Activity rhythms linked with foraging behaviour in insecticide-resistant and susceptible strains of Oryzaephilus surinamensis (Coleoptera: Silvanidae)

0022-474X/91 $3.00 + 0.00 Pergamon Press plc

J. stored Prod. Res. Vol. 27, No. 3, pp. 171-177, 1991 Printed in Great Britain

ACTIVITY RHYTHMS LINKED WITH FORAGING BEHAVIOUR IN INSECTICIDE-RESISTANT AND SUSCEPTIBLE STRAINS OF OR YZAEPHILUS SURINAMENSIS (COLEOPTERA: SILVANIDAE) CHRISTOPHER H. BELL ADAS Slough Laboratory, Ministry of Agriculture, Fisheries and Food, London Road, Slough SL3 7HJ, Berkshire, England (Received /or publication 14 March 1991)

Abstract-Cycles of locomotory activity of adults of Oryzuephilus surinumensis (L.) were examined under different lighting conditions in an open arena containing a refuge surrounded by an outer ring of split wheat grains. Three strains were tested, two of which were resistant to malathion, one specifically to malathion and fenitrothion and the other resistant to a number of other compounds including pirimiphos-methyl, chlorpyrifos-methyl, etrimphos and fenitrothion. This latter strain shared a common pattern of activity with the third strain which was susceptible to malathion, the number of beetles in the arena being controlled by an endogenous oscillator entrained primarily by “lights off’ with a lesser contribution from “lights on”. The number rose to a peak 4-8 hr after the start of the scotophase with minimum activity occurring 8-l 1hr after the start of the photophase, and this cycle persisted in continual darkness (DD). In the strain resistant to malathion and fenitrothion, the rhythm was of low amplitude in lightdark cycles but, with some loss of distinction, did persist in DD. Activity tended to increase throughout the photophase to peak at the onset of darkness after which it declined. In 24 hr cycles, with photophases of 12 hr or less, minimum activity was recorded towards the end of the scotophase. There was thus no obvious link between resistance and the nature of the activity rhythm.

INTRODUCTION

Many features of the physiology, growth and behaviour of animals show a circadian periodicity. The success of an organism within a dynamic ecosystem depends greatly on the fine adjustment of such rhythms to fluctuations in the environment. The group of insects which are associated with stored products are renowned for their cosmopolitan distribution, travelling with produce across international boundaries from one type of store to another, and exploiting situations where quantities of food are gathered together in one place, often quite independently of local climate. Many of the insects which infest grain bulks are of tropical origin. The saw-toothed grain beetle Oryzaephilus surinamensis (L.) (Col., Silvanidae), currently the most serious pest of grain stored in the U.K., is one such example. The adult is a small active beetle which although winged, rarely flies. A locomotory rhythm associated with foraging behaviour was recently observed in this species (Bell and Kerslake, 1986). The activity and wandering behaviour of insects has long been known to contribute to the success of treatment of surfaces with insecticide. It has been shown in Tribolium castaneum (Herbst) that biochemical resistance to a chemical can be accompanied by behavioural changes such as a positive avoidance of a treated surface (Pinniger and Wildey, 1979; Wildey, 1984). While such avoidance or refuge-seeking behaviour in the presence of an insecticide may bear no relation to the individual’s normal level of activity or wandering behaviour, the retention of a circadian rhythm of activity may tend to reduce the protection conferred by the refuge by encouraging excursions on the treated surface at particular times of day. In the current experiments, the foraging rhythm observed in an insecticide-susceptible strain of 0. surinamensis (Bell and Kerslake, 1986) was compared with that in two other strains of known resistance status to investigate how widespread the rhythm was in this species. 171

