P H Y S I O L O G I C A L A S P E C T S O F I N S E C T R E S I S T A N C E T O I N S E C T I C I D E S
L . E . C H A D W I C K
Medical Laboratories, Army Chemical Center, Maryland
I. Introduction I3 3
II. The Genetic Basis of Acquired Resistance 134 III. Types of Physiological Mechanisms in Resistance 134
A. Behavior 135
B. Structure I3 6
C. Penetration I3 6
D. Storage 1 37
E. Excretion 1 38
F. Detoxification 9 i 3
G. Decreased Sensitivity 141 H. By-pass Mechanisms 1^2
IV. Summary 1 43
References
I. Introduction
The ability of insects and other arthropods to develop tolerance toward chemical measures intended for their control has been recog
nized in agriculture for at least 50 years (Babers, 1949, 1953; Babers and Pratt, 1951). The problem is not, therefore, merely an unwelcome by-product of the recent widespread use of synthetic insecticides. One may note too that there is invariably a great range in tolerance for any given poison among different insect species or populations, some of these being from the first so resistant that control with the selected agent is impractical. From this point of view, one might say that resistance has always been with us.
Nevertheless, current interest is focused on those instances in which previously susceptible populations have become uncontrollable by one or more of the new synthetics. Examples with serious medical implica
tions are already numerous (Simmons, 1954). A comparable situation is developing in economic entomology (Babers, 1953), and many other examples, not yet encountered in actual control operations, have been provided by laboratory selection.
133
134 L . Ε . CHADWICK
The phenomenon of resistance to insecticides has, however, a broader significance than is mirrored in its immediate impact on medical and economic problems. It provides an experimental approach, on a scale hardly to be duplicated elsewhere, to the study of evolution in multi
cellular organisms. The geneticist, the ecologist, and the student of animal behavior all may discover here much to engage their best effort, plus an unusual opportunity of correlating their findings with fundamental physiological mechanisms, the elucidation of which will keep an army of physiologists and biochemists occupied productively for many years. To some, resistance forebodes only disaster; but it has also its virtues. Not the least of these lies in the impetus it is already giving to basic research in all the numerous disciplines on which it impinges.
It is only through such basic advances that we may hope ultimately to attain not merely a firmer control of harmful insects than we enjoy today, but also a deeper understanding of many more far-reaching biological problems.
II. The genetic basis of acquired resistance
All instances of acquired resistance that have been analyzed geneti
cally appear to depend on a shift that has occurred in the average genetic make-up of the population concerned. That is to say, resistance is the consequence of selection pressure exerted by the presence of the toxicant in the environment, rather than the result of a build-up of tolerance in the individual following repeated or continuous sublethal exposure. Moreover, the inheritance pattern is usually, but not always, consistent with the view that multiple genetic factors are concerned (Hough, 1934; Dickson, 1941; Yust et al, 1943; Bruce and Decker, 1950, 1951; Harrison, 1951, 1952; La Face, 1952; Oopenorth and Dres
den, 1952; Cochran et al, 1952; Crow, 1952; March, 1952; Busvine, 1953; Harrison, 1953; Keiding, 1953; Newman, 1953; Norton, 1953;
Tsukamoto and Ogaki, 1953; Harrison, 1954, Milani, 1954; B. J. Har
rison, personal communication).
III. Types of physiological mechanisms in resistance
Paralleling this diversity in the genetics of resistance is a multiplicity of physiological mechanisms. These may be separated into two princi
pal categories: (1) mechanisms that enable the insect to avoid accumu
lating a lethal dose; and (2) mechanisms that allow the insect to cope successfully with an otherwise lethal dose once it has been acquired.
INSECT RESISTANCE TO INSECTICIDES 135 The following outline indicates the order in which these mechanisms will be considered in the discussion below:
Types of Physiological Mechanisms in Resistance
Avoidance Disposal
a. Behavior d. Storage ( ? )
b. Gross structural changes ( ? ) e. Excretion ( ? ) c. Reduced absorption f. Metabolism
g. Lowered sensitivity h. By-pass systems ( ? )
Proof that certain types of mechanisms actually participate in resist
ance is not conclusive, in the opinion of the writer, and these are indicated with the question marks ( ? ) .
