Resistance of the grain mite Acarus siro L. (Acarina, Acaridae) to unfavourable physical conditions beyond the limits of its development

Agriculture, Ecosystems and Environment, 11 ( 1 9 8 4 ) 3 1 9 - . 3 3 9 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

319

R E S I S T A N C E O F T H E G R A I N M I T E A C A R US S I R O L. ( A C A R I N A , ACARIDAE) TO UNFAVOURABLE PHYSICAL CONDITIONS BEYOND THE LIMITS OF ITS DEVELOPMENT

A.M. CUNNINGTON

Ministry of Agriculture, Fisheries and Food, Slough Laboratory, Slough, Berks. SL3 7HJ (Gt. Britain) (Accepted for publication 27 March 1984)

ABSTRACT Cunnington, A.M., 1984. Resistance of the grain mite Acarus siro L. (Acarina, Acaridae) to unfavourable physical conditions beyond the limits of its development, Agric. Ecosystems Environ., 11: 319--339. The resistance of the grain mite Acarus siro to unfavourable physical conditions was studied to determine how long some members of a population might survive so that development and increase could continue if conditions became favourable. Preliminary experiments showed that: (i) the eggs formed the most resistant stage, both to extreme temperatures and to desiccation; (ii) the most resistant eggs were those in an early stage of development, and that resistance declined with age; and (iii) environmental conditions prior to oviposition influenced resistance, resistance being greater in eggs from populations reared at near threshold conditions than in those reared at ecologically more favourable temperatures and humidities. Experiments with eggs exposed to lethal high and low temperatures and to both favourable and desiccating humidities show that above 30°C and below 0 ° C, relative humidity ceased to affect survival, the eggs being killed by heat or cold before the effects of desiccation became important. Eggs were fairly susceptible to high temperatures; none survived longer than 10 h at 35°C or 35 rain at 40°C. They showed greater tolerance to low temperatures, surviving up to a maximum of 12--14 days at - 1 0 ° C and 24--26 days at -5°C. At 0°C, the maximum survival period varied from 80 days at 20% RH to 180 days at 90% RH. For eggs exposed at temperatures within the developmental temperature range to relative humidities from 20 to 50%, survival appeared to be directly related to humidity and inversely to temperature, maximum survival periods ranging from 20 days (20% RH) to 45 days (50% RH) at 5°C, and from 1 day (20% RH) to 3 days (50%RH) at 30°C.

INTRODUCTION In an earlier paper (Cunnington, 1965) the physical limits for complete d e v e l o p m e n t o f A c a r u s siro w e r e d e f i n e d a n d its d i s t r i b u t i o n a n d s t a t u s as a p e s t o f s t o r e d g r a i n , in r e l a t i o n t o t h e s e l i m i t s , w e r e d i s c u s s e d . Beyond the limits of development, different stages of the mite can survive

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320 exposure to unfavourable conditions for various limited periods of time. Because resistance of the animal to unfavourable physical conditions has an important bearing on its survival and distribution, it seemed desirable to explore more closely the resistance of the mite to unfavourable conditions beyond these limits. In this context, the chief point of interest is how long some members of a population might survive so that development and increase could continue if conditions became favourable. This study was therefore planned to secure estimates of the resistance of the most hardy stage in the life cycle of A. siro to combinations of temperature and relative humidity (RH) that were either slowly or rapidly lethal to the animal when it was exposed to them. EXPERIMENTAL METHODS The methods used both for the mass culture o f Acarus siro and for the micro-culture of this species, the latter in order to study isolated individuals, eggs or small groups of mites, have been fully described elsewhere (Solomon and Cunnington, 1964; Cunnington, 1965) and need only briefly be stated here. Single pairs or small groups o f mites were reared on wheat germ flakes in micro-cells under controlled conditions o f temperature and humidity. These mites were used to provide the eggs and other stages required for experimental use. Different sizes of rearing cell were used depending upon the needs of particular experiments or cultures. Parent mites used to initiate these populations were taken from laboratory cultures of A. siro similarly reared on wheat germ flakes in small glass flasks tightly plugged with nonabsorbent c o t t o n wool maintained at 17.5°C and 75% RH. Experimental temperatures ranged from - 1 0 ° C to +40°C. For zero and sub-zero temperatures a domestic refrigerator and small deep freeze unit were used. Desiccators held at these temperatures were fitted with thermocouples connected to a temperature recorder, and with low temperature mercury thermometers used to check the recorder readings. For temperatures from 5 to 15 ° C, domestic refrigerators were used. These were modified by incorporating more sensitive thermostats and internal fans to increase air circulation within the cabinets. Experiments at 20, 25 and 30°C were carried out in constant temperature rooms. For experiments above 30°C, a water bath was used. A metal frame inside the bath held a flat-topped screw-cap glass jar, approximately 9.5 cm in diameter and 9.0 cm deep, immersed to within 1 cm of its upper surface. The metal cap supported a small t h e r m o m e t e r to record temperatures inside the jar and a rubber stopper from the b o t t o m of which micro-cells or other vessels containing eggs or mites were suspended on thin wire; a glass capillary tube was also fitted to allow for air pressure adjustments. Relative h u m i d i t y inside jars and desiccators was controlled by solutions of potassium hydroxide or sulphuric acid (Solomon, 1951}.

