Ann. appl. Biol. (1g70), 66, 313-321 Printed in Great Britain

313

The action of some systemic aphicides on the nymphs of Anthocoris nemorum (L.) and A. confusus Reut.

BY W. M. ELLIOTT" Department of Zoology and Applied Entomology, Imperial College,

London, S. W. 7

(Accepted 9 May 1970)

S U M M A R Y

The aphicides phorate, dimethoate and menazon were compared to elucidate. the different pathways by which they can affect Anthocoris nymphs and their aphid prey.

When nymphs were caged in contact with deposits on bean leaves phorate and dimethoate had contact LC 50s of 20 and 3 ,ug/cm2 respectively to Antho- coris nemorum and 46 and 6 ,ug/cmZ to A. confusus. When the nymphs were confined on treated leaves on the opposite surface to the deposits, neither phorate nor dimethoate killed them. Menazon did not kill anthocorids at any dosage. All three aphicides killed over 50 yo of Acyrthosiphon pisum (Kalt.) on bean leaves at 1.6,ug/crn2 whether the aphids were on the treated or untreated surface.

Experiments with 3SS-labelled phorate showed that anthocorids confined on phorate-treated bean plants, with or without insect food, accumulated the insecticide or its labelled derivatives. In field experiments in which A. nem- orum were caged on plants treated with phorate, many were killed on young newly treated plants but not on older plants. A. confusus was relatively unaf- fected.

Anthocorids were reared from 2nd-instar nymphs to adults on aphids killed systemically with phorate, dimethoate or menazon without ill effects, despite evidence that 35S-labelled phorate was ingested from the aphids and excreted in the faeces.

In the field, fewer large A. nemorum nymphs were found in August in plots of tick beans treated with phorate granules at 6 lb/acre (6.7 kg/ha) when sown, than in plots treated at 1-5 lb/acre (1.7 kg/ha) with phorate or menazon or untreated plots.

I N T R O D U C T I O N

The selectivity of aphicides, some examples of the value of anthocorids as predators and the effects of insecticides on them have been reviewed (Elliott & Way, 1968). Phorate, but not dimethoate or menazon, is toxic to the eggs of A. nemorum by systemic action, though it does not affect eggs of A. confusus in the same plants, probably be- cause the two species use different oviposition sites (Elliott & Way, 1968).

This paper follows the effects of these three aphicides on the developing anthocorid

* Present address: Canada Department of Agriculture, Research Branch, Research Station, Harrow, Ontario, Canada.

3 I4 W. M. ELLIOTT nymph. The experiments are aimed at separating the different ways in which the nymphs are likely to encounter insecticides in treated crops.

Sprays of dimethoate are toxic to anthocorids and so reduce the nunibers avail- able to prey on psyllids and mites in orchards (Madsen, 1961; Gratwick, 1965). Phorate is not used as a contact insecticide, since it is volatile and very toxic to mammals, so the experiments on contact action are most relevant to dimethoate and menazon.

Anthocorids occasionally imbibe plant sap and some species can be reared on plants alone (Barber, 1936) ; consequently they may be poisoned directly on systemically treated plants.

Where predators eat aphids on treated plants, food-chain toxicity may occur. Small syrphid and coccinellid larvae feeding on systemically-killed aphids can acquire enough demeton to kill themselves, but large larvae and adult coccinellids arc less susceptible (Ahmed, Newsom, Emerson & Roussel, 1954; Bonnemaison, 1962). Aphids killed by contact action with ekatin (thiometon) are toxic to coccinellids but aphids killed sys- temically are not (Zeleny, 1965). The rearing experiments reported here allow a long exposure period to increase the chance of insecticide poisoning via the prey.

Insecticides and acaricides reduce populations of A. nemorum and other predators by contact action (Collyer, 1953) or in some cases by destruction of the prey (van de Vrie & de Fluiter, 1958). The aim of population counts in this work is to show whether systemic effects from root treatment can also affect A. nemorum.

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

Anthocorids and aphids for experiments were reared as described previously (El- liott & Way, 1968). Laboratory experiments were done in rooms kept at 2o°C and lit by fluorescent tubes for 16 h-day.

Laboratory tests on contact toxicity of aphicides to anthocorid nymphs Bifoliate leaves were cut from broad bean seedlings (Viciu faba L.), each with a

section of stem attached, and placed with the cut stem in a container of water so that the adaxial leaf surface faced upwards. Aqueous solutions of phorate, menazon and dimethoate were made, using Cellosolve as an intermediate diluent, and applied to leaves as 50 ,ul drops which dried forming deposits about 5 inm in diameter. The con- tact toxicity of these deposits was tested by caging single 1st and 2nd-instar antho- corid nymphs or groups of ten 4th-instar aphids on them (Burt, Bardner & Etheridge, 1965). In some experiments the insects were caged on the abaxial surface of the leaf (opposite the deposit), in which case the leaf was secured with the abaxial surface uppermost so that the cage’s orientation to the light source would be the same.

