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Mini Review
Sunlight-activated insecticides: historical background and
mechanisms of phototoxic activity
Thameur Ben Amor, Giulio Jori
*Department of Biology, University of Padova, Via U. Bassi 58B, Padova 35121, Italy
Received 21 January 2000; received in revised form 12 March 2000; accepted 14 March 2000
Abstract
Several photosensitizing agents, which are activated by illumination with sunlight or artificial light sources, have been shown to be accumulated in significant amounts by a variety of insects when they are administered in association with suitable baits. The subsequent exposure of such insects to UV/visible light leads to a significant drop in survival. Of the photosensitizers tested so far, xanthenes (e.g. phloxin B) and porphyrins (e.g. haematoporphyrin) appear to be endowed with the highest photoinsecticidal activity. In particular, porphyrins absorb essentially all the UV/visible light wavelengths in the emission spectrum of the sun; hence they are active at very low doses. Thus, 1 h irradiation of Ceratitis capitata, Bactrocera oleae(also known as Dacus oleae) or
Stomoxys calcitrans which ingested a few nanomoles of porphyrin per fly with light intensities of the order of 1000µE s21m22 causes about 100% death in laboratory tests. Present evidence suggests that such photosensitizers act on the membranes of the midgut with consequent feeding inhibition, as well as on the neuromuscular sheath. No apparent onset of photoresistance has been observed. The rapid photobleaching of xanthenes and porphyrins when illuminated by visible light, as well as the lack of significant toxicity of such compounds in the dark, minimizes the risk of an important environmental impact of such photoinsecticidal agents.
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1. Introduction
The use of photochemical processes as a tool to con-trol the population of several types of insects has been repeatedly examined in both laboratory experiments (Heitz, 1987; Rebeiz et al., 1991) and field studies (Pimprikar et al., 1980a; Lenke et al., 1987). Most inves-tigations have been performed by using photoactivatable polycyclic aromatic dyes that absorb near-UV light wavelengths, including thiophenes, furocoumarines and quinones (Robinson, 1983; Cunat et al., 1999). The use of xanthene derivatives such as eosin and its analogues absorbing selected light intervals in the visible spectral range has also been proposed (Yoho et al., 1971; Fondren and Heitz, 1978a; Heitz, 1997a). All of these dyes require the presence of molecular oxygen to express their phototoxic action; hence the overall photoinsectici-dal process appears to be of the photodynamic type (Jori,
* Corresponding author. Tel.:+39-049-8276333; fax: + 39-049-8276344.
E-mail address:[email protected] (G. Jori).
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1996). In addition, furocoumarines, upon photoexcit-ation, can generate various types of addition products with DNA bases, which often results in genotoxic effects (Jori, 1985).
More recently, Rebeiz and co-workers proposed the use of porphyrins as photoinsecticides (Rebeiz et al., 1987). In particular, these authors tested protoporphyrin IX and its Zn(II) derivative, which appear to be especially promising photoinsecticidal agents since these compounds absorb essentially all the UV–visible wave-lengths, that is to say these molecules can be efficiently excited by natural sunlight. Along the same line, we recently demonstrated that haematoporphyrin is an efficient phototoxin to several insects (Ben Amor et al., 1998a). Subsequently, we extended our investigations to some meso-substituted porphyrins (Ben Amor et al., 1998b) in order to identify possible relationships between the chemical structure and the photoinsecticidal activity of this class of compounds.Ceratitis capitata, a Mediterranean fruit fly, was selected as an experi-mental model.
the use of photoactivatable insecticides, as well as dis-cussing the limitations, scope and potential of this novel technique. The interest in this topic is enhanced by safety and environmental considerations. The use of sunlight as the promoter of the phototoxic activity is in line with the growing trend to utilize and validate natural resources. Moreover, a careful selection of the chemical structure of the photosensitizing dye can modulate the nature of the subcellular photodamaged sites, which is quite important for optimizing the efficiency of the cyto-cidal effects and minimizing the risk of selecting photor-esistant insect species.