321

Temperatures in the water bath were controlled to within + 0.1°C. That inside the desiccator at - 1 0 ° C was virtually constant throughout the duration of the experiments, no detectable variations being recorded. During the short period of disturbance at the beginning and end of each experiment, caused b y the insertion or withdrawal of mites from the desiccator, the temperature rose slightly but never exceeded a maximum of -8.8°C; the experimental temperature was quickly regained when both desiccator and cabinet were closed. Control was less precise at other temperatures but the range of fluctuation was rarely more than + 10C and often did not exceed + 0.5 ° C. Acarus siro typically passes through an egg, larval and two nymphal stages before becoming adult. A few individuals may pass through an extra instar -- the h y p o p u s -- between the first nymphal and penultimate stages of the life cycle. Both larval and nymphal stages undergo a resting phase before entering the next active stage. Although Jones (1950), working with some related species of Acaridae, found that the separation of the old cuticle from the epidermis and new cuticle occurred at the beginning of each resting phase and that the latter was properly part of the succeeding active phase, I have followed the older convention of referring these resting phases to the preceding active stage and of describing them as the resting phase of this stage, e.g. resting larva and not pre-protonymph. In this paper, the various stages in the life cycle and the symbols designating them are described as: egg (E), active larva (AL), resting larva (RL), active p r o t o n y m p h (AN1), resting p r o t o n y m p h (RN1), active tritonymph (AN3), resting tritonymph (RN3), and adult (A). Resistance to lethal conditions

The first step was to determine which stage in the life cycle of A. siro was the most resistant to adverse physical conditions or whether different stages were most resistant according to conditions. Some earlier workers (Schulze, 1923; Kozulina, 1940; Ushatinskaya, 1954) had indicated that the eggs and particularly hypopi of a number of Acaroid mites were more resistant to unfavourable physical conditions than were other stages of the life cycle. To confirm these observations, in so far as they applied to A. siro, and to secure more precise experimental evidence, a series of preliminary experiments was carried o u t to determine resistance of the different stages to heat and cold, by exposing them to temperatures of 35°C and - 1 0 ° C at 90% RH, and to dryness by exposing them to a relative humidity of 50% at 20 ° C. The h y p o p u s of A. siro was not tested either in these or in later experiments. It occurs infrequently in stored product habitats and attempts to rear it in the laboratory under a wide range of physical conditions and on a variety of foodstuffs during the present study were unsuccessful. Similar failures to rear it experimentally have been recorded by Polezhaev (1940)

322

and Boczek (1957). Griffiths (1966) described two strains of A. siro, one of which produced a few hypopi while the other failed to produce hypopi under any o f the experimental conditions tested. He concluded that A. siro had a poor potential for hypopus formation and suggested that many of the published records of the occurrence and distribution of the A. siro h y p o p u s were based on misidentifications of the field species A. farris Ouds.

Preliminary studies to determine the stage most resistant to heat, cold and dryness To provide the numbers of eggs and other stages required for these experiments, several micro-cultures of A. siro were set up at 10 and 30°C at 90% RH, and at 20 ° C at 70% RH. Each culture was initiated with known-age eggs laid by adults maintained under similar conditions to the experimental groups. As soon as sufficient mites reached the required stage o f developTABLE I

Proportion of adult, egg and juvenile stages surviving exposure to: (a) 35°C, 90% RH, for 3 h (b) -10°C, 50% RH, for 24 h (c) 20°C, 50% RH, for 24--48 h Stage

Exposure

(a) No. exposed A

152

(b) Per cent survival 6.6

(c)

No. exposed

Per cent survival

No. exposed

Period (h)

Per cent survival

100

17.0

50 50 -50

24 48 -24

nil -nil

66 50 40 36 43 42 46 50 55 55

24 48 24 48 24 48 24 48 24 48

77.3 4.0 12.5 5.6 88.4 2.4 8.7 10.0 100.0 43.6

E AL

356 320

34.0 nil

435 100

57.7 28.0

RL

292

nil

213

7.0

AN1

280

1.4

196

26.5

RN1

312

1.3

210

11.0

AN 3

236

15.7

176

35.2

RN~

271

1.5

200

8.5

8.0

323

ment, they were removed from the culture and exposed in groups of 20--100 as follows: (a) mites from cultures maintained at 30 °C, 90% RH, to 35°C, 90% RH, f o r 3 h; (b) mites from cultures maintained at 10°C, 90% RH, t o - 1 0 ° C , 90% RH, for 24 h; and (c) mites from cultures maintained at 20 ° C, 70% RH, to 20 ° C, 50% RH, for 24--48 h with eggs exposed from 1 to 10 days. All eggs were 0--24 h old when used and were exposed in batches of 50--100. Immediately after exposure, the different groups o f mites or eggs were examined and then returned to their respective culture conditions for further observation. Post-oval stages were examined after 24 h and again, if necessary, after 36 and 48 h. Resting stages recorded alive were those which completed ecdysis after exposure. Observations on eggs were continued until hatching was complete. The results (Table I) and Fig. 1 are summarized below. 100

80

~ 60 \ \\\ u

\

\

40

\\\

20

0

@----~D

o

L 2

i 4 Exposure period

I 6 (days)

I 8

I lO

Fig. i. Determination of the most resistant stage: resistance of A. siro eggs to desiccation.

Resistance to heat. Eggs, 34% of which hatched after incubation at 30°C and 90% RH, showed a far greater resistance to heat than any other stage in the life cycle of A. siro. Larvae were the most susceptible of all the postoval stages and all were killed by the treatment. Active and resting protonymphs and resting tritonymphs were only slightly less susceptible with survival rates of 1.5% or less, while adults, 6.6% o f which survived, were slightly more resistant. Active tritonymphs were the most resistant of all the post-oval stages with a survival rate o f nearly 16%.