172

CHRISTOPHER

H. BELL

METHODS

Insect stocks Three strains of 0. surinamensis were reared for the experimental programme. One of these, obtained from a shipment of Iranian sultanas in August 1980, was the malathion susceptible strain in which a circadian rhythm of activity was first demonstrated (Bell and Kerslake, 1986). A second strain (MSR) had been collected from a warehouse in Bradford in December 1979 and showed resistance to malathion in a filter paper discriminatory dose test (Anon., 1974). Further tests conducted concurrently with the present programme (Muggleton, pers. commun.) revealed that whereas resistance was also evident to fenitrothion ( x 29 compared to x 14 for malathion) there was little resistance to a wide range of other compounds including chlorpyrifos-methyl, etrimfos, pirimiphos-methyl and permethrin. This strain was reared with the Iranian susceptible (S) strain in a 9 hr light, 15 hr dark cycle (LD 9: 15) at 25°C 65% r.h. from 1982, but for the previous 2 yr was reared in an unlit room at 25°C. From 1984 after completion of the earlier experiments in LD 9:15, the two stocks were reared under LD 12: 12 in preparation for the present programme. The third strain was collected from a farm in North Yorkshire in October 1984 and a line was bred for the current programme from 1985 at 25°C 65% r.h. in LD 12: 12. This strain (HR) proved resistant to malathion and all the compounds listed above in filter paper discriminatory dose tests. To establish each culture, about 100 adults of mixed age and sex were added to a prepared 750 ml glass jar containing 100 g of rolled oats and with a coating of “fluon” suspension on the upper part of the inside to prevent beetles scaling the sides. The culture was sealed by waxing a sporeproof glass fibre paper disc to the rim. Further details of rearing are given by Bell and Kerslake (1986). Emergence in cultures commenced at about 6 weeks but only cultures aged between 10 and 18 weeks of age were used to provide adults for tests. Activity tests Activity rhythms were investigated under three different 24 hr-phase light/dark (LD) cycles (LD 9 : 15, 12: 12 and 15 : 9), and in an ensuing period of continual darkness (DD). The intensity of illumination during the light phase, measured using a photographic light meter, was 800-1000 lx. Operations in darkness were performed in the dim red light provided by a filter-fitted fluorescent tube (wavelength 590 nm or higher). Prior to each investigation at a new photoperiod, culture lines were acclimatized to the experimental light cycle for at least 10 weeks. Tests were conducted in arenas comprising a 20 cm dia aluminium ring coated on the inside with “fluon” suspension and sealed to a 25 cm dia white filter paper with a quick drying cellulose-based sealant (Pinniger, 1974). The filter paper and ring were mounted on a 30 cm2 polystyrene tile for support. An artificial refuge comprising a metal tube 75 x 25 mm internal dia fitted with two 2 mm aperture gauze mesh stoppers was placed centrally in each area. The rim of each stopper had a flat region to prevent the refuge rolling across the arena. In the first series of tests, which was conducted on the MSR and S strains in LD 12 : 12, the refuge was empty, but in all subsequent tests a 5 x 5 cm muslin square was folded inside to provide a resting place for beetles. To stimulate foraging behaviour, about 20 wheat grains which had been split in a mortar were placed in a ring around the refuge. For each strain entrained to a particular photoperiod, 50 adults of mixed age and sex from a 10 to 18 week old culture were placed in each of three arenas the day before it was intended to start logging insect wandering. The first experimental run under each light cycle started from “lights out” at the beginning of the working day. In dim red light, the number of beetles in the arena outside the refuge was assessed at hourly intervals for 9 consecutive hours on this and the next 2 working days. To cover just over three full cycles (at least 73 hr from first day “lights out”), eight such experimental runs were performed. Between each experimental run, the time switch controlling the lighting was adjusted to delay the time for “lights out” by 3 hr, and cultures containing beetles for the next test were acclimatized to the new cycle for at least 10 days before setting up in arenas. Beginning at the new “lights out” time, the numbers of insects in the arena were counted as before until after “lights out” 3 days later. The time for “lights out” eventually moved beyond the period spanned by the working day. Readings were then started at the beginning of the next day. Following each run in LD, further adults from the cultures acclimatized to the time adjustment were set up in fresh arenas and the time switch was adjusted so that after the next “lights out”

Foraging behaviour of Oryzaephilus surinamensis

173

lights were not switched on again. Readings were taken as in LD cycles to observe whether the rhythm persisted in DD. After this time of continual darkness, the light cycle was resumed to start acclimatization to the next “lights out” time. If in a test run mortality in any arena rose to 10% by the third day, the run was repeated. Assessment of results Because it was recognised that there was potential for the baseline level of activity in different batches of insects to vary, for each strain, the 8 experimental runs of 3 working days (or 2 days plus 2 part days) with hourly readings for a particular photoperiod were brought together and examined for consistency. Each hour of the 73 hr observation period was represented by 3 readings taken in different runs. If anomalies were evident between the readings taken in a particular run and those for the same hours of the cycle taken in others, the run was repeated with a fresh batch of conditioned insects and an average of these two sets was used for the calculation of hourly means with the other readings. When each set of results was complete, the mean values of hourly readings were used to calculate three-point averages for each hour from the start of the cycle. From these, plots of activity against time were prepared for each strain and light condition.

