The susceptibility of the developmental stages of Sitophilus granarius (L.) (Coleoptera, Curculionidae) to methyl bromide

J. stored Prod. Res., 1966, Vol. 2, pp. 13-26.

The Susceptibility (Coleoptera,

Pergamon Press Ltd.

Printed in Great Britain.

of the Developmental Curculionidae)

to Methyl Bromide R. W. HOWE and B. D. HOLE Pest Infestation Laboratory,

Slough, Bucks, England

(Received 30 March, 1966) Abstrac&-Developing individuals of pithily gmzmius, cultured at 25’C and 70 per cent r.h. and with ages known to within a day, were fumigated with methyl bromide at regularly-spaced dosage levels between log concentration-time (c.t.) products of 0.74 and I.58 at the same temperature and r.h. The most susceptible age was 9 days, the middle of the first larval instar. Susceptibility decreased up to ages 30-31 days, the early pupal stage and then increased again. Eggs were about as susceptible as larvae of about 23-days old, but on hatching individuals rapidly increased in susceptibility. Free-living adults were slightly less susceptible than eggs and less susceptible than developmental stages outside the range 28-32 days. The LD 50 of the least susceptible age group was approximately 29 mg hr/l., about ten times that of the most susceptible group. Comparable values for adults and eggs were 20 and 15 mg hr/l. The mortality for a given c,t. product used against a given age group was higher when the gas concentration was higher. The development period of surviving individuals was apparently increased by the damaging influence of poisoning. The variation of s~ceptibility with stage of development sometimes masked and sometimes enhanced the effects of this on the emergence curves. It is estimated that a concentration time product of 50 mg hr/l. would kill over 99 *9 per cent of a population of S. gramrius.

INTRODUCTION

METHYL BROMIDE is one of the most satisfactory of the fumigants available for the treatment of stored cereal grains infested by insects. It penetrates readily into bulks or stacks of bags and is highly toxic to the insects, but is known to kill adults more readily than some of the developmental stages (BROWN, 1954; WHITNEY and WALKDEN, 1961). Unfortunately the larvae and pupae of several of the most typical pests of stored whole cereals live entirely inside the grains so that surviving individuals of the least susceptible stages of these species cannot be found immediately after a fumigation. For this reason, and because the detection of small numbers of free-living insects in large stacks or bulks is very difficult, a routine procedure in the Fumigation Department of the Pest Infestation Laboratory, is to introduce I3

14

R. W.

HOWE

and B. D.

HOLE

small test cages of weevil-infested grain into bulks that are subjected to experimental fumigation. After the fumigation the samples of grain from these cages are withdrawn and kept at 25°C together with untreated samples and examined regularly for periods up to 7 weeks for the emergence as adults of any individuals that may have survived the fumigation and continued to develop. The general inference from the practical use of these test samples was that the least susceptible stages of development had a fairly narrow age range somewhere around 4-weeks old at 25”C, and were either fully grown larvae or young pupae. KROHNE and LINDGREN (1958) compared the susceptibility of the four developmental stages of Sito@‘ziZus ory.~~e (L.) to a number of fumigants, with developmental groups that covered an age span of 3 days; they found that the half-grown larva was the most susceptible to methyl bromide, followed by adults 2-4 weeks old and by eggs. Their least susceptible age group was predominantly in the pupal stage. This technique was satisfactory for the egg and pupal stages that are completed in about 5 days in their experimental conditions and for adults of the age used. It is unlikely to have been satisfactory for the larval stage which persists for about 20 days and increases in The susceptibility of an individual in the weight considerably during this time. larval stage is likely to change markedly as it ages. A detailed investigation of the change of susceptibility with age was therefore undertaken, with the expectation that it would provide data that could be used to predict the dosage levels of methyl bromide required to give kills of more than 99 per cent in practical fumigations. MATERIALS AND METHODS