324 Resistance to cold. Proportions of all stages survived exposure to -10°C. Eggs were again found to be most resistant; 57.7% hatched after incubation at 10 ° C, 90% RH, compared with a survival rate of 35.2% for active tritonymphs which were again found to be the most resistant of the post-oval stages. Active larvae, which were most susceptible to heat, were among the more resistant groups when exposed to cold and 28% survived exposure to the experimental conditions. Active larvae, p r o t o n y m p h s and tritonymphs appeared to be more resistant than their corresponding resting phases. This was particularly noticeable among larval and tritonymphal groups, where the ratio of survival o f active to resting stage was 28.0--7.0% for larvae and 35.2--8.5% for tritonymphs. Adults, with a survival rate of 17%, appeared to o c c u p y an intermediate position in order of cold resistance. Resistance to dryness. In these experiments, active larvae were exposed for 24 h, with observations on some o f them being made after 31/~, 7 and 22 h. Nymphs and adults were exposed for 24 and 48 h. Eggs (0--24 h old) were exposed in groups of 50 for periods from 1--10 days. Active larvae were the least resistant of all stages and none survived 24 h exposure; some were dead after 7 h. A majority of active p r o t o n y m p h s and tritonymphs were also dead after 24 h. The survivors were all individuals approaching the resting phase, easily distinguishable by their characteristically swollen appearance. Only 6% o f p r o t o n y m p h s and 10% of tritonymphs, all of which underwent final ecdysis during the exposure period, survived 48 h. Newly emerged adults were equally susceptible and 46 o u t of 50 were either dead or dying after 24 h. Resting nymphs and larvae were virtually unaffected by 24 h exposure, but when exposed for 48 h invariably completed ecdysis during this period, the newly emerged active stages dying quickly unless transferred to 70% RH or above. Eggs were far more resistant: more than 60% were still alive after 3 days and a small proportion survived exposure for 7 days (Fig. 1). Preliminary studies having thus established that the egg was the stage most resistant to heat, cold and desiccation, a comprehensive series of experiments was planned to determine the rates o f survival o f A. siro eggs at a number of lethal high and low temperatures, respectively just above and below the upper and lower developmental temperature limits for the species, and to a range of lethal low relative humidities below the minimum level for development of A. siro at each of a number of favourable temperatures. Before carrying o u t these experiments, however, it was necessary to determine whether the resistance of the egg to unfavourable physical conditions was affected b y its age at the time o f exposure, and also to investigate whether such resistance was influenced by the environmental conditions at oviposition prior to exposure. Hora (1934), working with Glycyphagus domesticus de Geer., found that eggs in an early stage of development were more resistant to dry conditions than those at a later stage, while the results of some earlier laboratory studies of A. siro indicated that environmental conditions

325 during rearing had some effect on the subsequent biological performance of the species when exposed to a different physical environment. EXPERIMENTS WITH EGGS

Factors affecting resistance Effect of age at time o f exposure. To determine whether age and resistance to unfavourable physical conditions were related, groups o f 0--1-day-old eggs were removed from cultures maintained at 10, 20 and 30 °C, 90% RH, and arranged in groups of 50--100 in micro-cells in which they were allowed to develop under the same conditions as their respective parent adults for periods up to 10 days, depending upon the mean oviposition period at each temperature, before being exposed as follows: (a) eggs reared at 30°C, 90% RH, to 35°C, 90% RH, for 3 h; (b) eggs reared at 10°C, 90% RH, to - 1 0 ° C , 90% RH, for 48 h; and (c) eggs reared at 20°C, 90% RH, to 20°C, 50% RH, for 24--48 h. After exposure, each group of eggs was returned to its original culture conditions and observed for hatching. The results, each point based on 50, 100 or 250 eggs and corrected for control mortality, are summarized in Table II and Fig. 2. They show that where, as at 10 and 20°C, 90% RH, the incubation period of the egg permitted several age groups to be examined, eggs less than 24 h old were considerably more resistant to cold or to dryness than were older eggs, and that resistance declined rapidly with age. This effect was less marked when eggs reared at 30 ° C, 90% RH, were exposed to heat. Under these conditions (i.e. 30°C, 90% RH), the incubation period of the egg is short (mean 3.5 days) which, since observations were made daily, did not permit more than three age groups -- mean ages: (i) 0.5 days, (ii) 1.5 days, and (iii) 2.5 days -- to be examined. Although fewer eggs hatched in group 2 than in group 1, this trend was obscured in group 3 where exposure to heat of eggs already in an advanced stage of development appeared to accelerate eclosion, resulting in the hatching or attempted hatching of several eggs during the exposure period and in the rapid death of emerging T A B L E II Effect of age of the egg on resistance to heat (35°C, 90% RH, for 3 h). Each point based on 250 eggs Mean age (days

Per cent hatch a

0.5 1.5 2.5

27.0 16.4 28.3

aCorrected for control mortality.

326 larvae. With such a short incubation period, the age differences between groups were t o o large to show the relationship between age and resistance clearly. Nevertheless, the results do not conflict with the general conclusion that the most resistant eggs were those in an early stage o f development and that resistance declined with age. 100

8O

60 •

U ~40 20

~

~

0

Resistance Io cold

es,santeoescica ,on

~ 0

~ - e

I

J

I

I

I

2

4

6

8

IO

Mean age of eggs (days) at time of exposure

Fig. 2. Effect of age of the egg on resistance to cold and desiccation.