A. B E H A V I O R
The first report of resistance due to an alteration in normal behavior was that of Hough (1928), who observed that arsenic-resistant codling moth larvae entered treated fruit in such a way as to ingest less poison from the surface than was consumed by normally susceptible larvae.
Behavioral changes have also been asserted to play a part in the resistance of scale insects to HCN fumigation (Gray and Kirkpatrick, 1929; Hardman and Craig, 1941), but these conclusions and some of the data on which they were based have been questioned (Quayle, 1942; Yust, 1952), and evidence for another sort of mechanism has been produced (Yust and Shelden, 1952).
An interesting example of a protective change in behavior has been reported for hive bees by Newell (1946). Some stocks of bees are resistant to the bacterial disease known as American foulbrood because of the promptness with which they remove from the comb the bodies of larvae that have succumbed. Sporulation and further spread of the disease within the hive are thus prevented, whereas colonies that are more sluggish about their sanitation are decimated. Perhaps one should draw a moral from this case.
Reports from the field that changes in behavior are contributing to the resistance to DDT * of flies and mosquitoes are numerous (flies:
Bruce, 1949; King and Gahan, 1949; Decker, 1950; Bruce and Decker, 1950; Decker and Bruce, 1951; Morrison, 1951; Decker, 1952; Silverman and Mer, 1952; Wiesman, personal communication; mosquitoes: Muir- head-Thompson, 1947a, 1947b; Hadaway, 1950; Ludvik et al, 1951;
Trapido, 1951, 1952, 1953; Hess, 1953). In general these statements assert
136 L . Ε . C H A D W I C K
that the insects now tend to avoid surfaces of the sort on which they formerly rested, so that they fail to make contact with the insecticidal residues that have been applied. Although few of these conclusions are supported by convincing data, there seems to be little doubt that such shifts in behavior do occur and that the effectiveness of residual treat
ments is impaired thereby. No physiological investigation of the be
havioral changes has been attempted.
B. S T R U C T U R E
Wiesmann (1947) reported structural differences of the legs, involv
ing shape and size of the distal segments, degree of pigmentation, and the thickness of tarsal cuticle, between a resistant and a susceptible strain of housefly; he suggested that the resistance was dependent on a decreased rate of penetration of DDT. Comparison of other resistant and susceptible strains failed to establish a correlation between the small structural differences found and the degree of resistance (D'Alessandro et al, 1949; March and Lewallen, 1950); and Bettini (1948) and many subsequent investigators showed that resistance was still conspicuous when the integumental barrier had been by-passed, as in injection experiments. On these grounds, the present writer (1952) and others rejected Wiesmann's conclusion that reduced penetration of DDT is an important factor in the resistance of flies to DDT. However, more recent information dictates a revision of this judgment, although the significance of visible structural modifications, such as Wiesmann described, still requires clarification.
C. P E N E T R A T I O N
For a general discussion of factors concerned in the penetration of chemicals through the insect cuticle, and a review of the earlier litera
ture on the penetration of insecticides, see Richards (1951) or Brown (1951).
Data on the rate of absorption of DDT and similar compounds by flies and other insects are given by many investigators (Ferguson and Kearns, 1949; Sternburg et al., 1950; Sternburg and Kearns, 1950; Win- teringham et al, 1951; Perry and Hoskins, 1951b; Lindquist et al, 1951;
Sternburg and Kearns, 1952a; Vinson and Kearns, 1952; Fisher, 1952;
Kearns, 1952; Perry, 1952; Hoskins, 1952; Lindquist, 1952; Wintering- ham, 1952; Perry et al, 1952; Hoffman and Lindquist, 1952; Hoffman et al, 1952; LeRoux and Morrison, 1953; Tahori and Hoskins, 1953;
Perry et al, 1953; Chang and Crowell, 1953; Babers and Roan, 1953;
I N S E C T R E S I S T A N C E T O I N S E C T I C I D E S 137 Perry and Sacktor, 1955; Lewis, personal communication). These studies have brought to light a number of variables that influence absorp
tion rates, but unfortunately the existence of these variables, together with the failure of most investigators to report the magnitude of their experimental error, preclude many of the useful quantitative com
parisons that might otherwise be attempted.