Dosages were calculated as ,ug/cm2, using the average area of the dried drops (20 mm2). Dosages were adjusted by altering the concentration of insecticide on 50 pl drops of solution.

Control leaves were treated with 50 ,ul of an aqueous solution of Cellosolve equiva- lent to that used to make the highest concentration of insecticide.

Dead anthocorids and aphids were counted after 24, 48 and 72 h, and at the same time aphids were given to the anthocorids as food.

Systemic aphicide action on Anthocoris nymphs 31.5

Laboratory tests on toxicity of systemically-treated plants and aphids Bean seedlings were treated with phorate, menazon and dimethoate by the cut-

taproot method to give a concentration of 10 ppm (Elliott & Way, 1968). An aqueous solution of phorate labelled with 35S was made by diluting a 95 7' solution of phorate in acetone. This was applied to bean seedlings to give a concentration of 10 ppm and a specific activity of 0.1 mc/g fresh weight of plant.

Direct ingestion of phorate from the plants was investigated by caging anthocorids on the plants with and without insect food. Ten A. nemomnz and ten A. confusus 4th- instar nymphs and ten A. nemorum females were caged on treated bean seedlings with- out any aphid prey, and three similar groups were caged on treated seedlings with Myxus persicae Sulz. and Acyrthosiphon pisum (Kalt.) supplied as food. After 17 days all the anthocorids were autoradiographed by sticking them to adhesive tape and ex- posing them to X-ray plates for 24 days.

Anthocorid nymphs were fed on an exclusive diet of M. persicae and Aphis fabae Scop. killed by 35S-labelled phorate to establish whether they take up systemic aphi- cides from their prey and so may suffer the effects of food-chain toxicity. Two-leaf bean plants, treated with 35S-labelled phorate at 10 ppm, were each fitted with a de- vice to collect dead aphids. This was a cellulose acetate funnel with its mouth under one of the leaves and its stem leading down into a flat-bottomed 2 x I in (2.5 x 5.0 cm) glass tube. One anthocorid nymph was put in the tube under each funnel and pre- vented from climbing out by a smooth polytetrafluorethane coating painted on the inside of the tube.

When aphids (Aphis fubae Scop. or M. persicae) were put on the bean plants they moved under the leaves to feed, were killed by the insecticide and fell down the funnel into the tube. The anthocorids were thus reared on a diet of insecticide-killed aphids. Excrement was collected from the anthocorids by fitting a 22 mm diameter glass cover- slip into the bottom of each tube and removing it for autoradiography.

The plants were replaced every 7 days, since their toxicity began to decline. Control anthocorids were fed on aphids killed by heating to 40 "C. After different periods, or if they died, the nymphs were assigned random numbers and autoradiographed as described above. The dark traces of individual insects on the X-ray plates were graded by eye into four intensities and these grades were then compared with the treatment records of the individuals by reference to the random numbers. This procedure was intended to eliminate subjectivity in grading.

A further series of tests was done to determine whether unlabelled phorate, menazon and dimethoate affected survival via prey. Anthocorids were reared from 2nd-instar to adulthood on aphids killed 'systemically with phorate, menazon and dimethoate. The numbers dying or completing development on the aphids killed by different insecticides were recorded, controls being fed as before.

316 W. M. ELLIOTT

Field experiments on the toxicity of systemically-treated plants The tick bean field experiments described by Elliott & Way (1968) were used to

assess the survival of nymphs on treated plants. In 1964 plants were treated by applying phorate at 6 and 1.5 lb/acre (6.75, 1*7kg/ha) in the seed drills. In 1965 the plants were treated with menazon at 1-5 Ib/acre in addition to the phorate treatments.

The toxicity of the soil treatments was assessed by caging anthocorids on the plants and, in 1965 only, by counting the A. nemorum population which had developed on the plots. Experiments on survival of caged anthocorids were done in 6 x 2 in (IS x 5 cm) cylindrical muslin sleeves and with 0.1 in (0.254 cm) mesh Terylene net sleeves on 14 x 8 in (35.56 x 20.32 cm) cylindrical wire frames around whole plants. To reduce cannibalism and starvation Microlophium evansi (Theo.) an aphid which does not survive on beans, was supplied as food. Deaths were recorded when the cages were opened after 7-10 days; in these conditions differences between deaths on treated and control plants could be attributed to insecticides and plant feeding would be minimized. Variable results were obtained, so the experiment was repeated under different conditions as shown in Table 2. In addition, in June 1965, sixteen A. confusus nymphs were confined in leaf cages for 10 days, without aphid food, on the 4th (topmost) leaf of untreated plants and others treated at 6 lb/acre with phorate. The leaf cages were in full sunlight, so the anthocorids would experience water stress and starvation and therefore probably feed on the plants.