Effect o f environmental relative humidity on egg viability and resistance to desiccation. Experiments were planned to investigate the possibility that the environmental humidity at oviposition had some bearing on the subsequent viability of eggs incubated at different humidities after oviposition. Adult mites were bred at 20°C, 90% RH, and their eggs removed every 24 h. Groups of 100 eggs were exposed to a series of relative humidities ranging from 60 to 80% in steps o f 2% % and from 80 to 100% in steps o f 5%. They were examined periodically to determine the percentage hatch at each humidity (Fig. 3 -- transferred eggs). In further experiments, the viability of eggs laid at 62.5, 65, 70, 80 and 90% RH, 20°C, by adults reared at these humidities, was also determined (Fig. 3 -- conditioned eggs). The numbers of eggs exposed at each humidity varied with each experiment, the minimum number being approximately 100. The results show some striking differences. Transfer of eggs from 90% RH to higher humidities or to lower humidities down to 77.5% RH had no significant effect on the percentage o f eggs hatching. Transfer to lower humidities in the 62.5--75% RH range severely reduced the viability of the eggs, the reduction being highly significant (P < 0.01) even for those at 75% RH. Eggs bred at 62.5--70% RH showed a much greater viability at these humidities than did those transferred from 90% RH. These results indicated that within certain limits the environmental

327

relative humidity at the time of oviposition affected the subsequent percentage hatch and furthermore that eggs from females reared at low relative humidities close to the developmental limits for the species were likely to be more resistant to desiccation than those from females reared at the higher more favourable relative humidities. This view was supported by a further experiment in which day-old eggs from adults breeding at 65% and 90% RH were exposed to drying at 50% RH for periods o f 1--5 days, after which the eggs were returned to their original culture conditions for hatching. The 100

80

.c

o -r c

6O

6

20

0

~

A

60

70 Relative

Fig. 3.

80 Humidity(%)

I 100

910

Effect o f environmental relative humidity on egg viability.

100

75

• •

~

65°/° RH

o

•

~ 50

a_

25

0

O

I

I

I

1

2

3

4

5

Exposure period (days) Fig. 4. Effect of environmental relative humidity on the resiAtance of eggs to desiccation.

328

results, each point based on 50 eggs, are shown in Fig. 4. Eggs bred at 65% RH were more resistant than eggs bred at 90% RH, showing considerably greater viability in the groups exposed for 3 days (P < 0.05), 4 days (P < 0.01) and 5 days (P < 0.01). Effect o f environmental temperature on resistance to heat and cold. Preliminary studies indicated that the resistance of A. siro to heat was, to some extent, dependent on its previous thermal history. These experiments, however, did not include the egg, which this investigation shows is the most resistant stage in the life cycle of the grain mite to unfavourable physical conditions. To remedy this omission and to determine the effect of environmental temperature on the resistance o f A. siro eggs to both heat and cold, several micro-cultures were set up at 90% RH at 10, 15, 20, 25 and 30°C. From each of these cultures, numerous newly-emerged mating adult pairs were transferred to fresh cells from which the eggs were removed daily and groups totalling several hundred 0--1-day-old eggs exposed in batches of approximately 50 to 40°C, 90% RH for 5 min, or in batches of 100 or more to -10°C, 90% RH for 24 h. Approximately equal numbers and groups of eggs were kept as control at each of the four environmental temperatures until each exposure was completed, when both treated and control groups were transferred either to 25 °C, 90% RH for those batches previously exposed to 40°C, or to 150C, 90% RH for those previously exposed to - 1 0 ° C , and all groups examined periodically for hatching. Environmental temperature affected the resistance of A. siro eggs to both heat and cold (Table III). Within the limits of the developmental temperature range, the resistance to conditions beyond these limits was clearly greater in populations reared at near threshold temperatures than in those reared at the ecologically more TABLE III Effect of environmental temperature on the resistance of eggs to heat (40°C) and cold ( -10 ° C ) Environmental temperature (° C)

