- Email: [email protected]
Phosphine resistance in immature stages of a laboratory selected strain of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)
J stored Prod. Res. Vol. Prlrited m Great Bra&n
17. pp.
43 to 52,
1981
0022-474X%1
WJO43-iOSO2.00/0 Pergamon Precc Lid
PHOSPHINE RESISTANCE IN IMMATURE STAGES OF A LABORATORY SELECTED STRAIN OF TRIBOLIUM CASTANEUM (HERBST) (COLEOPTERA: TENEBRIONIDAE) H. NAKAKITA* CSIRO.
Division
(Received
and R. G. WINKS
of Entomology, P.O. Box 1700. A.C.T. 2601. Australia
in final
firm
17 December
1980)
Abstract-Responses to phosphine of immature stages of an adult-selected. phosphine-resistant strain of Tribolium castaneum (Herbst) were studied and compared with those of a phosphinesusceptible strain. Larvae (15 and 20-day old) pre-pupae and pupae (early-, mid- and late-) were examined and found resistant to phosphine. The highest resistances were present in early- and mid-pupae at levels much higher than those selected in the adults. Latent toxic effects were observed following treatment of all stages except 15-day larvae and were most pronounced with 20-day larvae. Respiration rates in a normal atmosphere were similar in the two strains on a body-weight basis and differed in terms of oxygen consumption per individual in accordance with the weight differences between the two strains. Normal respiration rates did not account for. or contribute towards. the observed tolerance differences between the two strains. The oxygen consumption of all examined stages of the resistant strain was reduced less by phosphine than that of the corresponding stages of the susceptible strain but the significance of this in terms of phosphine uptake is not known.
INTRODUCTION
A RECENT global survey of pesticide resistance in stored grain insects revealed that a number of the major pest species, in several countries, has developed resistance to the fumigants phosphine and methyl bromide under field conditions (CHAMP and DYTE 1976). The numbers of phosphine-resistant strains and the countries from which phosphine resistance was recorded were almost twice those for methyl bromide, despite the fact that phosphine has been used as a grain fumigant for a much shorter period than methyl bromide. This strongly suggests that the potential for insects to develop phosphine resistance is greater than that for methyl bromide resistance. Although immature stages such as eggs and pupae are generally more tolerant of fumigants than the corresponding adults, very few data are available that indicate the relative changes in tolerance of the developmental stages resulting from the development of resistance. UPITIS et al. (1973) reported that, following laboratory selection of the adult stage of Sitophilus granarius (L.) with methyl bromide, the tolerance of the immature stages changed very little compared with the change in tolerance of the adult with the result that the adult became the most difficult stage to control. Moreover, laboratory studies of phosphine resistance in a strain of S. granarius indicated that when adults were the stage selected for phosphine resistance, some resistance occurred in the pupal stage but not in other immature stages (ANON. 1974). By contrast, in field strains of Rhyzopertha dominica (F.), BELL et aI. (1977) recently reported resistance in the egg stage of nine out of ten strains in which resistance had been detected in the adult stage. The present study was undertaken to examine differences between a phosphine-resistant and a susceptible strain of Tribdium custuneum (Herbst) in responses of the immature stages to phosphine. * Present
address: Ken 305, Japan.
National
Food
Research
Institute.
2-l-2
43 SPR
17.:2-
A
Kannondai.
Yatabe-Machi.
Tsukuba-Gun.
Ibaraki-
H. NAKAKITA and R. G. WINKS MATERIALS
AND METHODS
Insects The insects used in this study were a phosphine-resistant and a phosphine-susceptible strain of 7Ycastaneum. The phosphine-resistant strain (TCP,A) was derived from laboratory selection of six generations of the adult stage following which resistance in the adult stage was approximately x 5 at the LD 50 and approximately x 7 at the LD,,., level when compared with the susceptible strain (WINKS 1973). The resistance of adults of this strain was determined at the start of the present study and found to be x 4.8 at the LD,, and x 5.6 at the LD99.9. The susceptible strain (CTCJ originated from a collection in 1965 in a produce merchant’s store in Brisbane, Australia. Both strains were reared in a wheat-flour medium containing 5% yeast by weight. In order to obtain immature stages of the required ages, batches of 2000 adults were allowed to oviposit for 24 hr in 200 g of medium in a 700 ml glass jar. Cultures started in this manner were maintained in a controlled-environment room at 25°C and 60% r.h. Larvae, 15 and 20 days old, prepupae and early-, mid- and late-pupae were used in the study. The identification of pre-pupae, early-, mid- and late pupae followed the method of RYAN and NATHANSON (1969). This method is based on the stage of development rather than age, which presumably favours a more discrete tolerance distribution of the developmental stage selected. Larvae of required age and uniform size were collected with a small brush. Fumigation experiments Batches of 100 larvae were weighed and placed in fumigation chambers consisting of 270ml polystyrene tissue culture bottles, each sealed with a rubber septum placed in a perforated screw cap. Pre-pupae and pupae were collected from cultures 25 days after oviposition and held to obtain the required age and stage. Weighed batches of 50 prepupae and of early-, mid- and late-pupae were treated in the same way as for the larvae. For each experiment, three batches were used for each of five concentrations of phosphine and for untreated controls. All experiments were repeated three times at 25°C. The aim of experiments was to examine differences between particular stages of development. Because of this, a fixed exposure period of 6 hr was used in all experiments. This period was chosen to minimise changes in tolerance associated with continued development during treatment and, at the same time, to avoid excessively high concentrations with correspondingly short exposure periods. Phosphine gas was prepared from Phostoxin@ tablets according to the method of KASHI and BOND (1975). The required amounts of phosphine were withdrawn from the source using gas-tight syringes and injected into the fumigation chambers. The concentration of phosphine in the chambers was determined by gas chromatography using the peak-height response of a flame photometric detector. A 1650 x 6 mm OD glass column filled with 80/100 mesh Porapak Q was used at 100°C with N, as the carrier gas. Mortality assessment Following fumigation, the insects were transferred to 30ml jars containing wholewheat flour and held in the rearing room at 25°C and 60% r.h. Larval mortality was assessed at 7-day intervals until all surviving adults had emerged. Newly-formed pupae were removed and placed in separate jars to avoid cannibalism by larvae. Mortality of pre-pupae and pupae was assessed at 3-day intervals following treatment. During final counts of adults that developed from the treated immature stages, deformed adults were recorded. Adults without morphological deformities were considered to be unaffected by phosphine. With the exception of the assessment of latent toxicity, estimates of LCSO and LC99.9 were derived by probit analysis (FINNEY 1971) of the final counts of emerged adults. Respiration measurements The oxygen consumption
of the insects in air in the presence or absence of phosphine
Phosphine resistance in
Tribolium
castaneum
45
was measured with Warburg manometers using 16 ml capacity flasks at 25°C. Carbon dioxide produced by the insects was absorbed by filter paper strips impregnated with KOH (20%) that were placed in the centre well of the flasks. One of the side arms of each Warburg flask was fitted with a silicon rubber septum to permit injection of phosphine into the respirometer. The numbers of insects used in these experiments were: 30 of 20-day larvae, early- and late-pupae and 50 of 15day larvae, pre- and mid-pupae. From 4 to 8 replicates were used for each measurement of oxygen consumption in a normal atmosphere and generally 2 replicates for each dosage of phosphine.