Counts were made of the anthocorids and aphids present in the field bean plots on 11 and 25 August 1965, by placing a I m2 beating tray against the outside plants of each plot. These plants were then beaten with a stick and the dislodged anthocorids werc separated and counted. On the second occasion aphids were also counted. The heights of two randomly selected plants from each plot were measured when the plants were full grown.

R E S U L T S

Contact toxicity of aphicides to anthocorids Preliminary experiments to determine the range of dosage where survival occurred

showed that menazon did not affect anthocorid nymphs at doses killing aphids. For instance, at 1.6 ,ug/cm2 anthocorids were unaffected but 93 yo of Acyrthosiphon pisum were killed.

Phorate and dimethoate killed both species of anthocorid. Probit analysis on the 72 hour data, after correction for control mortality, (6% or less) showed that the mortality dosage regression lines for the two species and the two insecticides were parallel with a common regression coefficient (b) = 0.89 & 0.13.

The LC 50 for phorate against A. nemorum was 20.2 f 6.4,ug/cm2 and against A. confusus was 46.4 +_ 22.6 ,ug/cm2. The LC 50 for dimethoate against A. nemorum was 3.1

Phorate and dimethoate were more toxic to aphids than to anthocorids. Phorate killed 100% of 4th-instar Aphis fabae and Acyrthosiphon pisum in 72 h at 6.4 ,ug/cm2 which was lower than the LC 50 for either anthocorid. I n a separate test dimethoate

1.1 and 6.1 k 3.0 ,ug/cm2 against A. confusus.

Systemic aphicide action on Anthocoris nymphs 317 killed IOO % of Acyrthosiphon pisum and 87 % of Aphis fabae at 1.6 ,ug/cm2 which was also lower than LC 50 for either anthocorid.

When phorate and dimethoate were applied to leaves at 102 pg/cm2 and A. nemorum nymphs were caged on the untreated surface opposite the deposit, all the nymphs survived for 4 days, whereas 4th-instar A. pisum nymphs caged on the untreated sur- face were all killed in 48 h.

Ingestion of phorate from systemically-treated plants and prey Adults and nymphs of both species of anthocorid became radioactive on plants

treated with 35S-labelled phorate, whether aphids were supplied as food or not, but the traces produced on X-ray plates by the anthocorids with no aphids to feed on were much fainter than those produced by anthocorids supplied with aphids.

Table I . Number of anthocorids reared (n) and mortality (yo) on aphids killed systemically by aphicides and heat

Phorate Men a z o n Dimethoate Heat - - * +- n x n % n % n /U

19 I5 20 24 29 28 18

01

- - Anthocoris nemoruin 61 23 47 A. confusus 24 21 23 26

Table 2. Effect of caging anthocorids for 7-10 days 012 tick beans treated when sown with phorate granules at 6 lblacre

Control Days Number corrected after Leaves/ Sleeve and mortality

Date treatment plant type" stage % A. nemorum

2. vi. 65 11. vi. 65 11. vi. 65 24. vi. 65 13. vii. 64 24. vii. 65 20. vii. 64

33 4 S 16 9 90 42 7 L 89 I00

42 7 L 16 N t 8.3 L 50 N 17'5

84 20 S 16 9 14'7 21 L I Z ? 0

91 - S 16 9 0 85

- 55

A. confusus 12. vii. 65 74 L 200,6 0 -

* I, = 4 in x 8 in sleeve of 0.1 Terylene mesh on a wire frame, S = 6 x 2 in muslin sleeve. 1- N = nymphs.

Fifty-eight anthocorids, consisting of adults and nymphs of both species, were reared for various periods on prey killed by radioactive phorate applied to the roots of bean seedlings. Factors such as the species of anthocorid, the species of prey aphid (Aphis fabae or Myxus persicae), whether the individual was an adult or nymph and whether it was alive or dead before autoradiography, did not affect the level of radioactivity acquired, as judged by the intensity of the traces on the X-ray plates.