Outline The aims of the experiment were to fumigate with methyl bromide, samples of wheat containing developing weevils of an age known to within a day and to cover the entire developmental cycle from 1 to 36 days old at 25°C. Every age group was fumigated at 7 dosage levels and a regression line relating the percentage mortality expressed as a probit to the dosage level expressed as the logarithm of the product of concentration in mg per 1. and time in hours was calculated for each. The susceptibility of the age groups varied so much that three ranges of dosage were needed and these were obtained by using 9, 6 or 3 mg/l. of methyl bromide in the experimental chamber. The tests were carried out with successive generations of the same laboratory stock of Sitophilus granarius (L.) (Table 1) and every set of fumigations was spread over three days. An incomplete randomized block design provided replication and a means of checking that variability, both between cultures and between dates of fumigation, was not statistically significant. Preparation of samples for fumigation The routine method of breeding S. granarius at 25°C in the Fumigation Department of this Laboratory is to place 200 adults on 300 g of wheat conditioned to 70 per cent r.h. and remove them 3 weeks later. This method can be relied upon to yield about 100 weevils per 10-g sample, so it was calculated, assuming a comparable rate of oviposition, that cultures made up with 500 adults on 160 g of wheat for one day only would yield at least 25 weevils of age known to within a day per 10-g sample. This was confirmed experimentally and all the cultures in this series

The Susceptibility

of Sitophilus granaries

to Methyl

15

Bromide

of experiments were of this size. Every 24 hr, the adults from the current cultures were removed and placed on a fresh lot of humidity-conditioned wheat to give a series of consecutive 160-g cultures differing in age by one day. Whenever possible, a fumigation experiment with a series of cultures containing young stages was quickly followed by one with a series containing older stages so that both could be covered by a single series of successive cultures from a single batch of parent weevils (e.g. B, C, D, in Table 1). TABLE

1.

CONCENTRATION FUMIGATION

Age at fumigation (days)

OF METHYL OF EACH

BROMIDE

SERIES OF AGE

Series of culture

USED

IN EXPERIMENTAL

GROUPS

Concentration of methyl bromide (mg/l.)

1-12

D

7-18

C

3

14-19 18-29 20-25 25-36 29-34

B A D B C

3 9 3 9 9

6

Each 160-g culture provided two 80-g ‘half-cultures’ that were fumigated at different ages. Either 4 or 8 age groups were fumigated simultaneously on each of 3 days in each experimental series so that 12 or 24 half-cultures, i.e. 6 or 12 cultures, were used in each experiment. Every 80-g age group was split into 8 samples weighing approximately 10 g; 7 were fumigated with a graded series of dosages and 1 was kept as a control. The splitting was done as soon as possible after the adults had been removed from the 160-g culture. The culture was passed through a 2-way divider (HOWE,1963) to give 2 fractions and this sub-division was continued to give 16 samples weighing about 10 g. These 16 random samples of the original culture were shared between the 2 ‘half-cultures’ at the final divisions of 20 g into 10 g, 1 sample from each pair being allocated to each ‘half-culture’. The 10-g samples were placed in numbered 3 x l-in (7 -5 x 2 *5-cm) glass tubes closed by muslin held in place by short lengths of thin-walled plastic tubing and returned to 25°C and 70 per cent r.h. On the day before fumigation, the 10-g samples were transferred to numbered fumigation cages, cylinders of brass gauze about 3-in long and 0.5 in. in diameter closed at each end by tightly fitting copper caps. The 8 cages from each halfculture were each attached to a different piece of slightly concave metal strip by pieces of sticky tape placed at each end around the caps and the strip. Eight cages could be conveniently attached to each strip. The strips were kept overnight at 25°C. Technique of fumigation Fumigations were carried out in a 3000-l. chamber at 25°C and 70 per cent r.h. On the day before the first test sufficient methyl bromide liquid to give the requisite concentration was weighed in a flask and vaporized into the slightly evacuated bin.

R. W.

16

HOWE

and B. D.