The photoinsecticides represent a possible alternative to traditional chemical insecticides. The latter com-pounds are known to cause several important problems, including widespread toxicity to plants and animals and in particular to humans, as well as the prolonged persist-ence in the environment which may cause severe pol-lution. As we will discuss in the present review, at least some of these issues can be adequately addressed by the use of photodynamic insecticides.
2. Historical background
The first scientific documentation that sunlight can be toxic to biological systems was provided by Marcacci in the late nineteenth century (Marcacci, 1888). This author reported that the fermentation of plant alkaloids and amphibian eggs becomes more important under UV/visible light than in the dark. Shortly thereafter, Raab (1900) observed that the presence of some exogen-ously added visible light-absorbing compounds, such as acridine orange, was necessary for sunlight to promote the death of paramecia. Acridine orange and other dyes with similar properties were defined as photosensitizers. Raab’s observations were repeated with a variety of multi- and unicellular organisms (Spikes, 1985). Finally, Jodlbauer and Von Tappainer (1904) demonstrated that the presence of oxygen is an essential requisite for pho-tosensitization to occur. The combined effect of the three elements, namely light, photosensitizer and oxygen, has been termed photodynamic action (Blum, 1941).
While most organisms have developed specific protec-tion or repair mechanisms to counteract the damaging action of light, several attempts have been made to con-trol the photoprocesses involving specific biological sys-tems with the aim to obtain beneficial effects. Thus, pho-totherapeutic techniques have been developed by taking advantage of the property of some photosensitizing agents to be accumulated in significant amounts by dis-eased tissues, such as tumours, atheromas or psoriatic plaques (Brown, 1997). At the same time, the photosen-sitized inactivation of yeasts and bacteria is used for the decontamination of microbially polluted waters by sun-light (Merchat et al., 1996).
The possibility of using photoactivatable compounds for controlling the pest population was first explored by Barbieri (1928) who used a combination of xanthene derivatives, including fluorescein, erythrosine and rose bengal, against Anopheles and Aedes larvae. Barbieri showed that light or photosensitizer alone had no detect-able toxic effects on insects.
This research line underwent no further developments until the early 1950s when Barbieri’s experiments on the photosensitized killing of insects were repeated by Schildmacher (1950), who concluded that the chemical structure of the xanthene dyes exerts a profound influ-ence on the degree of phototoxicity. Thus, the highest photoinsecticidal activity is displayed by rose bengal fol-lowed by eosin, erythrosine and fluorescein. The poten-tial of xanthenes as promoters of lethal effects on insects exposed to sunlight was explored in detail by Heitz and co-workers (Heitz, 1987; Heitz, 1997a), who investi-gated the role of various experimental parameters on the photoinsecticidal efficacy of this class of compounds. Of particular interest were the field studies based on several combinations of bait and xanthene dyes (Carpenter et al., 1981; Sakurai and Heitz, 1982). Xanthenes undergo rapid photodegradation in aqueous media by analogy with the well-known tendency to photobleach which is typical of many polycyclic aromatics when exposed to non-ionizing radiation (Spikes, 1992). This topic was the subject of intense discussions at a recent congress of the American Society of Photobiology (Heitz, 1997b).
3. Mechanisms of photodynamic sensitization
Upon absorption of UV or visible photons, a photo-sensitizer can be promoted to a variety of electronically excited states. However, the efficiency of the photosensi-tizing action is generally dependent on the photophysical properties of the lowest excited triplet state (3Sens),
which is reached via intersystem crossing from the initially formed excited singlet state (1Sens). The 3Sens
species is most often characterized by a lifetime in the microsecond to millisecond range, hence it can play a major role in diffusion-controlled processes (Carmichael and Huh, 1986). Several deactivation pathways are poss-ible for 3Sens; those which are of utmost importance
from a photobiological point of view can be schematized as follows.