The following general conclusions seem warranted:
1. If the limiting process in penetration is one of diffusion, as many assume, this is frequently obscured by complicating factors, especially as the time interval following application is increased.
2. Although the absorption rate is not affected by continuous anes
thesia with C 02 or cyclopropane, the rate of penetration of DDT is reduced by death, from whatever cause, and possibly by earlier events in the intoxication process.
3. The fractional dose absorbed in a given interval is less from higher doses, when the size and site of the area of application are kept constant.
4. Absorption rates may differ considerably in different regions of the body.
5. The rate of penetration is increased by an increase in temperature.
6. Absorption rates may differ significantly for different species, or for different strains and stages of a single species.
7. A low rate of penetration is a significant factor in the acquired resistance of some, but not all, DDT-resistant strains of flies, and in the natural resistance of some other insects.
Differences in the rate of absorption of HCN by various species of insects were found by Pradhan and Bhatia (1952) and appeared to be correlated to some extent with differences in tolerance.
D. S T O R A G E
Notwithstanding a slower absorption of DDT by some resistant strains of flies, they accumulate within them more than enough DDT to kill susceptible individuals. Because the compound is highly lipophilic, one is led readily to imagine a preferential storage in the fat depots as a means of reducing the concentration of the insecticide at the vital sites of action. The fact that roaches and other insects are able to withstand larger doses of DDT in oil solution than in other vehicles (Tobias et al., 1946; Ferguson and Kearns, 1949; etc.) also is sugges
tive of a possible role of the body fats in protecting the organism.
Munson (1953) was able experimentally to produce roaches whose
138 L . Ε . C H A D W I C K
body fat varied in degree of unsaturation, and to correlate this variable with their differing tolerance for DDT. Reiser et al (1953) similarly demonstrated a correlation between seasonal changes in the fat content of the boll weevil and the susceptibility of this insect to certain chlorin
ated hydrocarbon insecticides. However, as the authors cited have pointed out, the existence of such correlations does not necessarily prove a causal relationship between the variables in question; in fact, with the boll weevil, Reiser and colleagues concluded that the fat content was not related directly to resistance. There is, then, at present no clear proof that storage of DDT, etc., in fatty tissues constitutes a pro
tective mechanism, still less that variations in storage capacity con
tribute to differences in the susceptibility of different populations. The possibility is nevertheless a reasonable one that merits further explora
tion, especially in view of Munson's provocative findings.
E . E X C R E T I O N
Another potential factor in resistance is the ability to excrete the poison rapidly enough to prevent its internal concentration from reach
ing a critical level. For the chlorinated hydrocarbon insecticides, this possibility can be dismissed with the statement that there is no evidence for their excretion, if the term is used in the sense of a mechanism that tends to clear the circulating hemolymph of toxic agent. Some other poisons, such as arsenic, are removed from the blood by the Malpighian tubules (Patton, 1943), but even in these experiments no significant correlation was found between excretion rates and variations in toler
ance.
However, various insects that are naturally tolerant of ingested DDT and other organics do rid themselves of large amounts of these com
pounds unchanged in the feces (Sternburg and Kearns, 1952a; Kearns, 1952), and ingested rotenone is disposed of in this manner by Prodenia (Woke, 1938). This sort of excretion is obviously a reflection of the permeability characteristics of the gut, and the tolerance conferred by it should therefore be classed as due to slow penetration. Protection from arsenicals and various other poisons is also achieved, in some insects, by regurgitation of the ingested dose (Cook and Mclndoo, 1923;
Voskresenskaya, 1936, 1939). This too is a sort of excretion, but should probably be listed among the behavioral mechanisms for evading a toxic exposure; in fact, the behavioral resistance of the codling moth larva is of this type and affords protection against a variety of chemi
cally unrelated toxicants (Hough, 1934).
That the Malpighian tubes are not the only avenue for freeing the
I N S E C T R E S I S T A N C E T O I N S E C T I C I D E S 139 blood of foreign materials has been shown by experiments of Roan et al (1950). These investigators found a rapid accumulation of P3 2
in the foregut of the roach when radioactively labeled phosphate insecti
cides were injected into the hemocele. The observations reveal that substances may pass from the blood into the lumen of the foregut, a process that was previously unsuspected, but do not disclose whether the insecticides were excreted into the gut intact or only after they had been broken down elsewhere in the body. The same agents were quickly detoxified in the foregut when given per os.