The level of radioactivity was related to the time the individual had been held in the apparatus on the radioactive diet; thus individuals giving the most intense traces had

318 W. M. ELLIOTT been on the diet an average of 25.3 days, the next most intense 18.9 days, the faintest discernible marks for 10.4 days and those not giving traces 9-9 days. Excrement from the anthocorids on the radioactive diet was radioactive,

Toxicity of systemically-killed prey Table I shows that when both Anthocoris species were reared on aphids killed sys-

temically with phorate, menazon or dimethoate, mortality was similar to that of control insects reared on heat-killed aphids, i.e. 18-29 yo.

Survival on treated plants in the jield When anthocorids were caged on phorate-treated plants in the field, survival varied

in different experiments (Table 2). When A. nemorum females were tested, over 90 yo were killed in the test done 33 days after sowing and treatment, using the small sleeves, and also in the test done 42 days after sowing and treatment in larger sleeves using only eight females. In three other tests, done 80 or more days after treatment, mor- tality was less than 16 yo. In two tests with A. nemorum nymphs in large sleeves, 42 days or more after treatment, less than 18 yo were killed. I n the only sleeve test with A. confusus, 74 days after treatment, none were killed and in the leaf cage test where nymphs were starved for 10 days on plants treated 35 days previously, fewer died than on control plants.

Table 3. Average number of Anthocoris nemorum on plots treated with phorate and menazon and on untreated plots

Date ... I I . viii. 65 25. viii. 65

Treatments.. . Phorate Phorate Menazon Phorate Phorate Menazon I , r - 7

61b/ 1.5 Ib/ 1.5 lb/ 6Ib/ 1.5 lb/ 1.5 lb/ acre acre acre Control acre acre acre Control

Instars I 2'0 3'5 I '3 2'5 0 0 0 0

2 6.0 8.0 6.8 7'3 0.3 1'0 0 0

3 8.3" 16.3 16.0 18.0 3'3 I .8 2'0 2.8 4 4.5* 17.5 17.0 21.8 15'3 16.0 13'5 15.8 5 1.3" 3'5 4'3 7'3 13'3 22.8 18.0 30'3

Adult 1'0 1'0 0.5 1.5 1'5 2.3 I .8 5.5' Total 2 n * 49.8 45'9 58.4 33'7 43'9 35'3 54.4

* Asterisks indicate treatments with significantly different counts (P = 0 0 5 ) on each date (Duncan's Multiple range test).

Analysis of variance based on log. (n+ I) transformation.

Field plot counts (n) of A. nemorum from field bean plots in 1965 were transformed to log 10 (n -k 1) to normalize the variance and analysed by the analysis of variance (Table 3). In the count on I I August, 102 days after treatment, significantly fewer 3rd-, 4th- and 5th-instar nymphs and a smaller total of all stages (P = 0-05) occurred on the 6 Ib/ acre treatment compared with all the other treatments. The control plots had slightly more of most stages than the treated ones. In the 2nd count, 2 weeks later, no 1st- and very few 2nd-instar nymphs were found, since most of the insects were large nymphs or adults. There were significantly fewer 5th-instar nymphs on the 6 Ib/acre

Systemic aphicide action on Anthocoris nymphs 3=9

phorate plots than on the control plots ( P = 0.05). There were also more adults on the control plots than on all the other treatments. The totals of all stages on the different treatments did not differ significantly. On the 2nd count about twelve Acyrthosiphon pisum and seven Megoura viciae Buckt. were beaten from each untreated plot compared with three and one respectively from the treated ones.

The plants treated at 6 and 1.5 lb/acre phorate and 1.5 lb/acre menazon were 98 13, IOI f 6 and IOI rt 8 cm tall respectively, whereas the untreated ones were only 79 5 14 cm tall.

D I S C U S S I O N

Contact action of phorate, menaxon and dimethoate Dimethoate and phorate killed anthocorids by contact action whereas menazon did

not. Gratwick (1965) showed that a spray deposit of 0.023 % dimethoate killed 90- 100% of A. nemorum in 4 days. The probit equations show that this concentration, applied by our methods, would kill about 98 yo in 72 h which agrees well. All three insecticides were toxic to aphids and to a limited extent even dimethoate and phorate were selective, since any dosage killed more aphids than it did predators.

Dimethoate and phorate applied to the adaxial surface of bean leaves did not kill anthocorids on the abaxial surface though aphids were killed only slightly more slowly than on the treated surface. This implies that the predators did not ingest toxic quan- tities of sap when supplied with prey and that the insecticides did not emerge through the leaf surface in toxic quantities.

A . confusus nymphs were twice as tolerant as A. nemorum nymphs to both insect- icides. The interspecific difference in larval tolerance is considerably less than the difference in egg tolerance since A . nemorum eggs laid in leaves are killed by phorate whereas A . confusus eggs in the stems of the same plants are not (Elliott &Way, 1968).