RESULTS
Lethal effects of phosphine
Results of regression analyses, including estimates of the LC5e and the LC99.9r and resistance ratios are given in Table 1. Most data satisfied a linear model based on probit-transformed mortality and log-transformed concentration with heterogeneity due to random deviation about the linear model. The data obtained from mid-pupae of the susceptible strain could be constrained to a linear relationship over most of the response range but, at response levels greater than 90’/& there was evidence of systematic deviation from the linear model (Fig. 1). This deviation, together with the slope and position of the fitted line, suggests that mid-pupae, as sampled, exhibited physiological variation from early-pupae to late-pupae. Complete kill of mid-pupae of the susceptible strain was obtained at dosages of 0.82 and 0.86 mg/l which were considerably lower than the LD99,9 calculated from the line. Mortality data obtained from early- and mid-pupae of the resistant strain showed systematic deviation from the linear model. These data, together with those obtained for the other pupal stages tested, are given in Fig. 1. The order of observed tolerances for both the susceptible and the resistant strain was early-pupae > mid-pupae > late pupae > pre-pupae > 15-day larvae > 20-day larvae. Early-pupae of the susceptible strain were 32 times more tolerant of phosphine than 20-day larvae at the 50% response level and 41 times more tolerant at the 99.9% level. TABLE 1. RESULTSOF ANALYSIS OF CONCENTRATION-MORTALITY DATA FOR IMMATURE STAGES OF A PHOSPHINE-RESISTANTSTRAIN(TCPIA)AND A PHOSPHINE-SUSCEPTIBLE STRAIN (CT&) OF Tribolium castaneum EXPOSED TO PHOSPHINE FOR 6 hr
Resistance factor (a) (b)
Strain
LCSO (mg/l)
Lc99.9 (mg/l)
Slope (kS.E.)
Chi-square*
d.f.
CTC, TCP,A
0.028 0.057
0.086 0.429
6.4 + 0.8 3.5 k 0.2
49 56
18 41
2.0
5.0
CTC., TCPrA
0.022 0.030
0.049 0.123
8.9 k 0.5 5.0 + 0.4
78 192
30 43
1.4
2.5
CTC., TCP,A
0.028 0.055
0.124 0.703
4.8 k 0.3 2.8 k 0.2
81 44
38 34
2.0
5.7
Early-pupae
CTC4 TCP,At
0.706
1.990
6.9 + 0.4
28
28
Mid-pupae
CTC,, TCP,At
0.115 3.5:
t
1.4 * 0.1
123
40
CTC., TCP,A
0.015 0.073
0.186 1.33
Stage 15day larvae
20-day larvae
Pre-pupae
Late-pupae
30.4$
2.8 & 0.2 2.5 + 0.2
* For goodness of fit. t Data indicate systematic deviation from linear model. $ Derived from eye-fitted line (Fig. 1). (a) At the 50% mortality level. (b) At the 99.9% mortality level.
133 68
40 33
4.9
7.2
46
H. NAKAKITA and R. G. WINKS
it Early pupae
l-CTC4 o-----TCP,A
Mid pupae S-
95a-
CTC.,
O--I.-
TCPl A
Late pupae A CT& A..-.-TCP,A’
A /
9( )8( Ig
7(I-
> $ g 5
6( l5( )4( I3(I-
0
,__&___..“--‘---f
2( ll( )-
d
0
l-
ii. 0 .OO’1
0.01
w
..I
0.1
1.0
Phosphine concentration
FIG. I. Concentration-mortality and a phosphine-susceptible
lines for pupal stages strain (CTCJ of Tribolium
10
100
(mg/l)
of a phosphine-resistant strain exposed to phosphine
castuneum
(TCP,A) for 6 hr.
All stages of strain TCP,A that were tested were resistant to phosphine, the early- and mid-pupal stages being highly resistant. The resistance factors for the late-pupal stage were similar to those reported for the adult stage of this strain. Latent efects of phosphine
The effect of phosphine on subsequent development was assessed following treatment of 15-day and 20.day larvae of both strains each with two different concentrations (Fig. 2). Assessment was based on the development to pupal and adult stages of the larvae that > 90
CT&
LPA
FIG. 2. Survival,
E
15 day larvae
100
LPA
TCP, A
LPA
20. day larvae
0)
LPA
2
CTC.q
LPA
LPA
TCP, A
LPA
LPA
through to the adult stage, of ISday and 20.day larvae exposed for 6 hr. Data corrected for mortalities in untreated batches.
to phosphine
FIG. 3. Examples of deformities produced by phosphine treatments of 20-day larvae, prepupae and pupae of a phosphine-susceptible and a phospine-resistant strain of Tribolium castaneum.
H.
48
and R. G. WINKS
NAKAKITA
TABLE 2. THE WEIGHTS OF THE DEVELOPMENTALSTAGES OF A PHOSPHINE-RESISTANTSTRAIN (TCPIA) PHOSPHINE-SUSCEPTIBLE STRAIN (CTQ OF Tribolium castaneum.