So far as present information goes, excretion of absorbed poisons affords some protection against certain insecticides, but these do not include many of the modern synthetics to which significant resistance has developed, and there is no indication that excretory mechanisms, sensu stricto, are concerned in such resistance.
F. D E T O X I F I C A T I O N
Chemical alteration of toxic materials within the body to produce nontoxic products is a major protective mechanism of all organisms, and the enhanced ability of some insects to deal in this manner with specific insecticides is beyond doubt a significant factor in certain cases of acquired resistance. Although the details of the process are in no in
stance fully understood, there is a confusing wealth of evidence for its existence.
Arsenic, for example, is perhaps detoxified in the roach by com- plexing with reduced glutathione, as suggested by the experiments of Forgash (1951). Ingested Pyrethrins are broken down into nontoxic materials in Prodenia, probably after absorption and transportation by the blood to the site of detoxification (Woke, 1939). Pyrethrins are also very rapidly converted, in an unknown manner, to harmless compounds by many other insects, unless they are applied in overwhelming doses or with adjuvants that prevent their metabolism (Swingle, 1934; Hock- enyos, 1936; Lindquist et al, 1947; Wilson, 1949; Page and Blackith, 1949; Chamberlain, 1950; Winteringham, 1953; Zeid et al, 1953).
Similarly, the insect attacks synthetic molecules such as DDT, chlordane and lindane, even without previous experience of them, and so alters them that their outstanding lethality is completely lost. That this is possible is in part a reflection of the highly specific nature of the toxic action of the compounds, which is such that even a slight structural change nullifies their toxicity. Simultaneously, the versatility of the living organism is revealed.
The natural tolerance of several species of insects for DDT is due
140 L. Ε . CHADWICK
in part to their ability to convert it into the nontoxic ethylenic deriva
tive, DDE, or into other harmless products, either in the gut or during passage through the integument (Ferguson and Kearns, 1949; Stern- burg and Kearns, 1952a; Vinson and Kearns, 1952; Kearns, (1952).
Chlordane-resistant roaches can also dehydrochlorinate DDT (Babers and Roan, 1953). All DDT-resistant strains of housefly that have been examined possess this ability in greater degree than susceptible strains (Perry and Hoskins, 1950, 1951a, 1951b; Sternburg et al, 1950; Stern- burg and Kearns, 1950b; March and Metcalf, 1950; Winteringham et al., 1951; Fletcher, 1952; Perry, 1952; Hoskins, 1952; March, 1952; Winter
ingham, 1952; Perry et al, 1952; Sternburg et al, 1953; Tahori and Hoskins, 1953; Perry et al, 1953a, 1953b; Babers and Pratt, 1953;
Perry and Sacktor, 1955), although the rate of detoxification is not perfectly correlated with the degree of resistance (Perry and Sacktor, 1955). It has also been demonstrated that application of compounds, such as piperonyl cyclonene or DMC, that diminish the rate of DDE formation, render the resistant flies more susceptible to DDT (Perry and Hoskins, 1950, 1951b; Fullmer and Hoskins, 1951; Tahori and Hoskins, 1953; Perry et al, 1953). Unfortunately, inhibition of detoxi
fication has not provided an answer to the practical problem of fly control, for two reasons. First, detoxification is only one of several elements in the resistance pattern, so that even complete inhibition of DDT metabolism leaves other resistance mechanisms, such as reduced penetration, intact. The other obstacle is that flies thus far have shown a capacity for developing tolerance for all combinations of DDT and synergists that have been tested, so that any advantage gained by the use of such combinations has been only temporary (March et al, 1952).
That some insects are able to detoxify insecticides other than DDT and its analogs has already been mentioned, and a few examples have been given. In addition, Hoffman and Lindquist (1952) have pro
duced evidence that toxaphene and chlordane are detoxified by flies.