1. Electron transfer to or from a substrate with suitable redox properties, e.g.
3Sens
1Sub→Sens·+1Sub·−
This pathway, defined as a type I mechanism (Jori and Reddi, 1991), leads to the generation of radical inter-mediates, which in turn can undergo further reactions with other substrates, solvent molecules, or oxygen. The latter process results in the formation of oxidized pro-ducts. A particular case occurs when oxygen acts as an electron acceptor:
3Sens1O
2→Sens·+1O2·−
The superoxide anion has a relatively low level of reac-tivity. However, under certain experimental conditions, it can be converted to very reactive and cytotoxic spec-ies, such as the hydroxyl radical and hydrogen peroxide (Bensasson et al., 1983).
2. Energy transfer to any substrate whose triplet state energy lies at a lower level compared with the photosen-sitizer triplet state; this pathway is defined as a type II mechanism (Jori and Reddi, 1991):
3Sens1Sub
→Sens13Sub
Most components of cells and tissues are not suitable acceptors of electronic energy from 3Sens, since their
triplet states are too energetic. One notable exception is represented by oxygen; this ubiquitous component of biological systems can be readily promoted to its excited singlet state, whose energy level lies at only 22.5 kcal above the triplet ground state and which is endowed with a high cytotoxicity:
3Sens
13O
2→Sens11O2
The high reactivity of 1O
2 is partly due to its long
life-time (3–4µs in aqueous media, several tens of microse-conds in lipid environments) which allows this species to diffuse over relatively long distances before being deactivated (Wasserman and Murray, 1989).
Both type I and type II photosensitization mechanisms
generate electrophilic species, hence the most photosen-sitive targets are represented by electron-rich biomolec-ules (Vilensky and Feitelson, 1999).
Table 1 shows that of the naturally occurring amino acids only those which possess aromatic or sulphur-con-taining side chains are readily photooxidized. Other rap-idly attacked moieties include carbon–carbon double bonds of unsaturated lipids and steroids, as well as the heterocyclic ring of guanosine nucleotides. At a cellular level the photosensitizing action is characterized by an additional selectivity, since the overall photoprocess is generally confined to the microenvironment of the pho-tosensitizer owing to the tendency of the photogenerated intermediates to react with a large variety of targets (Moan et al., 1995). Thus, it appears essential to control the subcellular distribution of the photosensitizing agent. In this connection, a critical role is performed by the chemical structure of the photosensitizer, and in parti-cular by its degree of hydrophobicity (Jori and Reddi, 1993). This parameter is usually measured by the par-tition coefficient between n-octanol and water. Thus, moderately or highly lipophilic dyes (partition coef-ficient.8–10) become preferentially associated with the cell membranes; at short incubation times the plasma membrane represents the main binding site, while at longer times significant photosensitizer concentrations are recovered from other subcellular membranes includ-ing the mitochondrial and lysosomal membranes, the Golgi apparatus and the rough endoplasmic reticulum. For in vivo administration, amphiphilic photosensitizers (which are sufficiently water-soluble, yet are charac-terized by the presence of a hydrophobic matrix facilitat-ing the crossfacilitat-ing of the lipid domains of cell membranes) proved to be particularly useful. Hydrophilic photosensi-tizers show a more complex pattern; although such com-pounds, especially if electrically charged, undergo ionic interactions with charged groups at the cell surface, the possibility exists of their internalization by both active or passive diffusion processes. Thus, cationic