Mean
Developmental
batch
Batch size
stage
15-day larvae 20-day larvae Pre-pupae Early-pupae Mid-pupae Late-pupae
weight
(mg) k S.D. batches)
CTC.,
100 100 50 :8
44.9 242.7 146.3 136.8 126.9 123.3
50
+ + + rt + k
(number
26.6 194.4 114.4 106.4 106.2 102.1
k f + k f +
A
of
Probability (t-test)
TCP,A
7.8 (17) 10.3 (45) 1.6(54) 9.8 (72) 3.4(56) 5.2(25)
AND
3.6(32) 20.0(54) 3.6 (48) 2.2 (31) 3.9 (54) 2.1 (24)
Weight ratio (CTC,/TCP,A) 1.7 1.2 1.3 1.3 1.2 1.2
survived the dosages of phosphine. Data were corrected for mortalities in untreated batches. Following treatment of 15day larvae of both strains, a small decrease in the number that subsequently pupated was observed but most of those that pupated completed their development to the adult stage. Treatment of 20-day larvae, on the other hand, produced a significant decrease in the proportion that pupated and in the proportion that subsequently emerged as adults. This was observed in both strains and at both concentrations chosen although the effect was more pronounced at the higher concentration. Of the 20-day larvae that survived the acute lethal effects of the low concentration of phosphine, 33.3% of CT& and 43.7% of TCPIA failed to emerge as adults. At the high concentrations the corresponding mortalities were 90.7% (CTC,J and 84.0% (TCPIA). Apart from the lower survival following treatment of 20-day larvae, it was observed that phosphine treatment of 20-day larvae, pre-pupae and all pupal stages of both strains produced a significant proportion of deformed adults. Examples of the deformities are illustrated in Fig. 3. Deformities were not observed in survivors of 15day larvae treated with phosphine. Weight relationship of the two strains The weight of each developmental stage of both strains was determined (Table 2). A t-test comparison of each stage indicated that all developmental stages of TCP,A were significantly lighter than the corresponding stages of CT&. Weight ratios (CTC4/TCP1A) ranged from 1.2 to 1.7. 100 _ CTC, _
---+I+-4 -
TCP,A
--C----e--
(15 .day I.rv.el *** (20
-A-
day Irrvreb
(Early
.-V-.-V--
A-
IMid.
-pupae) pupa4
E
.V
0.02
0.05
0.1
0.2
0.5
1.0
2.0
Phmphine concentration b’ns/l)
FIG. 4. Inhibitory phosphine-resistant
effect of phosphine on the oxygen consumption strain (TCP,A) and a phosphine-susceptible castaneum.
of developmental stages of a strain (CTC,) of Tribolium
strains
were not significant
between
Differences
+_ 122 k 140 f 7 f I * 9 f 10
3361 2530 724 824 530 852
O2 consumption f SE (jd/hr/g of insects) 1.52 6.15 2.12 2.26 1.35 2.10
O2 consumption per individual (Nhr)
(P > 0.30).
CTC4
15-day larvae 20-day larvae pre-pupae early-pupae mid-pupae late-pupae
Developmentalstage
Strain
TCP,A
3349 2559 136 834 547 835
f + f + + k
154 106 8 I 16 14
Oz consumption + SE (&M/g of insects)
Strain
TABLE 3. RESPIRATION RATESOF DEVELOPMENTALSTAGESOF A PHOSPHINE-SUSCEPTIBLE STRAIN (CTCJ OF Tribolium castaneum.
0.90 4.96 1.69 1.78 1.16 1.70
Ox consumption per individual MN)
1.7 1.2 1.3 1.3 1.2 1.2
Ratio of respiratory rates per individual (CTGWPIA)
AND A PHOSPHINE-RESISTANT STRAIN (TCP,A)
50
H. NAKAKITA
and
R. G. WINKS
Comparison of respiration rates of the two strains in a normal atmosphere
The respiration rates per gram of each stage in a normal atmosphere did not differ significantly between the two strains (Table 3). However, on the basis of oxygen consumption per individual, the respiration rates of all stages differed markedly between the two strains and respiratory ratios (CTC4/TCP1A) thus obtained were similar to the weight ratios for each stage. Eflects of phosphine on respiration rates
Fifteen-day larvae, 20-day larvae and early- and mid-pupae were exposed to a range of phosphine concentrations and their oxygen consumption per gram (t) measured over a 6 hr period. Results thus obtained for each concentration were compared with the oxygen consumption per gram of control batches (c) of each stage in a normal atmosphere and expressed as percent respiratory inhibition (100 (l-t/c)). Marked differences in the level of respiratory inhibition were observed between the two strains over the range of phosphine concentrations (Fig. 4). In all of the stages examined, the degree of respiratory inhibition was much less in TCPIA than in CTC4. DISCUSSION
Mortality data for the larval and pupal stages of the phosphine-resistant strain TCPIA indicate that these stages are also resistant to phosphine. The pupal stage is clearly the most resistant stage with resistance levels well in excess of those of the adult. The data for early- and mid-pupae of this strain, plotted in Fig. 1, show curvature that suggests a protective mechanism which is enhanced with increasing concentration. In both strains, the tolerance of the pupal stage decreased markedly with age and in late-pupae approached levels obtained for adults (WINKS 1973). The resistance level of pupae of TCP,A also decreased with age and the resistance level of late-pupae was similar to that obtained for the adult stage. In practice, phosphine exposure periods are normally in excess of four days. During this time pupae may be expected to continue development towards the adult stage and become less tolerant of phosphine. Thus the higher tolerance levels of early-and mid-pupae and the greater resistance of early- and mid-pupae of resistant strains such as TCP,A, may not be as important in practice as they would be if short exposure periods were used e.g. 1 or 2 days. The deformities observed following treatment of 20-day larvae, pre-pupae and all pupal stages were similar to those reported following treatment of 4th instar larvae of Tenebroides mauritanicus (L.) with phosphine (BOND and UPITIS 1973). They suggested that phosphine affected an aspect of larval metabolism involved with the formation of pupal and adult tissues. Thus, if pupal and adult tissue had either not developed or had not developed to a vulnerable stage in 15-day larvae of 7: castaneum, a similar explanation might account for the absence of deformities following treatment of these larvae in the present study. It is also possible, however, that repair of sub-lethal damage to 15day larvae occurs during subsequent larval development. Respiration of insects has been shown to have an important role in the uptake and subsequent toxicity of fumigants (COTTON 1932; E~OND and MONRO 1967). Moreover, in some species, differences in tolerance between developmental stages and between species are correlated with respiration rates. Some fumigants such as hydrogen cyanide and phosphine at sublethal concentrations, suppress oxygen consumption but it is not clear what effect this has on the uptake of fumigant. Respiratory rate, however, is thought to be the chief factor controlling the amount of fumigant absorbed and hence fumigant toxicity and any secondary effect such as the level of respiratory inhibition. This being so, strains with lower metabolic rates, e.g. less active strains, would be more tolerant than those with higher rates. This could account for small resistance factors. Respiratory rates of the two strains were compared for this reason. Respiratory rates per individual of the resistant strain in a normal atmosphere were lower, for all stages examined, than those of the susceptible strain. If uptake of phosphine