Myers and Smith (1953) have reported that the migratory locust detoxifies phenols by converting them to glucosides. Data as yet un
published, from several laboratories, show that resistant flies can metabolize heptachlor, lindane, aldrin and dieldrin.
Little or nothing is yet known of the mechanism of most of these reactions, although they are usually presumed to be enzymic in nature.
Sternburg et al. (1953) have succeeded in preparing from resistant flies tissue extracts and powders that are highly active in converting DDT to DDE and have partially characterized the enzyme concerned.
I N S E C T R E S I S T A N C E T O I N S E C T I C I D E S 141 Similar work is being carried out with preparations that metabolize several of the other insecticides mentioned above, and considerable progress will undoubtedly be seen in this field during the next few years.
The metabolism of some insecticides to nontoxic products has its counterpart in the metabolic conversion of certain other compounds, themselves relatively inert, to highly virulent poisons. Known examples include OMPA (Duspiva, 1951; Martin, 1951; Stahmann et al, 1953;
O'Brien and Spencer, 1953), and various organic thionophosphates (Chamberlain and Hoskins, 1951; Metcalf and March, 1953). These agents, on absorption into the body, serve as substrates for enzyme systems that convert them into active anticholinesterases, which disrupt the functions of the insect nervous system. Such findings re-emphasize the desirability of a fuller understanding of all metabolic processes in insects, for there is here an obvious likelihood of being able to turn such information to practical advantage.
G. D E C R E A S E D S E N S I T I V I T Y
The resistance mechanisms previously mentioned all operate to reduce the internal concentration of the toxicant. Yet, even in situa
tions where it can be shown that one or more of these mechanisms is active, the quantity of agent recoverable internally may amount to several times the lethal dose for a susceptible individual. Unless one can assume that the insecticide is detoxified at the site of toxic action and that it is released to this site very slowly from a storage depot in which it remains harmless, one must reckon among the mechanisms of resist
ance a decreased affinity of the vital acceptors for the poison (Fletcher, 1952). Concrete evidence concerning this hypothesis is scanty. How
ever, Pratt and Babers (1953) have shown that DDT-resistant flies respond less readily than susceptible flies to DDT applied directly to the exposed thoracic ganglion. Smyth (personal communication), work
ing in Roeder's laboratory, observed that brief exposure of susceptible flies to a DDT residue lowered their chemosensory threshold for sugar solutions, whereas such treatment was without effect on individuals from a resistant strain. Following these experiments, Weiant (personal communication) has demonstrated that injection of DDT emulsions into the legs of susceptible flies produces a characteristic abnormal activity in the ascending neurons, such as was noted some years earlier with the roach (Roeder and Weiant, 1946), but that similarly treated flies from a resistant strain ordinarily maintain the normal pattern of nervous
142 L . Ε . C H A D W I C K
discharge. Winteringham (1952) found that Trogoderma larvae ab
sorbed large amounts of DDT, failed to metabolize it, and nevertheless were entirely unaffected. These various bits of evidence justify the hypothesis that tolerance for DDT is a function not only of the internal concentration öf the agent, but also of the properties of the acceptors at the site of action.
A similar situation in regard to HCN poisoning is suggested by the observations of Pradhan and Bhatia (1952); and O'Brien and Spencer (1953) have shown that the roach readily converts injected Schradan into an active anticholinesterase, but that the insect is nevertheless resistant. They suggest that the inhibitor formed may fail to penetrate the nerve sheath.
From these few examples, the conclusion is warranted that changes in acceptor characteristics may contribute significantly to resistance.
The recognition and elucidation of such mechanisms is impeded at present, however, by dearth of precise information about the site and mode of action of many insecticides.
H. B Y - P A S S M E C H A N I S M S
The living organism is dependent for its continuing existence upon the integrity of certain physiological systems, for example, series of biochemical reactions that provide energy in usable form. Toxicants are poisonous because they interfere with, or block, one or another of the essential steps in these activities. Fortunately, or perhaps neces
sarily, the evolution of the living substance has been such that there are frequently several paths to the vital goal. Such diversity provides insurance for the continuance of indispensable functions under adverse conditions, which will seldom affect all mechanisms to an equal extent.