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Fig. 1. Wandering activity of adults of three strains of 0. surinamensis in arenas held in LD 12: 12 or in an ensuing 72 hr period of darkness (DD). A: unbroken line HR strain and broken line S strain (empty refuge) in light cycle. B: MSR strain, unbroken line muslin in refuge, broken line empty refuge, in light cycle. C: as A but in DD following conditioning in LD 12: 12. D: as B but in DD following conditioning in LD 12:12.

CHRISTOPHER H. BELL

174

When the pattern of wandering activity appeared indistinct or was only weakly defined, in order to identify the presence or absence of a daily cycle, each day was divided into four 6-hr blocks, for which the mean activity level was calculated. An analysis of variance of the activities for blocks and days was then performed.

RESULTS Activity in LD 12:12 In the first run at this photoperiod, with an empty refuge, a strongly cycling rhythm which persisted in DD was obtained for the S strain [Fig. l(C), dotted lines]. Peak activity was recorded 6-8 hr after the start of the scotophase while minimum activity was evident towards the end of the photophase. The mean number of beetles in the arena was about 23 during the LD cycle and about 25 in DD. Results for the MSR strain in this run was less rhythmic than for the S strain, especially in DD [Fig. l(B) and (D), dotted lines]. Nevertheless, analyses of variance (Table 1) indicated differences between blocks aligned in days to be highly significant. Activity increased sharply at “lights on” and tended to decline throughout the scotophase, giving a strong visual impression of a cycle running in antiphase to that of the S strain [Fig. l(A) and (B)]. In a repeat run at this photoperiod with a piece of muslin folded into the refuge, the mean number of beetles in the arenas fell from 30 to 23/24 and there was even less variation throughout the cycle, peaks and troughs being separated by only 4 or 5 beetles [solid line in Fig. l(B) and (D)]. However, again in both LD and DD the cycles were significant (Table 1). The HR strain was included in this run and showed a very pronounced rhythm similar to that obtained for the S strain but peaking only 4-5 hr after the start of the scotophase [Fig. l(A) and (C), dotted lines]. The mean number of beetles wandering in the arena was higher than in other strains, averaging 29 in the LD cycle and 28 in DD, and a larger difference was evident between minimum and peak activity.

Table I. Analysis of results for MSR strain acclimated in LD 12: 12, (null hypothesis: no cycle of activity exists) Condition of refuge in arenas

Light conditions following LD 12:12

Day from start at the beginning of a scotophase

Empty

LD 12:12

1 2 3 I 2 3 I 2 3 1 2 3

DD

With folded muslin

LD 12:12

DD

Analysis of variance Empty refuge

LD 12:12

DD

Total Days Blocks Error Total Days Blocks Error

Muslin in refuge

LD 12:12

DD

Total Days Blocks Error Total Days Blocks Error

Mean numbers of beetles wandering in arena from 6 consecutive hourly readings Block of hours of each day: 1-6 7-12 13-18 1924 30.2 30.6 29.8 32.7 32.1 31.0 23.9 24.5 24.4 25.6 26.3 25.0

27.5 27.4 28.4 29.1 29.4 29.1 21.6 22.2 21.7 23.1 24.2 24.9

31.1 30.0 29.8 30.2 29.9 29.9 24.0 23.5 23.0 23.1 24.5 24.5

Adjusted SOS

Mean square

ratio

21.72 0.84 18.31 2.57 14.92 0.53 13.35 1.04 12.01 0.36 10.85 0.80 18.16 2.21 13.53 2.42