Phosphineresistancein Tribolium castaneum
51
was due largely to normal respiration activity, the resistant strain would absorb less phosphine in a given period of time by a factor equal to the ratio of respiratory rates per individual. However, since the respiration rates per gram were similar between the two strains for all stages examined the amount of phosphine absorbed by each stage for a given period of time, expressed as a dose per unit weight, would also be similar between the two strains. Thus the respiration rates in normal atmospheres do not account for, or contribute to the resistance of strain TCP,A but they do provide a basis for assessing differences in respiratory inhibition due to phosphine. When both larval and two pupal stages of both strains were exposed to a range of phosphine concentrations the respiration of the resistant strain was inhibited less than that of the susceptible strain. It is not known what effect the observed inhibition would have on the amount of phosphine absorbed nor on the observed tolerance differences between the two strains. However, since cytochrome oxidase appears to be an important site of phosphine action (KASHI and CHEFURKA 1976; NAKAKITA 1976) the differences in respiratory inhibition may indicate that a component of resistance is due to reduced mitochondrial sensitivity. The present study indicated also, that when adults of T castuneum are selected for phosphine resistance, the larval and pupal stages may become resistant to phosphine with the highest levels of resistance occurring in the early stages of pupal development. The highest level occurring in other than the stage selected differs from the findings of GEORGHIOU et al. (1966) who separately selected larvae and adults of Cule.u ,fatigans with propoxur and obtained the highest levels of resistance in the stage selected. The results of the present study also differ from those obtained from a phosphine-resistant strain of S. ~ranarius which suggested that when adults are the stage selected, some resistance may occur in the pupal stages but not in other immature stages (ANON. 1974) and further differ from the results of UPITIS et al. (1973) in which the level of methyl bromide resistance in pupae was less than that in adults of a strain of S. yranarius. This latter strain arose from 50 selections of the adult stage with methyl bromide. over 69 generations. Thus, the pattern of resistance in phosphine-resistant strains appears, at this stage, to vary. However, in spite of the differences among the results of the various studies it seems likely that some or all stages of phosphine-resistant strains in which resistance is either detected or selected in the adult, will be resistant and possibly more so than the adult. Moreover, where high levels of phosphine resistance occur in pupal stages similar to those found in early- and mid-pupae of TCP,A these levels of resistance could pose a threat to the usefulness of this fumigant especially where exposure periods are short either by design or as a result of gas leakage. .4ckr~owledgrmrnts_We would like to express our thanks this study. One of the authors, Dr H. Nakakita, would Fellowship which enabled him to pursue these studies in to Dr D. F. Waterhouse. Mr S. W Bailey and Dr B. R.
to Mr G. P. Smith for his technical like to express his gratitude for the the CSIRO, Division of Entomology Champ for their encouragement and
assistance during CSIRO Research and in particular support.
REFERENCES ANON, (1974) Fumigants-Mode of action. use and residue analysis. Res. Inst. Loudon. Ot~f.. Rex Brmch Rep. 1973. p. 186. BELL, C. H.. HOLE, B. D. and EVANS, P. H. (1977) The occurrence of resistance to phosphine in adult and egg stages of Rhpzopertha domirlica (F.) (Coleoptera: Bostrichidae). J. srorrd Prod. Res. 13, 91-94. BOND,E. J. and MONRO. H. A. U. (1967) The role of oxygen in the toxicity of fumigants to insects. J. stored. Prod. Rex 3. 295-3 IO. BOND, E. J. and UPITIS, E. (1973) Response of three insects to sublethal doses of phosphine. J. stored Prod. Rex 8.307-313. CHAMP, B. R. and DYTE, C. E. (1976) ‘Report of the FAO Global Survey of Pesticide Susceptibility of Stored Grain Pests’ FAO PIunt Prod. Prof. Serv. No. 5 (FAO. Rome). COTTON. R. T. (1932) The relation of respiratory metabolism of insects to their susceptibility to fumigants. .I. rcon. E,lf. 25, 1088-l 103. FINNEY, D. J. (1971) Probit Analysis 3rd Edn. Cambridge Univ. Press, Cambridge. GEORGHIOLJ,G. P.. METCALF. R. L. and GIDDEN. F. E. (1966) Carbamate resistance in mosquitos. SelectIon ot C&x pipiens futigans Wiedemann(= C. quinquefisciutus Say) for resistance to Baygon. Bull. Wld Hkh Org. 35, 691-708. \ P.R 17Q R
52
H. NAKAKITA and R. G. WINKS
KASHI, K. P. and BOND, E. J. (1975) The toxic action
of phosphine: Role of carbon dioxide on the toxicity of phosphine to Sitophilus granarius (L.) and Tribolium confisum DuVal. J. stored Prod. Res. 11, 9-15. KASHI, K. P. and CHEFURKA, W. (1976) The effect of phosphine on the absorption and circular dichroic spectra of cytochrome c and cytochrome oxidase. Pesticide Biochem. Physiol. 6, 35C-362. NAKAKITA. H. (1976) The inhibitory site of phosphine. J. Pesticide Sci. 1, 235-238. RYAN, M. F. and NATHANSON, M. (1969) A relationship between handling and a developmental abnormality in the flour beetle, Tribolium spp. Bull. ent. Rex 59, 435-440. UPITIS,E., MONRO,H. A. U. and BOND, E. J. (1973) Some aspects of inheritance of tolerance to methyl bromide by Sitophilus granarius (L.). J. stored Prod. Res. 9. 13-17. WINKS, R. G. (1973) Some aspects of the response of Tribolium castaneum (Herbst) to phosphine. Ph.D. Thesis, University of London.
