922 T. Ben Amor, G. Jori Insect Biochemistry and Molecular Biology 30 2000 915–925
Fig. 7. Percent survival of Ceratitis capitata upon exposure to white
light at a fluence rate of 1220 µ
E s
21
m
22
in the presence of 8 µ
M haematoporphyrin: irradiated control s; 60 min exposure j; 90
min exposure I; 120 min exposure G. Data from Ben Amor et al. 1998a.
light intensity. Such conclusions are likely to be of gen- eral validity since essentially identical results were
obtained with other photosensitizers, including rhoda- mines Respicio and Heitz, 1981, rose bengal Fondren
and Heitz, 1978a; Carpenter and Heitz, 1980 and meth- ylene blue Lavialle and Dumortier, 1978. These effects
have been interpreted as suggestive of an intrinsic capacity of the insect to repair the photodamage; such
capacity would be more efficiently overcome as the light intensity is enhanced Fondren et al., 1978; Ben Amor
et al., 1998a,b.
It is interesting to note that several insects are insensi- tive to even high light intensities in the absence of the
photosensitizer see Ben Amor et al., 1998a and Fig. 7. On the other hand, it is difficult to compare the relative
efficiency of different dyes since the overall photoinsec- ticidal effect is dependent on the overlap between the
emission spectrum of the light source and the absorption spectrum of the photosensitizer. Most frequently, the
photoinsecticides are used in open fields, hence they are activated by sunlight. As shown in Figs. 6 and 7 and
reported by other authors Clement et al., 1980; Heitz, 1987, natural sunlight is generally more efficient than
artificial sunlight, probably because of its significantly greater intensity. From this point of view, as mentioned
earlier, porphyrins have the distinct advantage of absorb- ing almost all the light wavelengths in the sun’s emission
spectrum. As a consequence, porphyrins are expected to express an efficient photoinsecticidal action at lower
concentrations than many other dyes see Fig. 7.
In some cases the degree of insect mortality was shown to increase with increasing accessibility periods
of the insects to the photosensitizer-loaded bait before exposure to light Robinson, 1983. These findings were
confirmed by studies with porphyrins performed in our laboratory Ben Amor et al., 2000. However there
appeared to be little advantage in prolonging the “dark” exposure of the photosensitizer beyond 24 h. Most
importantly, we observed that at least in the case of porphyrins the insects display a significant photosensi-
tivity for about 48 h after having taken up the dye Ben Amor et al., 1998a,b.
A few experiments suggest that the pH of the formu- lation in which the photosensitizer is offered to the
insects may play an important role. Thus the free acid forms of xanthene dyes were found to be about tenfold
more effective as photosensitizers than the correspond- ing salt derivatives against both Aedes Pimprikar and
Heitz, 1984 and Culex Carpenter et al., 1984; Respicio et al., 1985 mosquito larvae. Since the carboxylic func-
tional groups of xanthenes have pK values around 5–5.5, one can infer that a slightly acidic dietary preparation of
the photosensitizing agents should be preferred.
Lastly, some attempts have been made to protect insects against photodynamic action, a typical oxidative
process, by the administration of antioxidizing agents, such as carotenes, tocopherol and ascorbic acid
Robinson and Beatson, 1985; Heitz, 1987. In all cases, no appreciable protection was obtained, even though
these compounds are powerful inhibitors of photooxid- ative reactions in vitro Jori, 1996. The lack of photop-
rotection in the insects could reflect either a rapid meta- bolization
of the
antioxidants or
a different
biodistribution as compared with the photosensitizing agents.
7. Mechanisms of the insecticidal action performed by photodynamic sensitizers
The possibility of controlling pest population by means of photodynamic action has been investigated so
far by using a variety of photosensitizers, target insects and irradiation conditions. In spite of the large differ-
ences in the experimental protocols, a comparative analysis of the whole set of results reported by the vari-
ous authors allows one to draw some general conclusions regarding the mode of action of photodynamic-type
insecticidal agents.
These can be summarized as follows: 1. The membranes of the midgut wall appear to be
among the
primary photodamaged
sites Schildmacher, 1950; Robinson, 1983; Ben Amor et
al., 1998a, leading to feeding inhibition Fondren et al., 1978. Other lipoidal membranes, in particular the
neuromuscular sheath, become involved in the photo- process as the photosensitizer diffuses to other sites
Robinson, 1983. This is confirmed by the early pho- toinactivation of enzymes such as acetylcholinesterase
Callaham et al., 1977a; Ben Amor et al., 2000 which represents the neurotransmitter enzyme. A gen-
923 T. Ben Amor, G. Jori Insect Biochemistry and Molecular Biology 30 2000 915–925
eralized oxidative modification of the membranes takes place, as suggested by ultrastructural studies
Callaham et al., 1977b. 2. Changes in membrane permeability are also demon-
strated by the presence of altered potassium levels in the hemolymph Weaver et al., 1976. The hemo-
lymph volumes decrease significantly upon photosen- sitization and the hemocoel fluids undergo a rapid
transfer from the body cavity to the alimentary canal with a consequent increase in crop volume.
3. Photosensitized insects also show large differences from controls as regards weight, levels of water and
protein mass, suggesting the occurrence of a lethal energy stress in the insect Broome et al., 1976.
Other consequences reported for photosensitized flies involve a lowered fecundity Pimprikar et al., 1980b.
4. The photosensitized induction of physiological and morphological abnormalities was detected at the lar-
val, pupal and adult stage Pimprikar et al., 1979; Fairbrother et al., 1981. Thus, adults deposit fewer
and less-viable eggs, treated eggs are less likely to hatch, and larvae exhibit a strongly reduced prob-
ability of ultimate adult emergence. In particular, there often appears to be an incomplete extrication of
the pupal stage from the larval cuticle Pimprikar et al., 1979, while several adults are stuck to the chitin
inner lining of the puparium Fairbrother, 1978. Moreover, pupae injected with the photosensitizer fre-
quently develop into adults that are especially suscep- tible to photodynamic action Sakurai and Heitz,
1982. In general, earlier instar larvae appear to be more
photosusceptible than
later instar
forms Heitz, 1987.
5. The possible onset of photoresistance in the xanthene- sensitized house fly was studied Respicio and Heitz,
1985. A wild strain of this insect developed a 48- fold resistance after 32 generations that underwent
exposure to increasing levels of erythrosin plus light. Upon removal of the selection pressures for 20 gener-
ations, the resistance remained at a fairly constant level. No evident induction of photoresistance was
observed in porphyrin-phototreated flies Ben Amor et al., 2000. Thus, this specific aspect of the photoin-
secticidal action appears to be strictly connected with the photosensitizer used and the irradiation con-
ditions. It is important that no cross-resistance was detected for erythrosin-photosensitized flies that were
exposed to chemical pesticides, such as permethrin and propoxur Respicio and Heitz, 1985.
6. Some photodynamic sensitizers, including rose bengal and other xanthenes, also exert a toxic effect towards
selected insects in the dark Carpenter and Heitz, 1981. While this toxic mechanism occurs with adult
flies, it has been observed that boll weevils fed with rose bengal during the larval development exhibit a
decrease in body weight, and in protein and lipid lev- els Broome et al., 1976. The dark process appears
to be markedly less efficient than the corresponding light-induced reactions. Moreover, it seems to be pec-
uliar to xanthene dyes, since no similar effects were observed with furocoumarines,
α -terthienyl Cunat et
al., 1999; Guillet et al., 2000 and porphyrins Ben Amor et al., 2000, at least at doses which are photo-
chemically active.
8. Conclusions