F

31.9 30.2 31.0 31.2 31.1 31.2 24. I 24.2 23.1 22.4 23.3 22.3 P VdW

0.42 6.10 0.43

0.98 14.22

Not sig.
0.27 4.45 0.17

1.55 25.58

Not sig. to.001

0.18 3.62 0.13

1.39 27.14

Not sig. to.001

1.10 4.51 0.40

2.74 11.19

Not sig. to.01

Foraging behaviour of Oryzaephilus surhamensis

175

Activity in LD 9:lS and 15:9 The S strain yielded a strongly rhythmic pattern in both photoperiods which persisted in DD (Figs 2 and 3). Peaks of activity occurred 6-7 hr after the start of the scotophase in LD 9: 15, as in LD 12: 12, but in LD 15:9 they occurred 3 hr earlier. From “lights on” in this photoperiod activity dropped sharply to reach a minimum about 10 hr later [Fig. 3(A)]. The cycle in LD 15 : 9 was of greater amplitude than in other photoperiods, while the general level of activity was lower with a mean of only 13 beetles in the arena during the period observed, compared with 15 in LD 9 : 15 and 23 with the empty refuge in LD 12: 12. Results for the MSR strain in LD 9: 15 and 15:9 gave a somewhat clearer rhythm than in LD 12: 12 [Figs 2(B) and 3(B)] but again the rhythm, though statistically significant (Table 2), was not well defined in DD with some evidence of delay in reaching peak activity [Figs 2(D) and 3(D)]. The mean number of beetles in the arena increased with photoperiod, reaching about 27 in LD 15:9 compared with only 20 in LD 9: 15. In all experiments mortality was low. DISCUSSION An activity rhythm controlled by a circadian clock has been shown to be present in three different strains of 0. surinamensis, though the rhythm was shallow in a strain with a resistance specific to malathion and fenitrothion, which showed a peak of activity towards the end of the photophase. In the other two strains tested, the endogenous oscillator was entrained primarily by “lights out”. In short photoperiods (LD 9: 15 or LD 12: 12) the activity peak of the S strain occurred 4-8 hr after the start of the scotophase, which was a little later than in earlier tests (Bell and Kerslake, A 18

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Fig. 2. Wandering activity of adults of 0. surinaI5 or in an ensuing 72 hr period of darkness (DD). A: S strain in LD 9:15. B: MSR strain in LD 9:15. C: S strain in DD after conditioning in LD 9: 15. D: MSR strain in DD after conditioning in LD 9:15. mensis in arenas held in LD 9:

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Fig. 3. Wandering activity of adults of 0. surinamends in arenas held in LD 15 : 9 or in an ensuing 72 hr period of darkness (DD). A: S strain in LD 15: 9. B: MSR strain in LD 15:9. C: S strain in DD after conditioning in LD 15:9. D: MSR strain in DD after conditioning in LD 15:9.

CHRISTOPHERH.BELL

176

Table 2. Analysis of results for the MSR strain acclimated in LD 9: 15, or LD I5 :9 (null hypothesis: no cycle of activity exists)

Photoperiod of acclimation LD9:15

Light conditions following acclimation LD9:15

DD

LD IS:9

LD 15:9

DD

Day from start at the beginning of a scotophase

1 2 3 I 2 3 I 2 3

I 2 3

Analysis of variance LD9:15

LD 9: 15

DD

LD 15:9

LD 15:9

DD

Mean numbers of beetles wandering in arena from 6 consecutive hourly readings Block of hours of each day: 14 7-12 13-18 19-24 22.2 21.1 20.5 21.4 22.2 22. I 28.8 28.9 21.5 26.8 29.6 27.3

19.1 19.4 17.9 20.5 21.1 20.8 26.0 25.2 23.8 24.8 26.9 25.5

20.6 19.5 19.7 20.6 18.9 18.3 25.4 25.4 25.1 .6 28.9 26.5

Adjusted SOS

Mean square

F

P

ratio

value

1.54 4.54

8.56 25.22

<0.05
22.1 21.3 20.8 19.3 19.3 I a.4 27.8 27.1 28.5 27.9 30.2 28.3

Total Days Blocks Error

17.77 3.07 13.62 1.oa

Total Days

20.25 0.71

0.35

0.67

Not sig.

Blocks Error

16.43 3.11

5.48 0.52

10.53


Total Days

32.64 1.17

0.58

1.18

Blocks Error

28.55 2.92

9.52 0.49

19.43

-Co.01

Total Days

31.17 15.28

7.64

84.0


Blocks Error

15.35 0.54

5.12 0.09

56.9


0.18

Not sig.