17. pp.
43 to 52,
1981
0022-474X%1
WJO43-iOSO2.00/0 Pergamon Precc Lid
PHOSPHINE RESISTANCE IN IMMATURE STAGES OF A LABORATORY SELECTED STRAIN OF TRIBOLIUM CASTANEUM (HERBST) (COLEOPTERA: TENEBRIONIDAE) H. NAKAKITA* CSIRO.
Division
(Received
and R. G. WINKS
of Entomology, P.O. Box 1700. A.C.T. 2601. Australia
in final
firm
17 December
1980)
Abstract-Responses to phosphine of immature stages of an adult-selected. phosphine-resistant strain of Tribolium castaneum (Herbst) were studied and compared with those of a phosphinesusceptible strain. Larvae (15 and 20-day old) pre-pupae and pupae (early-, mid- and late-) were examined and found resistant to phosphine. The highest resistances were present in early- and mid-pupae at levels much higher than those selected in the adults. Latent toxic effects were observed following treatment of all stages except 15-day larvae and were most pronounced with 20-day larvae. Respiration rates in a normal atmosphere were similar in the two strains on a body-weight basis and differed in terms of oxygen consumption per individual in accordance with the weight differences between the two strains. Normal respiration rates did not account for. or contribute towards. the observed tolerance differences between the two strains. The oxygen consumption of all examined stages of the resistant strain was reduced less by phosphine than that of the corresponding stages of the susceptible strain but the significance of this in terms of phosphine uptake is not known.
INTRODUCTION
A RECENT global survey of pesticide resistance in stored grain insects revealed that a number of the major pest species, in several countries, has developed resistance to the fumigants phosphine and methyl bromide under field conditions (CHAMP and DYTE 1976). The numbers of phosphine-resistant strains and the countries from which phosphine resistance was recorded were almost twice those for methyl bromide, despite the fact that phosphine has been used as a grain fumigant for a much shorter period than methyl bromide. This strongly suggests that the potential for insects to develop phosphine resistance is greater than that for methyl bromide resistance. Although immature stages such as eggs and pupae are generally more tolerant of fumigants than the corresponding adults, very few data are available that indicate the relative changes in tolerance of the developmental stages resulting from the development of resistance. UPITIS et al. (1973) reported that, following laboratory selection of the adult stage of Sitophilus granarius (L.) with methyl bromide, the tolerance of the immature stages changed very little compared with the change in tolerance of the adult with the result that the adult became the most difficult stage to control. Moreover, laboratory studies of phosphine resistance in a strain of S. granarius indicated that when adults were the stage selected for phosphine resistance, some resistance occurred in the pupal stage but not in other immature stages (ANON. 1974). By contrast, in field strains of Rhyzopertha dominica (F.), BELL et aI. (1977) recently reported resistance in the egg stage of nine out of ten strains in which resistance had been detected in the adult stage. The present study was undertaken to examine differences between a phosphine-resistant and a susceptible strain of Tribdium custuneum (Herbst) in responses of the immature stages to phosphine. * Present
address: Ken 305, Japan.
National
Food
Research
Institute.
2-l-2
43 SPR
17.:2-
A
Kannondai.
Yatabe-Machi.
Tsukuba-Gun.
Ibaraki-
H. NAKAKITA and R. G. WINKS MATERIALS
AND METHODS
Insects The insects used in this study were a phosphine-resistant and a phosphine-susceptible strain of 7Ycastaneum. The phosphine-resistant strain (TCP,A) was derived from laboratory selection of six generations of the adult stage following which resistance in the adult stage was approximately x 5 at the LD 50 and approximately x 7 at the LD,,., level when compared with the susceptible strain (WINKS 1973). The resistance of adults of this strain was determined at the start of the present study and found to be x 4.8 at the LD,, and x 5.6 at the LD99.9. The susceptible strain (CTCJ originated from a collection in 1965 in a produce merchant’s store in Brisbane, Australia. Both strains were reared in a wheat-flour medium containing 5% yeast by weight. In order to obtain immature stages of the required ages, batches of 2000 adults were allowed to oviposit for 24 hr in 200 g of medium in a 700 ml glass jar. Cultures started in this manner were maintained in a controlled-environment room at 25°C and 60% r.h. Larvae, 15 and 20 days old, prepupae and early-, mid- and late-pupae were used in the study. The identification of pre-pupae, early-, mid- and late pupae followed the method of RYAN and NATHANSON (1969). This method is based on the stage of development rather than age, which presumably favours a more discrete tolerance distribution of the developmental stage selected. Larvae of required age and uniform size were collected with a small brush. Fumigation experiments Batches of 100 larvae were weighed and placed in fumigation chambers consisting of 270ml polystyrene tissue culture bottles, each sealed with a rubber septum placed in a perforated screw cap. Pre-pupae and pupae were collected from cultures 25 days after oviposition and held to obtain the required age and stage. Weighed batches of 50 prepupae and of early-, mid- and late-pupae were treated in the same way as for the larvae. For each experiment, three batches were used for each of five concentrations of phosphine and for untreated controls. All experiments were repeated three times at 25°C. The aim of experiments was to examine differences between particular stages of development. Because of this, a fixed exposure period of 6 hr was used in all experiments. This period was chosen to minimise changes in tolerance associated with continued development during treatment and, at the same time, to avoid excessively high concentrations with correspondingly short exposure periods. Phosphine gas was prepared from Phostoxin@ tablets according to the method of KASHI and BOND (1975). The required amounts of phosphine were withdrawn from the source using gas-tight syringes and injected into the fumigation chambers. The concentration of phosphine in the chambers was determined by gas chromatography using the peak-height response of a flame photometric detector. A 1650 x 6 mm OD glass column filled with 80/100 mesh Porapak Q was used at 100°C with N, as the carrier gas. Mortality assessment Following fumigation, the insects were transferred to 30ml jars containing wholewheat flour and held in the rearing room at 25°C and 60% r.h. Larval mortality was assessed at 7-day intervals until all surviving adults had emerged. Newly-formed pupae were removed and placed in separate jars to avoid cannibalism by larvae. Mortality of pre-pupae and pupae was assessed at 3-day intervals following treatment. During final counts of adults that developed from the treated immature stages, deformed adults were recorded. Adults without morphological deformities were considered to be unaffected by phosphine. With the exception of the assessment of latent toxicity, estimates of LCSO and LC99.9 were derived by probit analysis (FINNEY 1971) of the final counts of emerged adults. Respiration measurements The oxygen consumption
of the insects in air in the presence or absence of phosphine
Phosphine resistance in
Tribolium
castaneum
45
was measured with Warburg manometers using 16 ml capacity flasks at 25°C. Carbon dioxide produced by the insects was absorbed by filter paper strips impregnated with KOH (20%) that were placed in the centre well of the flasks. One of the side arms of each Warburg flask was fitted with a silicon rubber septum to permit injection of phosphine into the respirometer. The numbers of insects used in these experiments were: 30 of 20-day larvae, early- and late-pupae and 50 of 15day larvae, pre- and mid-pupae. From 4 to 8 replicates were used for each measurement of oxygen consumption in a normal atmosphere and generally 2 replicates for each dosage of phosphine.