photosen-sitizers are often localized in mitochondria, while anionic dyes (e.g. carboxylated derivatives) are accumulated at the lysosomal level (Spikes, 1994; Jori, 1996). Lastly, some photosensitizers, such as furocoumarins, can reach the cell nuclei and bind to the DNA bases (Armitage, 1998). The subcellular localization of a photosensitizer determines the efficiency, as well as the mechanism of photoinduced cell inactivation; in particular, it controls the relative weight of apoptotic and random necrotic pathways which are eventually responsible for cell death (He et al., 1994)
4. Main classes of photoactivatable insecticides
Table 1
Main biological targets of photosensitized processes
Cell constituent Target Modified chromophore Main photoproducts
Protein Tryptophan Indole Hydroxyindoles, kynurenine
Tyrosine Phenol Quinones, melanin-type pigments
Histidine Imidazole Endoperoxide
Methionine Thioether Methionine sulphoxide
Cysteine Thiol Cystine, cysteic acid
Unsaturated lipids Oleic, linoleic, linolenic, Carbon–carbon double bond Allylic hydroperoxides,
arachidonic acids endoperoxides
Steroids Cholesterol Carbon–carbon double bond 5α- or 7α-hydroperoxide
Nucleic acids Guanosine Purine Ketone-type degradation products
with either natural or artificial UV or visible light wave-lengths. A list of the main classes of photoinsecticides is given in Table 2. Undoubtedly, xanthene dyes have been most frequently and extensively studied largely due to the systematic investigations carried out by Heitz and co-workers (Heitz, 1987; Heitz, 1997a). Eventually, phloxin B, a polyhalogenated fluorescein (spiro-benzofuran-1(3H),(9H)-xanthen-3-one-29,49,5,79 -tetra-bromo-4,5,6,7-tetrachloro-39,69-dihydroxy disodium salt) has been developed for commercial use as a pestic-ide (Dowell et al., 1997; Heitz, 1997a). While fluor-escein can be considered as the parent compound of this class of photosensitizers, several derivatives can be syn-thetically prepared by the insertion of up to eight halogen atoms into selected positions of the aromatic macrocycle (Fig. 1A). All the xanthenes exhibit intense absorption bands in the green region of the visible spectrum; the maximal absorbance falls in the 480–550 nm interval, while the exact position of the peak shifts to the red as the number of the halogen substituents and their atomic weight increase. These parameters also affect the photo-dynamic efficiency of xanthenes, since the presence of heavy bromine or iodine atoms enhances the yield of
Table 2
Selected examples of photodynamic sensitizers which have been used as photoinsecticidal agents
Class Typical examples Targets References
Xanthenes Rose bengal House fly, Aedes larvae Carpenter et al. (1984) Erythrosin B House fly, face fly Fairbrother et al. (1981) Phloxin B Culex mosquito larvae Fondren et al. (1979) Rhodamin 6G Fire ant, House fly Fondren and Heitz (1978b)
Heitz (1987)
Phenothiazines Methylene blue Yellow mealworms Lavialle and Dumortier (1978) Cabbage butterfly
Furanocoumarins Xanthotoxin Black swallowtail larvae Cunat et al. (1999) Angelicin Swallowtail butterfly Robinson (1983) Thiophenes α-Terthienyl Aedes mosquito, Blackfly larvae Guillet et al. (2000)
Acridines Acridine red Acridine looper Robinson (1983)
Various Hypericin Fruit fly Armitage (1998)
Cercosporin Aedes mosquito larvae Bosca and Miranda (1998) Benzopyrene Black fly larvae Cunat et al. (1999) Polyacetylenes
intersystem crossing to the reactive triplet state of the dye (Jori and Reddi, 1991). Thus, other conditions being the same, the greatest photosensitizing activity is dis-played by tetraiodo xanthene derivatives, such as rose bengal and erythrosin B. As shown in Fig. 2, both these dyes are significantly more efficient than their tetra-bromo analogue (eosin yellow) in photosensitizing the killing of Musca domestica.