HOLE

Water was also added in this way to bring the r.h. up to 70 per cent. The gas concentration was measured with a thermal-conductivity meter (HESELTINE, PEARSON and WAINMAN, 1958) about half an hour before the test was due to start. At the same time a sample of the methyl bromide was absorbed in monoethanolamine in an evacuated flask for chemical estimation (BROWN, 1954). Three toxicity tests were made on different days each time the bin was prepared and the concentration was measured before each. The loss of fumigant during the week was always very small. Seven of the 8 strips prepared each day were placed in the fumigation chamber and the other kept as a control. Because the gas concentration in the chamber was constant, the dosage was varied by adjusting the length of time that each strip of cages was left exposed to this gas concentration. Equally spaced dosage levels on a log concentration-time (log c.t.) scale were chosen on the basis of earlier experience. In the first experiment these were 1.16 (= 14 -5 mg hr/l.) by steps of 0 -07 to 1.58 (=38 mg hr/l.). The lengths of exposure in minutes needed to give these log c.t. products were calculated for each fumigation from the meter-reading estimation of concentration, and a time-table prepared for inserting and removing the strips from the chamber. The fumigation chamber is fitted with a horizontal row of eight small ports normally closed with lead-seated screw caps, but closed by rubber bungs during the experiments. Inside the chamber a light alloy framework supports lengths of shallow channel which extend from each port along the length of the chamber. A strip of cages pushed through a port lies in a channel. Each strip has a short spike near each end, and can be removed by putting a hook round the one at the end near the port. A strip can be inserted or removed in less than half a minute and the time noted to the nearest minute. Thus the lengths of exposure in minutes were easily measured. Treatment of samples after fumigation After fumigation the strips were returned to 25°C and left undisturbed overnight. On the following day the samples were put back into their numbered glass tubes and were not examined until 35 days after the culture had been prepared. On this day and thereafter daily for 3 weeks and at the same time, the contents of each tube were spread on to a petri dish of 15-cm diameter and all the weevils that had emerged were counted and removed. A final examination was made on the 70th day. In the first series of experiments (A) with a gas concentration of 9 mg/l. there was a very high mortality among the younger stages even at the lowest dosage, so in subsequent experiments with larvae at these ages and younger, the concentration of methyl bromide was reduced to 3 mg/l. and the log c.t. range was 0 -74-l - 16. Later when the eggs were tested the concentration was raised to 6 mg/l. (Table 1) and the range of log c.t. raised to 0 -95-l -37 because a preliminary test had shown that eggs were less susceptible to methyl bromide than young larvae (see also KROHNE and LINDGREN, 1958). These 3 concentrations gave an overall range of log c.t. from 0 - 74 to 1.58 by 12 steps of 0 ~07, and all included the value l-16.

Adults To complete

the picture,

a test was done with adults,

1-5 weeks old from stock

The Susceptibility

of

Methyl

Bromide

17

cultures, exposed in groups of 100 on approximately 5 g of wheat to a concentration of 10 mg/l. of methyl bromide. Again the experiment was repeated 3 times on successive days. The shortest exposure was 90 min and the intention was to have 20 periods increasing successively by 7 min to 223 min, every level being replicated at least 12 times and some as many as 30 times. Some mistakes of timing spoiled this regular pattern. After fumigation the adults were kept at 25°C and 70 per cent r.h. in glass jars (7 x 5 cm) for a week before the numbers alive and dead were finally counted. TREATMENT

AND

EVALUATION

OF

RESULTS

The daily count of survivors that emerged as adults provided both the total number of survivors and an emergence curve for each sample. The former was examined by the probit analysis procedure described by WADLEY (1949) and by FINNEY (1952, pp. 203-205), b ecause the numbers exposed to the fumigant are estimated from parallel control samples. The emergence curves were examined to decide 8

7

6

-5 $ :4

x

x

x

x

3

2

FIG. 1. Probit lines relating mortality of Sitophilus gramrius to log dosage of methyl bromide in mg hr/l. for samples 7 days old (young first instar larvae). Percentage mortality for concentration of 6 mg/l. represented by + and for 3 mg/l. by x , each based on observed numbers of adults that emerged from relevant control samples. All probit lines are based on corrected estimates of numbers in control and here corrected mortality for 3 mg/l. is shown by smaller crosses. In Figs. 1-5 upwardpointing arrows represent complete mortality and downwardpointing arrows zero mortality. B

18

K. ii'.HOWE

and

B. D. HOLE

Log

ct

FIG. 2. Probit lines relating mortality of Sitophilus granarius to log dosage of methyl bromide in mg hr/l. for samples 6 days old (O-eggs close to hatching) at 6 mg/l., 9 days old (O-middle first instar larvae) at 3 mg/l. and 30 days old (+-predominantly young pupae) at 9 mg/l.