RESULTS
Lethal effects of phosphine
Results of regression analyses, including estimates of the LC5e and the LC99.9r and resistance ratios are given in Table 1. Most data satisfied a linear model based on probit-transformed mortality and log-transformed concentration with heterogeneity due to random deviation about the linear model. The data obtained from mid-pupae of the susceptible strain could be constrained to a linear relationship over most of the response range but, at response levels greater than 90’/& there was evidence of systematic deviation from the linear model (Fig. 1). This deviation, together with the slope and position of the fitted line, suggests that mid-pupae, as sampled, exhibited physiological variation from early-pupae to late-pupae. Complete kill of mid-pupae of the susceptible strain was obtained at dosages of 0.82 and 0.86 mg/l which were considerably lower than the LD99,9 calculated from the line. Mortality data obtained from early- and mid-pupae of the resistant strain showed systematic deviation from the linear model. These data, together with those obtained for the other pupal stages tested, are given in Fig. 1. The order of observed tolerances for both the susceptible and the resistant strain was early-pupae > mid-pupae > late pupae > pre-pupae > 15-day larvae > 20-day larvae. Early-pupae of the susceptible strain were 32 times more tolerant of phosphine than 20-day larvae at the 50% response level and 41 times more tolerant at the 99.9% level. TABLE 1. RESULTSOF ANALYSIS OF CONCENTRATION-MORTALITY DATA FOR IMMATURE STAGES OF A PHOSPHINE-RESISTANTSTRAIN(TCPIA)AND A PHOSPHINE-SUSCEPTIBLE STRAIN (CT&) OF Tribolium castaneum EXPOSED TO PHOSPHINE FOR 6 hr
Resistance factor (a) (b)
Strain
LCSO (mg/l)
Lc99.9 (mg/l)
Slope (kS.E.)
Chi-square*
d.f.
CTC, TCP,A
0.028 0.057
0.086 0.429
6.4 + 0.8 3.5 k 0.2
49 56
18 41
2.0
5.0
CTC., TCPrA
0.022 0.030
0.049 0.123
8.9 k 0.5 5.0 + 0.4
78 192
30 43
1.4
2.5
CTC., TCP,A
0.028 0.055
0.124 0.703
4.8 k 0.3 2.8 k 0.2
81 44
38 34
2.0
5.7
Early-pupae
CTC4 TCP,At
0.706
1.990
6.9 + 0.4
28
28
Mid-pupae
CTC,, TCP,At
0.115 3.5:
t
1.4 * 0.1
123
40
CTC., TCP,A
0.015 0.073
0.186 1.33
Stage 15day larvae
20-day larvae
Pre-pupae
Late-pupae
30.4$
2.8 & 0.2 2.5 + 0.2
* For goodness of fit. t Data indicate systematic deviation from linear model. $ Derived from eye-fitted line (Fig. 1). (a) At the 50% mortality level. (b) At the 99.9% mortality level.
133 68
40 33
4.9
7.2
46
H. NAKAKITA and R. G. WINKS
it Early pupae
l-CTC4 o-----TCP,A
Mid pupae S-
95a-
CTC.,
O--I.-
TCPl A
Late pupae A CT& A..-.-TCP,A’
A /
9( )8( Ig
7(I-
> $ g 5
6( l5( )4( I3(I-
0
,__&___..“--‘---f
2( ll( )-
d
0
l-
ii. 0 .OO’1
0.01
w
..I
0.1
1.0
Phosphine concentration
FIG. I. Concentration-mortality and a phosphine-susceptible
lines for pupal stages strain (CTCJ of Tribolium
10
100
(mg/l)
of a phosphine-resistant strain exposed to phosphine
castuneum
(TCP,A) for 6 hr.
All stages of strain TCP,A that were tested were resistant to phosphine, the early- and mid-pupal stages being highly resistant. The resistance factors for the late-pupal stage were similar to those reported for the adult stage of this strain. Latent efects of phosphine
The effect of phosphine on subsequent development was assessed following treatment of 15-day and 20.day larvae of both strains each with two different concentrations (Fig. 2). Assessment was based on the development to pupal and adult stages of the larvae that > 90
CT&
LPA
FIG. 2. Survival,
E
15 day larvae
100
LPA
TCP, A
LPA
20. day larvae
0)
LPA
2
CTC.q
LPA
LPA
TCP, A
LPA
LPA
through to the adult stage, of ISday and 20.day larvae exposed for 6 hr. Data corrected for mortalities in untreated batches.
to phosphine
FIG. 3. Examples of deformities produced by phosphine treatments of 20-day larvae, prepupae and pupae of a phosphine-susceptible and a phospine-resistant strain of Tribolium castaneum.
H.
48
and R. G. WINKS
NAKAKITA
TABLE 2. THE WEIGHTS OF THE DEVELOPMENTALSTAGES OF A PHOSPHINE-RESISTANTSTRAIN (TCPIA) PHOSPHINE-SUSCEPTIBLE STRAIN (CTQ OF Tribolium castaneum.