That insects are able, because of this flexibility, to make a way around some of their difficulties is suggested by the results of certain investigations of resistance mechanisms. Thus, Yust and Shelden (1952) found that HCN blocks the same principal respiratory pathways in both susceptible and resistant scale insects, but that an exceptional pro
portion of the respiration of the resistant scales is carried on via cyanide- insensitive systems, presumably the flavoproteins. For this reason, the resistant strains are better able to survive a temporary inactivation of their cyanide-sensitive cytochrome oxidase.
Sacktor (1949, 1950, 1951) has proposed that a similar mechanism may be concerned in the resistance of some houseflies to DDT. Having
INSECT RESISTANCE TO INSECTICIDES 143 demonstrated that DDT has an inhibitory effect on cytochrome oxi
dase, he showed that adults of a resistant strain possess more than normal cytochrome oxidase activity, and the pupae, which are sub
normal in this respect, are endowed with an unusually effective cya
nide-insensitive respiration. These observations led to the inference that the excess cytochrome oxidase activity of the resistant adults may permit them to carry on an essential fraction of respiration despite partial inhibition by DDT, whereas the pupae achieve a similar objective via a system that is unaffected by this toxicant. Although the correlations discovered here may yet turn out to be coincidental (Perry and Sacktor, 1955), rather than related as cause and effect, it seems logical to suppose that by-pass mechanisms will be found to play a greater role in the resistance of insects to toxicants than is apparent from the limited data now at hand.
IV. Summary
Acquired resistance to insecticides has appeared with a variety of species and to a variety of agents, to an extent that is a matter of concern in agricultural and medical entomology.
Resistance is usually transmitted in inheritance by multiple genetic factors, and several types of physiological mechanism are involved.
These include changes in behavior and in the properties of the integu
ment or other barriers, such that the chance of accumulating a lethal dose is diminished. Within the body, the insecticide may be sequestered by storage, excreted in unchanged form, or metabolized to nontoxic materials. There is also evidence, in some instances, for a reduced sensitivity or accessibility of vital acceptors, and for the development of alternate routes around functions that may have been blocked by the toxicant.
Not all of these mechanisms are involved in any given resistance situation, but in examples that have been analyzed most intensively, such as the DDT resistance of houseflies, it is apparent that two or more of these protective devices may be in operation simultaneously.
These complex phenomena pose many problems of fundamental interest in genetics, ecology, animal behavior, physiology, and bio
chemistry. The solution of the practical difficulties resistance is creating will require the coordination of contributions from all these disciplines with advances in the applied field,
144 L. Ε . CHADWICK APPENDIX I
LIST OF SYNTHETIC INSECTICIDES DISCUSSED IN TEXT 1. Aldrin
Chemical Name: 1,2,3,4,10,10-hexachloro-l,4,4a,5,8,8a-hexahydro-l,4,5,8- dimethanonaphthalene
2. Chlordane
Chemical Name: l,2,4,5,6,7,8,8-octochloro-3a,4,7,7a,tetrahydro-4,7-methanoidane 3. DDE
Chemical Name: 1,1-bis (p-chlorophenyl) -2,2-dichloroethylene 4. DDT
Chemical Name: 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane 5. DMC
Chemical Name: 1,1 -bis (p-chlorophenyl) -methylcarbinol 6. Dieldrin
Chemical Name: l,2,3,4,10,10-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8a-octahydro- 1,4,5,6-dimethanonaphthalene
7. Heptachlor
Chemical Name: l,4,5,6,7,8,8'-heptachloro-3a,4,7,7a-tetrahydro-4,7- methanoindane
8. Lindane
Chemical Name: 1,2,3,4,5,6-hexachlorocyclohexane, γ-isomer 9. OMPA (—Schradan)
Chemical Name: Octamethylpyrophosphoramide 10. Piperonyl cyclonene
Chemical Name: mixture of
3-hexyl-5- (3,4-methylenedioxyphenyl) -6-carbethoxy-3- cy clohexen-1 -one ( I )
and
3-hexyl-5- (3,4-methylenedioxyphenyl) -3-cyclohexen-1 -one (II) 11. Toxaphene
Chemical Name: mixture of polychlorinated bicyclic terpenes, Cl-content 67-69%
12. Schradan (see OMPA, number 9 above)
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