whether or not fumigation had influenced the subsequent rate of development of survivors. The probit lines for each age group, each replicate and various sequences of age groups were calculated by the Rothamsted Orion computer. For each the maximum likelihood estimate of slope, LD 50 and of the average number of individuals exposed in each sample was provided together with their standard errors and a value of x2-a measure of the heterogeneity of the observed mortality. The raw data from which the lines were computed was not always satisfactory, as for example, that illustrated in Fig. 1 for 7-day-old individuals fumigated with 3 mg/l. of methyl bromide. In calculating these probit lines, the greatest weight is placed on samples with an expected mortality of about 70 per cent (probit 5 *6). Expected mortalities below 15 or above 99.4 per cent (probits 4 and 7 ‘5) carry less than a tenth of the weight. This is because probits, based on the normal distribution so that estimates of high and low kills are relatively imprecise, are modified by errors in the estimate of the number treated. Variation from sample to sample in a culture divided successively at random into two is unimportant when few survive, but very important when many survive. There are several instances in the data obtained in these experiments (see Figs. 1, 2 and 4) of more beetles emerging from a fumigated sample than from the control. The probit lines calculated by the computer fit mortalities based on the adjusted control values as the number treated (Fig. 1). In all the figures the plotted values are based on the observed number of emerged beetles. This seldom has any noticeable effect since the adjustment of the control

The

Susceptibility

of Sitobhilus granarius to Methyl

IY

Bromide

value is usually small, but in Fig. 1, one line is below all the plotted points except one which represents a zero mortality. For this line the modified points are plotted as smaller crosses. The other line on Fig. 1 is clearly more satisfactory although This is because the remaining five there are two doses with complete mortality. points are all in the reliable range of expected probits (4 -5-6 - 7) for this method of analysis. Figure 2 gives the probit lines for the most susceptible and most resistant age groups and for a stage of intermediate susceptibility.

90 N 0-l c 50 2 2 IIO

25 20

I5

n + 3 0

10

2 \ I-

5 ) AGE

IN

DAYS

FIG. 3. The change with age in the susceptibility to methyl bromide of the developmental stages of Sitophilus granurius expressed (above) as probit of percentage survival estimated for a log c.t. product of 1.16, the only dosage level achieved with all three concentrations of fumigant and (below) as the estimated LD 50. The estimates obtained using 3, 6 and 9 mg/l. of methyl bromide were different and are given as separate lines.

20

R. W. HOWE and B. D. HOLE

Changes of susceptibility with age As a general rule, the c.t. product is an accurate way of measuring the dose of fumigant and the mortality caused by a given ct. product is about the same whatever the value of each factor. In this work, however, it is evident (in both Figs. 1 and 3) that for each c.t. product a higher mortality was caused when the gas In spite of this a fairly clear pattern of change emerges concentration was higher. (Fig. 3). Whether the calculated LD 50 or the mortality caused by a given c.t. product is the criterion for judgement the pattern is the same. The egg is fairly resistant, possibly most resistant when very young, but young larvae are extremely susceptible especially in the middle of the first instar. At g-days old only about 5 per cent survived the shortest exposures to 6 mg/l. of methyl bromide (g-14-5 mg hr/l.). Then the resistance of larvae increases with age at an accelerating rate and maximum resistance is reached at 30-31 days when young (2-3-day-old) pupae predominate in the populations treated. Older pupae and young adults still inside grains are progressively more susceptible but emerged adults with an LD 50 of about 20 mg hr/l. are more resistant than any stage other than the pupa. There was no significant heterogeneity of results until the late pupal stage was reached, x2 being significant at the 5 per cent level only for the 32- and 34-day age groups and nearly so for the 33-day group. By this time in the developmental cycle, a fairly wide spread of stages is probable and the susceptibility to fumigant is increasing rapidly with development. There is, however, no comparable heterogeneity for the prepupal stage where resistance is increasing and the probability of a spread of developmental stages is little less. Grouping together age groups in which each developmental stage is predominant (Figs. 3 and 4) leads to heterogeneity for second and third instar larvae when fumigated at 3 mg/l. and for pupae (Table 2). It is principally attributable to the large scatter of mortality at low dosages (Fig. 4)-these values being very unreliable, but it is possible that the best fitting line for pupal mortality is curved. Slope of probit lines It is evident from Fig. 2 that the probit line for the g-day group has a lower slope than either of the other two lines. This difference is statistically significant at the 5 per cent level. There is wide variation of probit regression against log c.t. among the daily groups but the value for the g-day group, 3 ‘9, is the lowest and those for eggs 2 and 3 days old ( 15 *3-15 -4) are the highest. Standard errors, however, are too large for there to be significant differences between successive days except between one (7.3) and two. There are two conflicting tendencies that are worth comment. The first is that For example, the slope of probit lines seems to be correlated with susceptibility. both the LD 50 and the slope are lowest for day 9, and both are high for days 30-31 (slope 9 *2-9 *3). Th e second tendency is for the probit slope to diminish with age. This is partly a result of heterogeneity-when samples contain stages of widely different susceptibilities, the presence of individuals killed by low doses and of others surviving the high doses reduces the probit regression. When the age groups are collected into approximate stage groups (Table 2, Fig. 4) there are still some signs of differences although heterogeneity reduces the The probit line for first instar larvae has a significantly regression coefficients.