Mean
Developmental
batch
Batch size
stage
15-day larvae 20-day larvae Pre-pupae Early-pupae Mid-pupae Late-pupae
weight
(mg) k S.D. batches)
CTC.,
100 100 50 :8
44.9 242.7 146.3 136.8 126.9 123.3
50
+ + + rt + k
(number
26.6 194.4 114.4 106.4 106.2 102.1
k f + k f +
A
of
Probability (t-test)
TCP,A
7.8 (17) 10.3 (45) 1.6(54) 9.8 (72) 3.4(56) 5.2(25)
AND
3.6(32) 20.0(54) 3.6 (48) 2.2 (31) 3.9 (54) 2.1 (24)
Weight ratio (CTC,/TCP,A) 1.7 1.2 1.3 1.3 1.2 1.2
survived the dosages of phosphine. Data were corrected for mortalities in untreated batches. Following treatment of 15day larvae of both strains, a small decrease in the number that subsequently pupated was observed but most of those that pupated completed their development to the adult stage. Treatment of 20-day larvae, on the other hand, produced a significant decrease in the proportion that pupated and in the proportion that subsequently emerged as adults. This was observed in both strains and at both concentrations chosen although the effect was more pronounced at the higher concentration. Of the 20-day larvae that survived the acute lethal effects of the low concentration of phosphine, 33.3% of CT& and 43.7% of TCPIA failed to emerge as adults. At the high concentrations the corresponding mortalities were 90.7% (CTC,J and 84.0% (TCPIA). Apart from the lower survival following treatment of 20-day larvae, it was observed that phosphine treatment of 20-day larvae, pre-pupae and all pupal stages of both strains produced a significant proportion of deformed adults. Examples of the deformities are illustrated in Fig. 3. Deformities were not observed in survivors of 15day larvae treated with phosphine. Weight relationship of the two strains The weight of each developmental stage of both strains was determined (Table 2). A t-test comparison of each stage indicated that all developmental stages of TCP,A were significantly lighter than the corresponding stages of CT&. Weight ratios (CTC4/TCP1A) ranged from 1.2 to 1.7. 100 _ CTC, _
---+I+-4 -
TCP,A
--C----e--
(15 .day I.rv.el *** (20
-A-
day Irrvreb
(Early
.-V-.-V--
A-
IMid.
-pupae) pupa4
E
.V
0.02
0.05
0.1
0.2
0.5
1.0
2.0
Phmphine concentration b’ns/l)
FIG. 4. Inhibitory phosphine-resistant
effect of phosphine on the oxygen consumption strain (TCP,A) and a phosphine-susceptible castaneum.
of developmental stages of a strain (CTC,) of Tribolium
strains
were not significant
between
Differences
+_ 122 k 140 f 7 f I * 9 f 10
3361 2530 724 824 530 852
O2 consumption f SE (jd/hr/g of insects) 1.52 6.15 2.12 2.26 1.35 2.10
O2 consumption per individual (Nhr)
(P > 0.30).
CTC4
15-day larvae 20-day larvae pre-pupae early-pupae mid-pupae late-pupae
Developmentalstage
Strain
TCP,A
3349 2559 136 834 547 835
f + f + + k
154 106 8 I 16 14
Oz consumption + SE (&M/g of insects)
Strain
TABLE 3. RESPIRATION RATESOF DEVELOPMENTALSTAGESOF A PHOSPHINE-SUSCEPTIBLE STRAIN (CTCJ OF Tribolium castaneum.
0.90 4.96 1.69 1.78 1.16 1.70
Ox consumption per individual MN)
1.7 1.2 1.3 1.3 1.2 1.2
Ratio of respiratory rates per individual (CTGWPIA)
AND A PHOSPHINE-RESISTANT STRAIN (TCP,A)
50
H. NAKAKITA
and
R. G. WINKS
Comparison of respiration rates of the two strains in a normal atmosphere
The respiration rates per gram of each stage in a normal atmosphere did not differ significantly between the two strains (Table 3). However, on the basis of oxygen consumption per individual, the respiration rates of all stages differed markedly between the two strains and respiratory ratios (CTC4/TCP1A) thus obtained were similar to the weight ratios for each stage. Eflects of phosphine on respiration rates
Fifteen-day larvae, 20-day larvae and early- and mid-pupae were exposed to a range of phosphine concentrations and their oxygen consumption per gram (t) measured over a 6 hr period. Results thus obtained for each concentration were compared with the oxygen consumption per gram of control batches (c) of each stage in a normal atmosphere and expressed as percent respiratory inhibition (100 (l-t/c)). Marked differences in the level of respiratory inhibition were observed between the two strains over the range of phosphine concentrations (Fig. 4). In all of the stages examined, the degree of respiratory inhibition was much less in TCPIA than in CTC4. DISCUSSION
Mortality data for the larval and pupal stages of the phosphine-resistant strain TCPIA indicate that these stages are also resistant to phosphine. The pupal stage is clearly the most resistant stage with resistance levels well in excess of those of the adult. The data for early- and mid-pupae of this strain, plotted in Fig. 1, show curvature that suggests a protective mechanism which is enhanced with increasing concentration. In both strains, the tolerance of the pupal stage decreased markedly with age and in late-pupae approached levels obtained for adults (WINKS 1973). The resistance level of pupae of TCP,A also decreased with age and the resistance level of late-pupae was similar to that obtained for the adult stage. In practice, phosphine exposure periods are normally in excess of four days. During this time pupae may be expected to continue development towards the adult stage and become less tolerant of phosphine. Thus the higher tolerance levels of early-and mid-pupae and the greater resistance of early- and mid-pupae of resistant strains such as TCP,A, may not be as important in practice as they would be if short exposure periods were used e.g. 1 or 2 days. The deformities observed following treatment of 20-day larvae, pre-pupae and all pupal stages were similar to those reported following treatment of 4th instar larvae of Tenebroides mauritanicus (L.) with phosphine (BOND and UPITIS 1973). They suggested that phosphine affected an aspect of larval metabolism involved with the formation of pupal and adult tissues. Thus, if pupal and adult tissue had either not developed or had not developed to a vulnerable stage in 15-day larvae of 7: castaneum, a similar explanation might account for the absence of deformities following treatment of these larvae in the present study. It is also possible, however, that repair of sub-lethal damage to 15day larvae occurs during subsequent larval development. Respiration of insects has been shown to have an important role in the uptake and subsequent toxicity of fumigants (COTTON 1932; E~OND and MONRO 1967). Moreover, in some species, differences in tolerance between developmental stages and between species are correlated with respiration rates. Some fumigants such as hydrogen cyanide and phosphine at sublethal concentrations, suppress oxygen consumption but it is not clear what effect this has on the uptake of fumigant. Respiratory rate, however, is thought to be the chief factor controlling the amount of fumigant absorbed and hence fumigant toxicity and any secondary effect such as the level of respiratory inhibition. This being so, strains with lower metabolic rates, e.g. less active strains, would be more tolerant than those with higher rates. This could account for small resistance factors. Respiratory rates of the two strains were compared for this reason. Respiratory rates per individual of the resistant strain in a normal atmosphere were lower, for all stages examined, than those of the susceptible strain. If uptake of phosphine