Assimilation from plants Laboratory experiments with 35S-labelled phorate proved that anthocorids ingested

phorate from systemically treated plants but no evidence was found that they could be killed by insecticides in laboratory experiments. Females of both species, caged on plants treated systemically via the taproot with the insecticides were not killed and laid eggs (Elliott & Way, 1968).

Nevertheless, results obtained in the field with A . nemorum show that anthocorids were killed by 6 lb/acre phorate taken up by the plants from soil, even when the need for plant feeding was reduced by supplying aphid food. Mortality is greatest on young recently treated plants; thus the 90 and IOO % mortalities (Table 2) were on plants with only four and seven leaves, 33 and 42 days after treatment respectively, whereas the two o yo mortalities were on plants with about twenty leaves, 85 and 91 days after treatment. Attempts to eliminate possible fumigant effects by using large mesh sleeves on wire frames did not prevent deaths.

In two tests A. confusus appeared to be unaffected by phorate-treated plants, even when caged without aphid food on plants only 35 days after treatment. This may be due to the greater tolerance of this species to phorate.

320 W. M. ELLIOTT

Assimilation from prey

labelled phorate in systemically treated plants they must assimilate phorate or a phorate metabolite. Some 35S was found in the faeces but traces of the insect's head and legs could bc seen on some of the radiographs, which indicates incorporation into parts of the body other than the gut.

Anthocorids feeding on systemically killed aphids from the second instar onwards grew into normal adults. This is a complete contrast to the situation where more than 80 yo of the predatory mite Phytoseiulus persirnilis Athias-Henriot died after feeding on systemically killed Tetranychus urticae Koch for only I day (McClanahan, 1967).

Since anthocorid nymphs became radioactive when fed on aphids killed by

Predator populations on treated plots Plants treated with 10 % menazon and phorate granules usually had lower popu-

lations of A. nernorum than control plants despite their greater size. This means that the number of predators/volume of vegetation was even lower on the treated plots than Table 3 suggests.

Laboratory results show that the population differences are unlikely to be due to food-chain toxicity and menazon-treated plants had no more anthocorids than those treated at the same dosage with the ovicidal phorate, so differences are not due to ovi- cidal action. This leaves the possibility of direct systemic or fumigant action, which is once again unlikely for menazon, but could account for the lower numbers at 6 Ib/acre phorate compared with 1.5 lb/acre phorate.

Possibly, differences in the supply of food can account for the differences between treatments, but aphids were scarce on all treatments when the predator counts were made. All the control plants became heavily infected with A. fabae which arrived in early June, but numbers had declined by mid-July. The anthocorids in the counts would have been from eggs laid from 24 July onwards.

Although the treated plots had fewer aphids than the untreated ones, on all the plots anthocorids outnumbered aphids and were probably not relying on them as food.

The findings in this work and in Elliott & Way (1968) show that phorate can affect A. nemorum via systemic ovicidal action, by direct contact action and by a direct effect (systemic or fumigant) which was demonstrated only in field experiments during the first 40 days after sowing and treatment. This effect could not be reproduced in the laboratory by the cut taproot method or by applying the insecticide on one side of a leaf and caging anthocorids on the other.

Dimethoate has contact action but no ovicidal action against anthocorids. It was not tested in the field.

Although menazon is not toxic to anthocorids, fewer anthocorids occurred on men- azon-treated plants than on untreated ones.

Food-chain toxicity from aphids to anthocorids does not seem to occur, although it occurs in food-chains involving mites, with both phorate and dimethoate.

Systemic aphicide action on Anthocoris nymphs 321

I am grateful to Professor M. J. Way and to Dr C.T. Lewis for advice and for help with isotope work and to Miss S. B. Vahrman for insect rearing. The research was financed by the Agricultural Research Council. The insecticides were donated by Plant Protection Ltd., Cyanamid of Great Britain Ltd. and Fisons Pest Control Ltd.

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MCCLANAHAN, R. J. (1967). Food-chain toxicity of systemic acaricides to predacious mites. Nature, Lond. 215, 1001.

VAN DE VRIE, M. & DE FLUITER, H. J. (1958). Some observations on the effect of insecticides and acaricides on the population of the European red spider mite (Metatetranychusulmi Koch) and its principal predators in commercial orchards in the Netherlands. P70C. 10th Int. Congr. Ent. 4, 603.

ZELENY, J. (1965). The effect of insecticides (fosfotion, intration, soldep) on some predators and parasites of aphids (Aphis craccivora Koch. Aphis fabae Scop.). Roxpr. &l. Akad. Vgd 75, 3.