The Susceptibility

of Sitophilus granariw to Methyl

Bromide

21

lower slope than that of the egg, but no other difference is significant because the standard errors are large. The fourth instar larvae have the lowest slope.

7-

+

6-

L

0.74

1.16

Log

1.58

c t

FIG. 4. Probit lines relating mortality of Sitophilus granarius to log dosage of methyl bromide in mg hr/l. for each instar, obtained by adding together the numbers of adults emerging from the relevant dosage levels, for days l-6 for eggs, 7-10 for I, 11-14 for II, 15-19 for III, 20-25 for IV, 26-28 for prepupae, 29-34 for pupae and 35-36 for adults inside grains.

R. W.

22 TABLE 2. INTO

GROUPS

and B. D. HOLE

SLOPE OF PROBIT LINES AND CORRESPONDING

Gas concentration

Stage*

Age (days)

HOWE

APPROXIMATELY

LD 50 (log mg hr/l.) i

SE

LD50

FOR OBSERVATIONSCOLLECTED

WITH

THE

DEVELOPMENTAL

Slope of probit line

x2

$_ SE

STAGES

Range of daily slopes for stage

(mg/l.) l-6 7-10 7-10 11-14 15-13 20-25 20-25 26-28 29-34 35-36

Egg

I I II III IV IV Prepupa Pupa Adult in grain Free-living adults

* Roman

numerals

6 6 3 3 3 3 9 9 9 9

1~144&0~008 0.855&0.022 0*972*0*025 0~904~0*013 1~040~0~007 1.284 0.918&0.041 1.244&0.011 1.375+0.014 1.232&0.019

10

1~309*0~001

9.39*0.53 5.6OkO.50 3.61kO.39 8.2710.85 7.15kO.49 3.26 4.48kO.50 7.18kO.40 6.94-10.30 6.37kO.58 13.80&0.16

3.48 10.52 5.89 15.57 15.53

5.79 7.13 24.63 8.93

7.3-15.4 6.3-6.8 3.9-9.7 6.6-8.6 6.5-8.9 -6.1 5.0-5.4 6.5-8.4 6.5-9.4 6.5-6.7

260

refer to larval instar.

The slope of the probit mortality line for free living adults (Table 2) is greater than for any developmental group more than 3-days old. The standard error is small because the calculation is based on very large numbers of insects but the goodness of fit is poor principally because all the replicates for certain dosages are surprisingly far from the line (Fig. 5). There is no evidence for curvature. hfiuence offumigation on developmental period Consideration of the effects of sub-lethal dosages of a fumigant on a population is complicated by the heterogeneity of the population. Weevil eggs laid over a 24-hr period develop at slightly different rates and adult emergence always spreads over a week to 10 days and as a rule there are stragglers for at least another week. Occasional individuals require twice as long as those that emerge first. Thus in every batch of fumigated individuals, especially those treated late in the cycle, there will be a spread in the precise stage reached by each. When susceptibility is changing rapidly, this spread will be extremely important because sometimes the older or younger individuals will be more susceptible and sometimes more resistant. At such times of selective mortality it will be difficult to establish whether or not the rate of development after fumigation is affected. The best age groups for investigating this matter are those with little spread or those where the susceptibility to fumigant is fairly constant-that is in the egg stage, possibly excluding day 1 and in the 2nd and 3rd larval instars. In the egg stage there is a slight delay of adult emergence for age 1 of about & day up to 2 days at most doses, and for the rest of the egg stage there is a delay of nearly a day at log c.t. 0.95 increasing progressively to 5 days at log c.t. l-30. For a given dosage level the delay of emergence is considerable when a larval stage

The Susceptibility

of Sitophilus granarius to Methyl

Bromide

23

8

l-2

lines

l-4 log ct

relating

l-5

l-6

1.7

of adult Sitophilus in mg hr/l. for concentration of 10 mg/l. Points represent mortality for daily replicates based on 400-1000 weevils ignoring deaths in control, but calculated line allows for control mortality by requisite weighting. FIG. 5.