Numbers exposed and per cent hatch 40°C

10 15 20 25 30

-10°C

No. exposed

% Hatch a

No. exposed

% Hatch a

250 250 250 250 250

0 0 0 7.2 35.5

139 660 620 426 150

64.0 45.5 35.2 27.1 11.5

aCorrected for control mortality

329 favourable middle range of temperatures. It was also clear that A. siro exhibited a greater tolerance to cold than to heat. Of the eggs exposed at 40 ° C, only a proportion o f those reared at 25 and 30°C survived exposure, in contrast to those exposed at - 1 0 ° C where some eggs in all groups hatched after exposure, the percentage hatch being inversely related to environmental temperature and reaching a maximum of 64% in the 10°C group. Since both age and environmental conditions were found to affect the resistance of the eggs to unfavourable physical conditions, it was clearly desirable that any further work should be carried o u t using only the most resistant eggs, i.e. those in an early stage of development reared under nearthreshold conditions. Under such marginal conditions, and particularly at low relative humidities, egg o u t p u t is poor and often erratic. Accordingly, to secure within reasonable time limits the relatively large numbers o f eggs required for experimental use, less resistant eggs from females reared under more favourable conditions had to be used. In evaluating the results of later experiments, therefore, it must be borne in mind that the estimated maxim u m survival times probably under-estimates the ability of the species to withstand unfavourable temperatures and humidities. Resistance to heat and cold Limits o f survival at lethal high temperatures. To determine a practical- upper limit to the range of lethal temperatures just above the developmental maximum o f 31--32°C, batches of 250 0--1-day-old eggs from females reared at 30°C, 90% RH, were exposed in groups of 50 to 40, 45 and 50°C for a minimum period of 5 min. Approximately equal numbers of control eggs were maintained for each group. After exposure, the eggs were returned to their original culture conditions and, together with the control groups, examined periodically for hatching. A small proportion o f those exposed to 40°C hatched b u t none of those exposed at either 45 or 50°C did so and no further experiments were carried o u t at these temperatures. To determine both the effect of relative humidity on resistance and the degree o f resistance at these temperatures, two further series of experiments were carried o u t at 35 and 40°C at both 20 and 90% RH, using various exposure periods. The numbers of eggs hatched at corresponding exposure periods at each relative humidity at either temperature did not differ significantly. It was concluded, therefore, that either relative humidity had no effect at these temperatures or that its effect was masked b y the overriding effect of high temperature. In consequence, relative humidity was ignored and the separate data combined to give a single figure for percentage hatch at each exposure period at each temperature. The results showed considerable heterogeneity (Table IV). Column (d) in the above table indicates the wide range of variation in percentage hatch at each exposure period. The percent survival figures given in columns (d) and (e) are corrected b y subtraction of control mortality. This also proved to be

330

somewhat variable and often heavy. The cause of this variable high mortality among control eggs was not determined but may have been due, in part, to the relatively high temperatures at which the cultures were maintained. Although A. siro is able to complete its life cycle at 30 ° C, this temperature is very close to the upper temperature limit for development o f the species and above the o p t i m u m for development. Under such conditions, continued breeding has a debilitating effect u p o n the population. Nevertheless, despite the heterogeneity o f the data, mean per cent survival appeared to be linearly related to time at 35°C and also at 40°C after an initial phase of disproportionately high mortality in the early stages o f exposure, the causes of which are not known. Regression equations for percent survival versus exposure period were calculated at each temperature. At 35°C, y = 98.89 11.68x, and at 40°C, y = 4 6 . 5 8 - 1.52x. From these regressions, estimates of the maximum survival period corresponding to 0% survival were made. These indicated periods o f approximately 10 h at 35°C and 35 min at 40°C. -

TABLE IV Resistance of A. siro eggs to lethal high temperatures

Exposure temperature (°C)

(a) No. of experiments

(b) Total number eggs exposed

(c) Exposure period

(d) Range of variationin per cent hatch a

(e) Mean per cent hatch a

35

11 11 11 14 25 20 20

550 550 550 700 1250 1000 1000

1 3 5 7 5 15 30

55.7--100 50.4--85.9 0--90.6 0--54.8 0--84.6 0--88.7 0--20.4

85.9 69.8 50.9 19.7 31.7 19.8 1.4

40

h h h h rain rain rain

aCorrected for control mortality.

Limits o f survival at lethal low temperatures. A similar series of experiments to those described above was carried o u t at temperatures just below the developmental minimum of 2--3°C. Batches of 100--200 0--1-day-old eggs from females reared at 10°C, 90% RH, were exposed for various periods to temperatures of 0, - 5 and - 1 0 ° C at 20 and 90% RH. As before, approximately equal numbers o f control eggs were maintained for each group. After exposure, the treated eggs were returned to their original culture conditions and together with the control groups examined periodically for hatching. Each experiment was repeated at least three times, the maximum exposure period being progressively increased, if necessary, in successive experiments until complete mortality was achieved. At temperatures below 0°C as at those above 32 ° C, no significant differ-

331 100

A

75

- 50 L) 25

0

4

8 12 16 Exposure period (days)

20

24

100

75

~o 5 0

25

2.

0 0

2

4

6 8 10 Exposure period (days)

12

l w 14

100

75

~

g

x

x

~

•

• 90 %RH

5o

u

25

25

50

75 100 Exposure period (days)

125

150

175

Fig. 5. Resistance of A. siro eggs to cold. (A) Exposure at -5°C. (B) Exposure at - 1 0 ° C (C) Exposure at 0°C at 20 and 90% RH.

332 ences were found between the numbers of eggs hatched at corresponding exposure periods at either 20 or 90% RH, which indicated that at sub-zero temperatures the eggs were killed by cold before the adverse effects of desiccation became important. The data for 20 and 90% RH were therefore pooled to give the percentage hatch at each exposure period a t - 5 and -10°C. At 0°C, however, the differences in percentage hatch were significantly different and the effects of relative humidity clear, the rate of survival falling sharply and rapidly at 20% RH, but more slowly and gradually at 90% RH, so that more eggs survived longer at the higher humidity. Although control mortality remained fairly constant at a relatively low level throughout the experiments, surival o f exposed eggs was often extremely variable. Despite this variability, mean values for the percentage survival at each exposure period, corrected for control mortality, when plotted at - 5 and - 1 0 ° C (Figs. 5A, B) and separately for each humidity at 00C (Fig. 5C), gave a series of points suggesting a sigmoid-shaped response at 0°C and a simple curvi-linear response at - 5 and -10°C. The maximum survival period at each temperature was, as before, taken as that point o n t h e curve corresponding to 0% survival. At 0 ° C, this was approximately 80 days at 20% and 175--180 days at 90% RH, at -5°C approximately 24---26 days, and at - 1 0 ° C approximately 12--14 days. Resistance to desiccation Limits o f survival at lethal low relative humidities at favourable temperatures. These experiments were carried o u t over 2--3 years using mites taken from different stock cultures but bred consistently on the standard food material (wheat germ) and under standard physical conditions (17.5°C, 75% RH). The experimental procedure was as follows. Adult mites were removed from the cultures and confined, two to three pairs per cell plus food, in micro-cells at 70 or 90% RH, at the required experimental temperature. These cells were examined daily, the eggs removed TABLE

V

R e s i s t a n c e o f A . siro eggs to d e s i c c a t i o n : r e p l i c a t i o n o f e x p e r i m e n t s and n u m b e r s o f eggs e x p o s e d to e a c h set o f c o n d i t i o n s Temperature (o C )

R H (%) 20

5 10 15 20 25 30 Totals

Total eggs 30

40

50

60

Exp.