1986). The rhythm obtained in the present tests was of greater amplitude although the mean number of beetles in the arena was lower in LD 9 : 15, probably as a result of including folded muslin in the refuge. In long photoperiods (Fig. 3), “lights on” also played a major role in controlling the rhythm in the S strain, activity increasing sharply from the tenth hour of the photophase. Again, the rhythm of activity was better established than in earlier work with an empty refuge, with deeper troughs and higher peaks. In many ways the storage environment is buffered from daily changes. Temperatures within grain bulks change little from month to month in the absence of aeration, humidity is constant and access to light is severely restricted. In such circumstances the retention of a functional circadian clock may be of limited value. Many species living in habitats with little environmental periodicity, such as cave dwellers, have apparently lost a circadian rhythm of activity (Lamprecht and Weber, 1977; Saunders, 1982). It is likely that laboratory rearing in constant darkness (or light) will also result in the loss of rhythms. Of the three strains tested here, it is noteworthy that whereas two (S and HR) were reared in LD cycles from well within their first year at the laboratory, the third (MSR) spent over 2 yr in DD, apart from periods of illumination when people entered the rearing room. This latter strain displayed a somewhat weaker endogenous rhythm. There was no evidence of any link between the presence of a well defined circadian rhythm and the resistance status of the strain. In summary, the circadian rhythm here examined, based on foraging behaviour (Bell and Kerslake, 1986), was obvious in two strains, but in a third displayed some typical hourglass characteristics (Lees, 1968) with activity simply increasing in light and decreasing in dark. However, some persistence of the rhythm in DD indicated the presence of an endogenous oscillator in this strain as in the others. With the possibility of such rhythms being out of phase, differences observed in the activity levels of different strains may vary with the time of day observations are made. The extent of the difference also depends on the general level of activity of the strain. Thus with the HR strain, which showed a higher tendency to wander in the arenas than the MSR strain, maximal differences will be expected during the scotophase when HR activity is at its height (Fig. l), while up to three times as many MSR beetles may be seen in the arena during the photophase in comparisons with the S strain (Figs 2 and 3).

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Such gross differences offer a possible explanation why results obtained for laboratory toxicity and other tests performed at different times of day may vary, although the behaviour of insects will also be altered by the presence of insecticide (Pinniger and Wildey, 1979). Also raised is the problem of comparative tests between strains. Currently, most tests for insecticide resistance depend on uptake of chemical from a treated surface, which in turn depends on activity of the insect. Clearly, strains with different activity cycles may give rise to misleading results if subjected to short exposures to insecticide starting at the same time of day. It is also apparent that care is needed in defining what constitutes “activity”. In the present tests, all of which were conducted in non-toxic arenas, both insecticide resistant strains yielded higher numbers of beetles wandering outside the refuge than the susceptible strain, but there is no way of correlating this effect with either the rate of frequency of movement of individuals in their natural environment, or with the proportion of individuals likely to survive exposure to an insecticide-treated surface. Acknowledgemenfs-The author is indebted to Mr M. Ledson, Mr M. Hutson, Mr J. G. Foster and Mr D. J. McGreevy who participated in this work while students at Liverpool Polytechnic, Byron Street, Liverpool. REFERENCES Anon. (1974) Recommended methods for the detection and measurement of resistance of agricultural pests to pesticides. Tentative method for adults of some major beette pests of stored cereals with malathion or Iindane. FAO method No. 15. F.A.O. Pl. Prot. Bull. 22, 127-137.

Bell C. H. and Kerslake P. R. (1986) A circadian rhythm influencing foraging behaviour in the saw-toothed grain beetle Oryzaephilus surinamensis. Physiol. Ent. 11, l-6. Lamprecht G. and Weber F. (1977) Die Lichtempfindlichkeit der Circadianen rhythmik dreier Hohlenkafer arten der Gattung Laemostenus. J. Insect Physiol. 23, 44-452. Lees A. D. (1968) Photoperiodism in insects. In Photophysiology (Edited by Giese A. C.), Vol. IV, pp. 47-137. Academic Press, New York. Pinniger D. B. (1974) A laboratory simulation of residual populations of stored product pests and an assessment of their susceptibility to a contact insecticide. J. srored Prod. Res. 10, 217-223. Pinniger D. B. and Wildey K. B (1979) Stored product insect behaviour as a factor in control and treatment assessment. Proc. 5th Brit. Pest Cont. ConjI, Stratford-upon-Avon 1979, Session 3, Paper 7, Spp. Saunders D. S. (1982) Insecf Clocks, 2nd edn. Pergamon Press, Oxford. Wildey K. B. (1984) Studies on the effects of repellency on the efficiency of residual insecticides against resistant and susceptible strains of Tribolium castaneum. Ph.D. thesis, University of Reading.