Probit

1.3

mortality

granaries to log dosage of methyl bromide

TABLE

3.

PROLONGATION UNIT

Age (days)

OF DOSE

IN DAYS (LOG

OF MEAN

C.T. OF 0.07)

Gas concn

EMERGENCE FOR

EACH

PERIOD STAGE

Stage

1

11-14 15-19 20-25 26-28 29-30 31-34 35-36

6 6 6 3 3 3 3 9 9 9 9

WEEVILS

Average emergence

Egg Egg

I I II III IV Prepupa Pupa Pupa Adult

PER

delay of per dose (days)

(mg/l.)

2-6 7-10 7-10

OF SURVIVING OF DEVELOPMENT

0.20 0.74 0.84 0.41 0.64 0.72 0.40 0.08 0.08 0.41 0.65

R. W. HOWE and B. D. HOLE

24

-

i

+ Age 24

Age 29

7

7

2

1

C 0

5

10

15

Days

0

10

5

15

Days

”

“4’.

Age 27

d

FIG. 6. Histograms of numbers of adult weevils emerging from fumigated samples and controls measured from day of first emergence, for samples of age 24 days (3 mg/l.-little mortality, some delay in emergence at high dosage) and ages 27 and 29 days (9 mg/l.-considerable mortality with much delay and none respectively). Very late emergences are omitted but were used to calculate mean periods represented by arrows.

-

-i

4

1

c-4 0

10

5

Days

15

l’he Susceptibility of Sitophilus gram&u

to Methyl Bromide

25

susceptible to the fumigant is treated, but when the more resistant late fourth, prepupal and early pupal stages are reached, the delay disappears and even becomes an acceleration at 28 and 29 days (Fig. 6). At this stage it is the most developed individuals that are most likely to survive fumigation. Thereafter, the mean period to emergence increases again with increased dosage. The slopes of the regression lines that relate the developmental period of survivors to the dosage levels used in the fumigation, but excluding the controls, were calculated for the results collected into age groups as in Table 3. These are given in the table for each dosage step; the values must be multiplied by 6 to give the difference in developmental periods of the lowest and highest dose for each series. Thus for the prepupae and pupae this difference is about half a day and for first stage larvae treated at 6 mg/l. it is 5 days and for eggs, as mentioned above, over 4 days. The slopes and their standard errors were calculated for the l-day-old eggs and for the ages shown in Fig. 6. They were O-201 f0.096 for age 1; 0.220f0.017 for age 24; 0*565&0*292 for age 27 and 0~086&0~123 for age 29. It therefore seems probable that there is a real check to the development of individuals that survive fumigation, although in the prepupal and pupal stages, age 28-31 days this is masked because the more advanced individuals are more easily killed. On the other hand, the effect is exaggerated on day 27 because only the more advanced individuals survive the higher dosages. DISCUSSION

One aim of this experiment was to fumigate groups of developing weevils all of which were as far as possible at the same point of development. This was nearly achieved perhaps for eggs which were all laid within 24 hr and which all hatched (in parallel samples) in about the same period. Although all the first instar larvae, therefore, appeared within little more than 24 hr about the 7th day, moulting into the second instar spread from the 10th to the 13th day. Third instar larvae were found on the 14th day so no treated sample consisted entirely of second instar larvae, nor did any sample contain more than about 85 per cent of third instar larvae. The 4th stage appeared on the 18th day and reached 90 per cent of the sample by the 21st and was still present on the 32nd, but 3rd stages persisted until the 24th day and on the 25th the first prepupa was found. This is a short stage which is not realistically demarcated in Tables 2 and 3 and Fig. 3, because when “dominant” it accounts for only 20-40 per cent of the sample population. Pupae first appeared on the 26th day and still accounted for about a quarter of the population on the 36th, having reached about 95 per cent on 31-32nd day. Adults first appeared on the 33rd day and usually began to emerge on the 37th, occasionally on the 36th. It is possible that the most susceptible age group fumigated on the 9th day was reasonably uniform in stage of development, but clearly the most resistant age groups were not. Presumably the most resistant stage of development is even more difficult to kill than these experiments suggest. The LD 50 for age 30 is already 6 times that for age 9. Further the c.t. product of the former was obtained at 9 mg/l. and of the latter at 3 mg/l. and it seems probable that from Fig. 3 the c.t. product needed to give an LD 50 for age 30 at the lower concentration would be at least twice that observed at the higher.

