 % R.H. Death of the scales was determined by the iodine test (Table I). I t can be seen that the Florida red scales gradually lose the ability to give a positive iodine reaction. On the third day after being placed in petri dishes, all 15 scales tested failed to give a reaction, although their bodies still

DEATH DETERMINATION IN SCALES 39

TABLE I

The longevity of female red scales after removal [rom the hostplant. The bodies of the Florida red scales were removed from their protective covers; those of the California red scale

were left in their shields

Days after removal

from plant

0 1 2 3 5 7 9

11

Positive iodine reactions divided by total number of scales

Florida red scale

14/15 6[t5 2]15 o/15

California red scale

15/15 14115

13/15 13/15 6/15 3/15 1/15

contained fluid. The California red scale females, which were removed from the plant host with the .shields intact (it is difficult to detach the body of the scale from its protective cover) lived longer than the Florida red scale. These results emphasize the good protection afforded by the California red scale's shields.

Exp. 2: This test was made to elucidate the nature of the enzymatic activity involved in the iodine test. The blue color film formed on the agar-starch block is a result of iodine-starch reaction. Hence, the disappearance of the color when a live scale is placed on the block could result from one or two causes: (a) digestion of the starch as a result of amylase activity and/or (b) raising or lowering the iodine oxidation number by reductive or oxidative enzymes, as a result of which the new iodine products are unable to produce a blue color with starch.

Three blue drops of solution, containing 2 % starch and 0.025 M iodine, were placed separately on a microscope slide; on each drop were placed: (a) two bodies of live Florida red scales, (b) two bodies of dead ones, or (c) nothing as a control. After about 1 5 min the blue color of the first drop disappeared. Addition of starch solution to the first drop did not affect the discolored state of the mixture. I t can be concluded, therefore, that the disappearance of the color after contact with a live scale is not directed by amylase activity, but may be due to the iodine's lack of ability to produce blue color with starch, probably due to a reduction in its oxidation value from I 0 - - I - L The possibility of iodine oxidation was excluded, since the live scale was not able to produce a blue color when potassium iodide was substituted for iodine in the iodine test.

Exp. 3: Mineral oil and organo-phosphorus compounds are generally used for controlling the armored scales. I t was therefore decided to determine the effect of these chemicals on the Florida and California red scales by the iodine test (Table II). Oil and methidathion treatment were found to have a rapid effect on the Florida red scale, with a mortality rate of 1000/0 and 80O/o seven days after oil and methidathion treatments, respectively. These chemicals were found to be less effect-

40 I. ISHAAYA AND E. SWIRSKI

TABLE II

The longevity o / f e m a l e scales after treatment with mineral oil or methidathion. Lemon fruit infested with the Florida or California red scale was dipped for 20 sec in 2% mhzeral oil (80%) oi" 0.3% methidathion (40% e.c.). The vitality o] the treated sca'es was compared to

that o / t h e non-treated ones by means of the iodine test

Scale Treatment Positive iodine reactions divided by total number of scales

Days after treatment

Florida red scale

California red scale

3 5 7 9 11 13

None 14115 14-/15 14/15 2% oil 2~15 3115 0[15 0.3% methidathion 6[15 7/15 3/15

None 15/15 15/15 14/15 14/15 14/15 15[15 2% oil 12[15 10115 8[15 6/15 5115 5/15 0.3% methidathlon 15/15 13115 8115 5/15 6/15 5/15

ive when tested on the Cal i fo rn ia r e d scale (Table II) , a p h e n o m e n o n which is

usual ly observed in the field when minera l oil is used. In a f ield expe r imen t car r ied

out in June 1969, 9 0 % of the F l o r i d a red scales on citrus leaves fai led to give a

posi t ive iodine react ion , three days af ter 2 % oil t rea tment ; these results resemble

those ob ta ined in the l abo ra to ry (Table II) . These da t a s t rengthen the possibi l i ty

tha t the iodine test can serve as a quick me thod for evaluat ing the insect ic idal

act ivi ty of var ious chemicals on red scales.

E x p . 4: This exper imen t was set up in o rde r to de te rmine whether the chemicals

tested in Exp. 3 could in themselves affect the iodine test (Table I I I ) . N o effect on

the iodine test results was no ted when live or dead scales were first d ipped in

minera l oil or meth ida th ion . Likewise , a d rop of oil o r me th ida th ion solut ion

a d d e d to the b lock had no effect on the b lue co lor of the iodine s ta rch react ion.

TABLE. I I I

Effect of mineral oil or methidathion on the iodine test reaction (Florida red scales were used for this test)

Treatment

Live scales Live scales dipped for 20 see in 2% oil Live scales dipped for 20 sec in 0.3 % methidathion Dead scales dipped for 20 sec in 2% oil Dead scales dipped for 20 sec in 0.3% methidathion Drop of 2% oil on the iodine-agar medium Drop of 0.3 % methidathion on the iodine-agar medium

Positive iodine reactions divided by total number of scales

15[15

15/15

15115

0,/15

0/15 no reaction

no reaction

DEATH DETERMINATION IN SCALES 41

These data confirm the reliability of the results obtained by the iodine test in the previous experiment.