Eggs

Exp.

Eggs

Exp.

Eggs

Exp.

Eggs

Exp.

Eggs

32 41 40 18 11 3

1 2 2 2

24 40 40 19 11 4

1 2 2 1

26 41 37 21 14 4

1 2 2 I

46 17 30 26 20 4

2 1 2 2 1

--24 21 16 14

--1 200 2 150 S00 700 4 850

600 030 950 000 550 150 9 300

200 000 950 600 550 200 8 500

300 050 800 700 700 200 8 750

300 350 650 200 000 200 9 700

6 7 12 9 3 1 41

400 450 550 650 600 450 100

333 and transferred to fresh cells in groups of 10--25. Batches of 50 or 100 eggs were then exposed to the experimental humidities at each temperature for different periods, the exposure periods being chosen to suit the physical conditions o f each experiment. The experimental temperatures ranged from 5 to 30 ° C in steps of 5 ° C. The relative humidity ranged from 20 to 60% in steps of 10% from 15 to 30°C, and from 20 to 50% at 5 and 10°C. After exposure, the eggs were returned to their original culture conditions and examined periodically for hatching. The results showed considerable heterogeneity which was most marked at the lower temperatures. In spite of frequent repetition of many experiments, involving many hundreds of eggs, this heterogeneity persisted, tending to obscure the relationship between the physical factors and survival and reducing the accuracy of determination of maximum survival times from the data. The necessity to repeat many experiments led, in consequence, to a certain quantitative imbalance in the data, estimates of mean survival being based on relatively few experiments for some conditions and on much larger numbers for others. The numbers of experiments performed and eggs exposed at each set of conditions are given in Table V. The aim of the experiments was to determine the minimum length of exposure to a given set o f conditions required to kill all the individuals exposed. It cannot be claimed that experiments on a few thousand individuals under each set of conditions give figures that will be valid for all members of the species. Indeed, no attempt was made to collect a representative set of strains of the species, although several different stocks were used. Nor was it possible to use probit analysis as a means of extrapolation, for the data were so heterogeneous that the slope of the probit lines could n o t be determined with enough accuracy to make such an operation realistic. Nevertheless, since the stocks were healthy and well cared for and the results show a broad overall consistency, t h e y provide a useful guide to characteristics of the species. Mean percentage survivals, corrected for subtraction of control mortality, were calculated for each exposure period for each set o f physical conditions. These values were then plotted against exposure period and a series of curves, one for each level of relative humidity at each temperature, was fitted by eye. From these curves, examples of which are shown in Fig. 6, and 7, values for 50 and 0% survival were determined and are given in Table VI. The m a x i m u m survival period is that corresponding to 0% survival except at 60% RH at 15 and 20°C (Fig. 8), where conditions permitted some eggs to develop and hatch during exposure but inhibited further development. The m a x i m u m survival period is here taken as that corresponding to the period o f m a x i m u m egg mortality. Maximum survival periods read from these graphs were then written on to a grid o f temperature versus humidity, and isoperiodic lines were drawn through convenient values determined by linear interpolation from the values plotted to indicate the relationship between survival, relative humidity and temperature (Fig. 9).

334 100

5oc

100

.5> >

20% RH

~

50 c U

30% RH 50

u

I 10

0

100

o-~.gp 20

6 30

,L 40

0

... ••

U

0 0

•

•

10

20

30

4O

100 >

40% RH

50

v

0

.~ o')

•

C

U

10 20 30 Exposure period (days)

~ 40

0

I

0

r

I

v

10 20 30 Exposure period (days)

'

40

Fig. 6. Resistance o f A . siro eggs to different desiccating h u m i d i t i e s at 5°C.

30°C

25°C 100

100

75

75

5> => ~o

>

50

k.2

~

• 60% RH • 50% RH • 40% RH 0 30% RH ~20%RH

! 60% RH 50% RH 40% RH

D

50 c

~J

== 25

25

4 Exposure period (days)

.' 6

0

--

0

i

,w

2 4 Exposure period (days)

-I 6

Fig. 7. Resistance of A. siro eggs to different desiccating humidities at 25 and 3 0 ° C

335 100

80

:iil

; 60

u

4O

20

i

0

I 5

0

~I • I/ I 10 15 20 Exposure period (days)

I ,. 25

I 30

Fig. 8. Resistance of A. siro eggs to desiccation at 60% RH, 15--30°C.

90

i

80

70 60

PhysicDalLi eveom lp~meitnstforComplete

~

/

/

/

z ~ 5o

40

I

30

8O O,~y,,

20

J

/

I

-10

-.5

0

J

/ I

/

/

/

/ /

/

16 8 4 2 Maximum survivalperiod--days J

/

I

/

/

/

32

/

/

/

/

/

/

/

/

O

/

/

/

/

i

I I

/

I

I

I

I

I I

/

/

I

/

/ /

I

10 15 20 Temperature(°C)

/ /

I

25

30

35

40

Fig. 9. Resistance of the grain mite A. siro to unfavourable physical conditions beyond the limits o f its development.