26

R. W.

HOWE and

B. D. HOLE

Although the hypothesis that the mortality caused by a given c.t. product is constant (EWES, 1965) is principally a practical rule (BROWN,1954) that cannot be applied below some minimum gas concentration, its failure in these experiments was surprising. The low concentration of 3 mg was expected to be a little above the minimum for the hypothesis to apply and differences between 6 and 9 mg/l. were not expected. The number treated at each dosage level in these age groups varied from 60 to 239, and usually exceeded 100, so the results should be reliable. To determine a practical dosage that will cause heavy mortality it is reasonable to consider the least susceptible age group. At the highest level of dosage used, a c.t. product of 38 mg hr/l., 10 per cent survived in the 30- and 31-day age groups. The probit line for 30 days must be extrapolated to provide estimates of the dosage required to give 99 and 99.9 per cent mortality and these are about 50 and 60 mg hr/l. respectively and the 31-day line gives values only a little lower. Extrapolation of any other line to these dose levels is probably invalid because less than 5 per cent survived the highest dosage level in any age group, but for the 2%day age group up to 0 -5 per cent might survive fumigation at a c.t. product of 50 mg hr/l. If a mixed population with every age group from 1 to 36 days were to be fumigated at this dosage, only about 1 per cent of each of the 30- and 31-day-old weevils, rather less of the 29- and 32-day-old weevils and no others would survive. It can be calculated, therefore, that about 0 -08 per cent of the whole mixed age population Since in a growing population the younger age would survive such a fumigation. groups would predominate, an even smaller percentage survival would be expected and a lower dosage should give a 99.9 per cent kill. Indeed the highest dosage level used in the experiments, 38 mg hr/l. should give a kill of over 98.5 per cent of a population in which all age groups are equally represented. The c.t. product obtained in this work for the LD50 of pupae is very close to that given by KROHNEand LINDGREN (1958) for pupae of S. oryzae and their value for larvae corresponds with that found in this work for larvae 15-20 days old. Their values for eggs and free living adults, however, were respectively higher (21.4 instead of 15) and lower (15 instead of 20) than those found here for S. granaries. Acknowledgers-Our thanks are due to Mr. J. WELCR who assisted with the preparation of cultures and the examination of samples for surviving weevils, and to Mr. G. J. S. Ross of Rothamsted Experimental Station who advised on statistical methods and supervised the analysis of results. REFERENCES BROWN, W. B. (1954) Fumigation with methyl bromide under gas-proof sheets. BulZ. D.S.I.R. Pest Infest. Res., No. 1, vi + 38 pp. ESTES, P. M. (1965) The effects of time and temperature on methyl bromide fumigation of adults of Sitophilus granarius and Tribolium confuum. J. econ. Ent., 58, 611-614. FINNEY, D. J. (1952) Probit analysis. A statistical treatment of the sigmoid response curve. Cambridge Univ. Press 2nd edn. xiv-j-318 pp. HESELTINE, H. K., PEARSON,J. D. and WAINMAN, H. (1958) A simple thermal conductivity meter for gas analysis with special reference to fumigation problems. Chemy Ind. 40, 1287-1288. HOWE, R. W. (1963) The random sampling of cultures of grain weevils. BUZZ.ent. Res. 54, 135-146. KROHNE, H. E. and LINDGREN, D. L. (1958) Susceptibility of life stages of ~~tophi~usoryzae to various fumigants. 3. econ. Et& 51, 157-158. WAIXEY, F. M. (1949) Dosage-mortality correlation with number treated estimated from a parallel sample. Ann. appl. Biol., 36, 196-202. WHITNEY, W. K. and WALK~EN, H. H. (1961) Concentrations of methyl bromide lethal to insects in grain. Mktg. Res. Rep. U.S. De& Agric., agric. Mktg. Serv., Mkt. Qual. Res. Div., No. 511, 25 pp.