921 T. Ben Amor, G. Jori Insect Biochemistry and Molecular Biology 30 2000 915–925
Fig. 4. Chemical structures of meso-substituted porphyrins: TMP,
meso-tetra4N-methylpyridylporphine tetratosylate; DDP, meso-[di- cis
4N-methylpyridyl] cis-diphenylporphine ditosylate; TRP, meso- tri4N-methylpyridyl, monophenylporphine tritosylate; TCPP, meso-
tetra4-carboxyphenylporphine tetrasodium salt. The counterions are not indicated in the structures.
Fig. 5. Percent survival of Ceratitis capitata after 1 h exposure to
white light at a fluence rate of 1220 µ
E s
21
m
22
in the presence of 3.0
µ M TCPP H, TRP I, DDP G and haematoporphyrin HP
j Ben Amor et al., 1998b.
Amor et al., 1998b. Such inertness must be related to the anatomical distribution of these porphyrins, since
both of them are accumulated in markedly large amounts by the flies; moreover, the two porphyrins are efficient
generators of singlet oxygen and exhibit phototoxicity towards a variety of biological systems both in vitro and
in vivo Spikes, 1994. As has been observed for other classes of photosensitizers, the photoactivity of porphy-
rins increases with hydrophobicity and is particularly large in the case of amphiphilic derivatives, such as the
meso-cis-diphenyl,
meso-cis-di-N-methylpyridyl- porphine Fig. 5; the latter is more phototoxic than the
tricationic analogue or the dianionic hematoporphyrin even in the presence of a smaller uptake of the dicationic
porphyrin by the insects Ben Amor et al., 1998b.
6. Factors affecting the photoinsecticidal activity
The efficiency of photodynamic sensitizers as insecti- cidal agents is affected by a variety of experimental para-
meters. Two obvious factors controlling the photosensi- tivity of insects are represented by the photosensitizer
dose and average light intensity. Typical examples of the interplay between these two factors are shown in Fig. 6
for the action of a xanthene dye eosin yellow on the house fly Musca domestica, Fondren and Heitz, 1978b;
Carpenter and Heitz, 1980 and in Fig. 7 for the action of a porphyrin dye haematoporphyrin on the fruit fly
Ceratitis capitata, Ben Amor et al., 1998a. In both cases the photoinsecticidal effect steadily increased with
increasing dose of photosensitizing agent in the bait and no saturation effect was detected at the highest photosen-
sitizer concentration examined by the investigators. Analogously, the rate and extent of the photosensitized
killing of insects appeared to increase with prolongation of exposure to light, as well as with an increase in the
Fig. 6. Effect of the concentration of Eosin Yellow on the percent
survival of Musca domestica irradiated with natural and artificial light for different time intervals. The fluence rate of the artificial light was
4300 foot candles. Data from Carpenter and Heitz 1980.
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