336 TABLE VI Exposure periods (days) for 50 and 0% survival levels at lethal humidities at favourable temperatures Temperature

50% Survival

0% Survival

RH (%)

RH (%)

(°c)

5 10 15 20 25 30

20

30

40

50

20

30

40

50

8.0 6.0 4.3 2.7 < 1.0 < 1.0

9.0 7.4 5.5 2.7 < 1.0 < 1.0

14.1 6.0 6.3 3.7 < 1.0 < 1.0

20.0 9.4 9.5 5.5 1.2 < 1.0

20.0 16.0 10.0 4.0 2.0 < 1.0

21.0 16.0 10.5 4.4 2.0 < 1.0

36.0 16.4 10.5 6.7 3.0 2.0

45.0 21.4 15.5 7.4 4.0 3.0

Although when grouped at each temperature the relative positions o f the survival curves for each humidity were, under some conditions, somewhat inconsistent, the broad effect of relative humidity was clear and distinct. Irrespective o f temperature, mean survival was directly related to relative humidity. This was particularly noticeable at 5°C where the estimates of the maximum survival period at each level of relative humidity were considerably greater than at other temperatures. At 25°C the relative positions o f the survival curves can be correlated directly with increase in relative humidity and this probably expresses the true relationship between the two factors which at other temperatures tends to be obscured by the irregular positions o f the survival curves, particularly at the lower humidities. The temperature effect was also clearly marked. Survival was found to be inversely related to temperature and to decrease sharply at the higher temperatures. For example, the exposure period required to give 50% survival at 5°C and 50% RH, was 20.0 days compared with only 1.2 days at the same relative humidity at 25 ° C. DISCUSSION

Among the many features that distinguish a successful pest species an ability to withstand unfavourable physical conditions b e y o n d the limits of its development is especially advantageous. It enables the animal to survive and develop when conditions become favourable and to increase in abundance. Howe (1963) noted that the survival of insect pests at temperatures and humidities b e y o n d those permitting development depended principally on the stage at which the insect was exposed, and further that for most storage insects the fully grown larva was usually the most resistant stage. The resistance of the grain mite to unfavourable physical conditions is

337

similarly dependent chiefly on the stage at which it is exposed, although the pattern of resistance of the different stages differs from that of most storage insects. Unlike the latter, the newly laid egg and not the fully grown larva is the stage most resistant to desiccation and temperature extremes although resistance declines with age. Larvae, nymphs and adults are fairly susceptible to heat and desiccation b u t show considerable resistance to cold. Active stages are more resistant to cold than resting individuals b u t the latter show a greater resistance to desiccation although each resting form rapidly transforms into a susceptible active stage. Active tritonymphs are usually t h e most resistant of the post-oval stages both to extreme temperatures and low humidity. That A. siro shows only a low resistance to desiccation is scarcely surprising. Like most Astigmatid species it is without specialist respiratory organs, the cuticle being the main respiratory surface. Because of its small size and proportionately large surface area, it loses water rapidly in proportion to its weight and hence is extremely vulnerable in dry conditions. Its critical equilibrium humidity -- the lowest relative humidity at which an animal without food can establish moisture equilibrium with the air and below which it rapidly loses water and eventually dies through desiccation -- is a b o u t 71% (Kniille, 1962), although on suitable f o o d at temperatures from 10 to 20°C it can develop and multiply at relative humidities down to 62.5% (Cunnington, 1965). The ability of eggs to restrict their water loss more efficiently than other stages is indirectly reflected in the mean maximum survival periods calculated for the range of conditions shown in Fig. 9 where the pattern of resistance is clearly demonstrated. Within the limits of the developmental temperature range resistance to desiccation increases steadily with falling temperature b u t decreases with falling humidity at each temperature as the effects of desiccation become more severe, although these differences become progressively less at the higher temperatures. Beyond the developmental limits, except at 0 ° C where eggs survived longer at 90 than at 20% RH, relative humidity either ceases to affect survival or its effects are suppressed or masked b y the overriding effects of lethal high or low temperature. The low resistance to high temperatures shown b y A. siro is also hardly surprising. Its upper temperature limit (31--32°C) compared with that of most stored product insects is relatively low and like most poikilotherms it is ill-adapted to survive prolonged exposure to temperatures much above its developmental maximum. Resistance to high temperatures for most storage pests, as Howe (1963) pointed out, is however relatively unimportant. Such temperatures are seldom met with in practice except when infested produce heats or surface layers are warmed b y the sun. Of far greater significance is resistance to cold. The minimum constant temperature threshold for A. siro is about 2.5°C (Cunnington, 1965), a threshold well below that of most stored product insects, few of which can develop at temperatures below 10 ° C. This relatively low developmental minimum suggests considerable cold