The following conclusions can be drawn regarding the advantages of the iodine test:

The test enables the toxicologist to determine the insecticidal activity o~ various chemicals shortly after treatment (within 1 - -7 days instead of the 2 - - 6 weeks required in the present commonly used methods. The longevity of the scales and the rate of mortality after treatment can be easily evaluated.

In studying the biology of the armored scale group, the iodine test can be of help in determining the life-span of the adults and the larval instars under various conditions of temperature, humidity and nutrition. Determining the vitality of the scale can be of help in evaluating some physiological aspects of this group of insects.

The iodine test may serve as a useful method for determining the death of similar insects.

Postscript An improved iodine test based on iodine test paper instead of the agar-starch

block has been recently developed: A sheet of regular, white, typing paper is dip- ped carefully from one side in a mixture of 2o/o starch and 0.02 M iodine. After drying, a blue color is formed. The scales are removed from their host and placed on the iodine test paper, which is then placed between two smooth iron plates and pressed firmly between two wooden boards with the aid of a carpentry clamp. About 15 min thereafter, clear discolored spots are seen only around the crushed bodies of the scales which were alive at the beginning of the test. The modified test, though based on the same chemical reaction as the original one, was found to be easier, more practical and better suited for large experiments than the original test.

RI~SUMI~

TEST D E L A B O R A T O I R E POUR D E T E R M I N E R R A P 1 D E M E N T L A M O R T C H E Z

Q U E L Q U E S C O C H E N I L L E S (COCCOIDEA : DIASP1DIDAE)

Un test bas6 sur l'activit6 enzymatique pour d6terminer rapidement la mort chez le pou de Floride (ChrysomphaIus aonidum L.) et la coehenille rouge de Californie (Aonidiella aurantii Mask.) est d6crit. Ce test repose sur la capacit6 des enzymes de la cochenille vivante de r6agir avec l'iode en donnant des produits iod6s ne pouvant plus colorer l 'amidon. Cette r6action semble due h la diminution du degr6 d'oxidation de l'iode de I~ par l 'entremise des enzymes r6ducteurs de la cochenille.

La long6vit6 des femelles, d6tach6es de leur h6te ou apr~s certains traitements insecticides usuels, a 6t6 d6termin6e au moyen de ce test.

REFERENCES

AVIDOV, Z. (1957). The Date-Palm Scale (Parlatoria blanchardi Targ.) in the Arava (Coccoidea: Diaspididae). Pt. I. Prelim. Rep. Agric. Res. Sta., Rehovot, No. 180 (in Hebrew).

42 L ISHAAYA AND E. SWIRSKI

BACHMANN, F. (1953). Untersuchungen an den gelben Obstbaumschildl~iusen Quadraspidiotus piri Licht. und Quadraspidiotus schneideri n. sp. Z. angew. Ent. 34 : 357--404.

BEN-AMOTZ, Y. (1939). Experiments with oil spray for the control 'of the mussel scale (Lepidosaphes pinnae[ormis Bch6.). Hadar 1 2 : 2 0 3 - - 2 0 6 (in Hebrew).

CUANOLER, S.C. & PORTER, B.A. (1925). How soon after spraying with oil may scale control be safely scored? J. econ. Ent. 18: 553--554.

CRESSMAN, A . W . & BROADBENT, B.M. (1944). Changes in California red scale populations following spray oils with and without derris resins. J. econ. Ent. 37 : 809--813.

CRESSMAN, A. W. & GILMORE, J. E. (1964). Studies of Dimethoate for the control of California red scale. J. econ. Ent. 57 : 322--324.

EBELINO, W. (1936). Effect of oil spray on California red scale at various stages of deve!op- merit. Hilgardia 10: 95--125.

GRiJN~ER6, A. (1967). Mode of action of mineral oils upon armoured scales (Diaspididae) in conjunction with low-volume application. Israel J. Ent. 2 : 179--186.

KEnAX, M. & SWIRSKI, E. (1964). Chemical control of the date-palm scale, Parlatoria blan- chardi, and the effect of some insecticides on the lady beetle Pharoscymnus aff. numidicus Pic. Israel J. agric. Res. 14 : 101--110.

KLEIN, H .Z . (1939). The chaff scale Parlatoria pergandei var. camelliae Comst. Hadar 12: 179--181 (in Hebrew).

MELANOER, A .L . (1925). Notes on oil sprays. J. econ. Ent. 18: 681--686. RIEHL, L .A. , LA DUE, J .P . & RODRIGUEZ, J .L . (1959). Efficiency of ethion in oil spray

against California red scale and citrus red mite. J. econ. Ent. 52 : 857--860. TASmRO, H. (1966). Improved laboratory techniques for rearing California red scale on lemon.

J. econ. Ent. 59 : 604---608. WooD, B. J. (1964). Trials with dimethoate, against the California red scale, Aonidiella attrantii,

and other citrus pests in Cyprus. Bull. ent. Res. 55 : 339--353.

Received [or publication: 6 August 1969.