Phosphineresistancein Tribolium castaneum
51
was due largely to normal respiration activity, the resistant strain would absorb less phosphine in a given period of time by a factor equal to the ratio of respiratory rates per individual. However, since the respiration rates per gram were similar between the two strains for all stages examined the amount of phosphine absorbed by each stage for a given period of time, expressed as a dose per unit weight, would also be similar between the two strains. Thus the respiration rates in normal atmospheres do not account for, or contribute to the resistance of strain TCP,A but they do provide a basis for assessing differences in respiratory inhibition due to phosphine. When both larval and two pupal stages of both strains were exposed to a range of phosphine concentrations the respiration of the resistant strain was inhibited less than that of the susceptible strain. It is not known what effect the observed inhibition would have on the amount of phosphine absorbed nor on the observed tolerance differences between the two strains. However, since cytochrome oxidase appears to be an important site of phosphine action (KASHI and CHEFURKA 1976; NAKAKITA 1976) the differences in respiratory inhibition may indicate that a component of resistance is due to reduced mitochondrial sensitivity. The present study indicated also, that when adults of T castuneum are selected for phosphine resistance, the larval and pupal stages may become resistant to phosphine with the highest levels of resistance occurring in the early stages of pupal development. The highest level occurring in other than the stage selected differs from the findings of GEORGHIOU et al. (1966) who separately selected larvae and adults of Cule.u ,fatigans with propoxur and obtained the highest levels of resistance in the stage selected. The results of the present study also differ from those obtained from a phosphine-resistant strain of S. ~ranarius which suggested that when adults are the stage selected, some resistance may occur in the pupal stages but not in other immature stages (ANON. 1974) and further differ from the results of UPITIS et al. (1973) in which the level of methyl bromide resistance in pupae was less than that in adults of a strain of S. yranarius. This latter strain arose from 50 selections of the adult stage with methyl bromide. over 69 generations. Thus, the pattern of resistance in phosphine-resistant strains appears, at this stage, to vary. However, in spite of the differences among the results of the various studies it seems likely that some or all stages of phosphine-resistant strains in which resistance is either detected or selected in the adult, will be resistant and possibly more so than the adult. Moreover, where high levels of phosphine resistance occur in pupal stages similar to those found in early- and mid-pupae of TCP,A these levels of resistance could pose a threat to the usefulness of this fumigant especially where exposure periods are short either by design or as a result of gas leakage. .4ckr~owledgrmrnts_We would like to express our thanks this study. One of the authors, Dr H. Nakakita, would Fellowship which enabled him to pursue these studies in to Dr D. F. Waterhouse. Mr S. W Bailey and Dr B. R.
to Mr G. P. Smith for his technical like to express his gratitude for the the CSIRO, Division of Entomology Champ for their encouragement and
assistance during CSIRO Research and in particular support.
REFERENCES ANON, (1974) Fumigants-Mode of action. use and residue analysis. Res. Inst. Loudon. Ot~f.. Rex Brmch Rep. 1973. p. 186. BELL, C. H.. HOLE, B. D. and EVANS, P. H. (1977) The occurrence of resistance to phosphine in adult and egg stages of Rhpzopertha domirlica (F.) (Coleoptera: Bostrichidae). J. srorrd Prod. Res. 13, 91-94. BOND,E. J. and MONRO. H. A. U. (1967) The role of oxygen in the toxicity of fumigants to insects. J. stored. Prod. Rex 3. 295-3 IO. BOND, E. J. and UPITIS, E. (1973) Response of three insects to sublethal doses of phosphine. J. stored Prod. Rex 8.307-313. CHAMP, B. R. and DYTE, C. E. (1976) ‘Report of the FAO Global Survey of Pesticide Susceptibility of Stored Grain Pests’ FAO PIunt Prod. Prof. Serv. No. 5 (FAO. Rome). COTTON. R. T. (1932) The relation of respiratory metabolism of insects to their susceptibility to fumigants. .I. rcon. E,lf. 25, 1088-l 103. FINNEY, D. J. (1971) Probit Analysis 3rd Edn. Cambridge Univ. Press, Cambridge. GEORGHIOLJ,G. P.. METCALF. R. L. and GIDDEN. F. E. (1966) Carbamate resistance in mosquitos. SelectIon ot C&x pipiens futigans Wiedemann(= C. quinquefisciutus Say) for resistance to Baygon. Bull. Wld Hkh Org. 35, 691-708. \ P.R 17Q R
52
H. NAKAKITA and R. G. WINKS
KASHI, K. P. and BOND, E. J. (1975) The toxic action
of phosphine: Role of carbon dioxide on the toxicity of phosphine to Sitophilus granarius (L.) and Tribolium confisum DuVal. J. stored Prod. Res. 11, 9-15. KASHI, K. P. and CHEFURKA, W. (1976) The effect of phosphine on the absorption and circular dichroic spectra of cytochrome c and cytochrome oxidase. Pesticide Biochem. Physiol. 6, 35C-362. NAKAKITA. H. (1976) The inhibitory site of phosphine. J. Pesticide Sci. 1, 235-238. RYAN, M. F. and NATHANSON, M. (1969) A relationship between handling and a developmental abnormality in the flour beetle, Tribolium spp. Bull. ent. Rex 59, 435-440. UPITIS,E., MONRO,H. A. U. and BOND, E. J. (1973) Some aspects of inheritance of tolerance to methyl bromide by Sitophilus granarius (L.). J. stored Prod. Res. 9. 13-17. WINKS, R. G. (1973) Some aspects of the response of Tribolium castaneum (Herbst) to phosphine. Ph.D. Thesis, University of London.