338 tolerance, a characteristic earlier noted by some Russian and Canadian workers. II'inskaya (1935) in the Northern Caucasus and R o d d (1939) in Eastern Siberia, where winter temperatures sometimes reach as low as - 4 5 ° C, both reported that A. siro survived the winter in grains in the ground. Ushatinskaya (1954) in Moscow reported that eggs survived for more than a year in grain at a b o u t 0°C, for nearly 6 months in that at a b o u t - 5 ° C and for nearly 2 months in that at about - 1 0 ° C. She noted that mobile stages were much less resistant than eggs although at 0°C a few individuals survived for about 15 months. In Canada, Sinha (1964) recorded a minimum winter temperature o f - 1 8 ° C in the surface layers of experimental grain bulks in Manitoba farms and reported that a few active stages survived exposure to this temperature for 7 days. Clearly, A. siro shows considerable cold tolerance, a characteristic which by setting the lower limits for its continued existence strongly influences its distribution and ecology. These limits require further investigation. Sinha reported that a few individuals survived exposure to - 1 8 ° C for 7 days. Ushatinskaya, on the other hand, observed that all stages were killed b y exposure t o - 1 5 ° C for 1 day while her estimates of the maximum survival periods of eggs exposed to temperatures of and below 0°C greatly exceed those obtained during the present study, even accepting that the latter probably under,estimate survival limits. Such differences in results m a y arise in part from differences in method or technique or to differences in strains b u t it is probable that adaptation or acclimatization to cold had a more important bearing on the results. That such acclimatization occurs is clear from the work of Sinha (1964) who found that populations o f A. siro reared at 6 ° (+2°C) were more resistant to cold than those reared at 21 ° (+ 2 ° C) and from the results o f the present study (Table III) which showed that resistance to cold increased as culture temperatures were lowered. It is relevant also to record that Ushatinskaya's observations were based on a long term study of infested grain bulks during which acclimatization to cold probably occurred as a result o f the low grain temperatures recorded during the winter months. This ability to acclimatize to cold and thus to endure lower temperatures than normal makes precise determination o f resistance limits difficult. Cold resistance in A. siro and other stored product mites has been little studied and there is a lack of information about most species and most aspects o f the subject including especially the physiology o f cold resistance. Virtually nothing is known, for example, of the relative effects of sudden as opposed to gradual falls of temperature or o f fluctuating as compared with constant temperatures. Nor o f the effects o f different conditions before exposure to cold or o f the powers o f recovery o f the mite from cold injury and related matters. Such knowledge is needed in order to understand and interpret fully the ecological data gathered from field studies and laboratory observations and to provide the background information necessary to devise more effective methods for the prevention and control o f infestation.

339 REFERENCES Boczek, J., 1957. The grain mite (Tyroglyphus farinae L.), morphology, biology and ecology, its depredations and tests of control measures. (In Polish, with English summary). Roczn. Nauk Roln. Ser. A, 75 (4): 559---644. Cunnington, A.M., 1965. Physical limits for complete development of the grain mite Acarus siro (Acarina, Acaridae), in relation to its world distribution. J. Appl. Ecol., 2: 295--306. Griffiths, D.A., 1966. Nutrition as a factor influencing hypopus formation in the Acarus siro speciescomplex (Acarina, Acaridae). J. Stored Prod. Res., 1: 325--340. Hora, A.M., 1934. On the biology of the mite Glycyphagus domesticus de Geer. (Tyroglyphidae, Acarina). Ann. Appl. Biol., 21: 483--494. Howe, R.W., 1963. The prediction of the status of a pest by means of laboratory experiments. World review. Pest Control, 1963, 2: 30--40. II'inskaya, L.L., 1935. On the survival of stored grain mites through the winter in field conditions (In Russian). Plant Prot., 4: 123--125. (English summary in Rev. Appl. Entomol., 1936, 24 A: 580.) Jones, B.M., 1950. Acarine growth: a new ecdysial mechanism. Nature, 166: 908--909. Kniille, W., 1962. Die Abh~/ngigkeit der Luftfeuchte-Reaktionen der Mehlmilbe (Acarus siro L.) vom Wassergehalt des KSrpers. Z. Vergl. Physiol., 45: 233--246. Kozulina, O.V., 1940. The effect of humidity and temperature on the development of the eggs of grain mites. (In Russian). Uch. Zap. Mosk. Gos. Univ., Zool., 42: 179-184. (English summary in Rev. Appl. Entomol., 1943, 31A : 71--72.) Polezhaev, V.G., 1940. The effect of atmospheric humidity and temperature on the formation of the hypopial stage in Glycyphagus destructor Schr., and Tyroglyphus farinae L. (In Russian). Uch. Zap. Mosk. Gos. Univ., Zool., 42: 185--196. (English summary in Rev. Appl. Entomol., 1943, 31 A: 72.) Rodd, V.E., 1939. On the survival of the corn mites (Tyroglyphidae) in the ground in East Siberia. Sb. Tr. Zasch. Rost. Vost. Sibiri, 5 : 5 0 - - 6 1 (In Russian with English summary). Schulze, H., 1923. Uber die Widerstandsf~/higkeit der Dauerformen yon wirtschaftlich wichtigen Milben. Naturwissensehaften, 11 (36): 763--765. Sinha, R.N., 1964. Effect of low temperature on the survival of some stored product mites. Acarologia, 6: 336--341. Solomon, M.E., 1951. Control o f humidity with potassium hydroxide, sulphuric acid or other solutions. Bull. Entomol. Res., 42: 543--554. Solomon, M.E. and Cunnington, A.M., 1964. Rearing Acaroid Mites. In: Proc. 1st Int. Congr. Acarology, F o r t Collins, CO, U.S.A., 1963. Acarologia, 6: 399--403. Ushatinskaya, R.S., 1954. The biological bases for the use of low temperature in the struggle against pests of stored grain (insects and mites) (In Russian). U.S.S.R. Academy of Sciences, Severtsov Institute of Animal Morphology, Moscow.