In general, xanthenes exert their phototoxic effects through the generation of reactive oxygen species (largely, singlet oxygen). However, one cannot rule out the parallel occurrence of radical-involving processes owing to the well-known photolability of covalent bonds between halogens and aromatic rings (Spikes and Mack-night, 1970). Xanthenes are typically localized in cell membranes, so that at a molecular level xanthenes mostly photosensitize the cross-linking of membrane proteins and the formation of hydroperoxides from unsaturated lipids, thereby markedly increasing the osmotic fragility of cells (Pooler and Valenzeno, 1979). Very similar considerations are found for acridines, which efficiently absorb light wavelengths around 550 nm and largely act via the generation of activated oxygen species (Rossi et al., 1981). One important difference is represented by the tendency of acridines to yield a heterogeneous subcellular distribution pattern, involving both the partitioning in specific organelles such as lyso-somes and the interaction with the phosphate groups in double-stranded DNA (Briviba et al., 1997), which enhances the possibility of photosensitized damage to the genetic material.
Both furocoumarines and thiophenes preferentially absorb near-UV light, which has a low penetration power into most biological tissues (Anderson and Parr-ish, 1992). Thus, these photosensitizing agents promote the damage mainly at the level of superficial tissue
lay-Fig. 2. Effect of different xanthene dyes on the percent survival of Musca domestica, upon exposure to sunlight after 12 h free access to a bait with 0.125% photosensitizer concentration. Eosin yellow (×); rose bengal (j); erythrosin B (I). Adapted from Fondren et al. (1978, 1979).
ers. However, the photodamaged area can become quite extensive since these compounds, once electronically excited, can promote radical processes which signifi-cantly amplify the initial damage through the induction of chain reactions. Furocoumarines may also intercalate among DNA bases with the formation of covalent pho-toadducts and the consequent inhibition of cell repli-cation (Armitage, 1998).
Lastly, phenothiazines (Boyle and Dolphin, 1996) and hypericin (Bosca and Miranda, 1998) are characterized by relatively intense absorption bands in the orange–red spectral region. Both these classes of photosensitizers therefore allow the direct damage of tissue compart-ments located at depths of several millimetres below the surface where light initially impinges. Moreover, several constituents of these classes produce the highly reactive singlet oxygen with a quantum yield greater than 0.6; hence, the overall photodamage is most often confined within the microenvironment of the photosensitizer. This makes it important to control the biodistribution of such photosensitizers as closely as possible.
5. Porphyric photoinsecticides
In recent years, increasing attention has been focused on the photosensitizing properties of porphyrins (Fig. 1B) and their analogues, such as chlorins and phthalocy-anines (Jori and Spikes, 1983). Porphyrins are natural compounds or close derivatives of such compounds, hence they are usually devoid of any appreciable intrin-sic cytotoxicity in the absence of irradiation. Further-more, porphyrins are characterized by specific features which make them especially useful photosensitizers of biological systems:
1. The ability to absorb essentially all the wavelengths of the solar spectrum in the UV and visible range. In particular, porphyrins exhibit an intense absorption band (the Soret band) in the blue spectral region, which represents the most intense component of the sun’s emission around midday (Svaasand et al., 1990). On the other hand, the red absorption bands of porphyrins are useful at dawn and sunset, when wavelengths greater than 600 nm represent an important component of sunlight.
pho-toactivity in biological systems which are charac-terized by a low oxygen pressure.
3. The chemical structure of porphyrins can be modified at different levels, including (i) the substituents pro-truding from the peripheral positions of the pyrrole rings or the meso-carbon atoms, (ii) the metal ions possibly coordinated at the center of the tetrapyrrolic macrocycle, and (iii) the ligands axial to the metal ion. In this way, it is possible to modulate the phys-ico-chemical properties of the porphyrin molecules and control their partitioning among subcellular com-partments.
4. Hydrophobic porphyrins are localized at the level of the cell membranes including the plasma, mitochon-drial and lysosomal membranes (Ricchelli and Jori, 1986). As a consequence, the genetic material is not involved in the photoprocesses leading to cell death. All the available evidence indicates that porphyrin photosensitization of cells does not promote the onset of mutagenic effects, thereby minimizing the risk of selecting photoresistant cell clones (Bonnett and Berenbaum, 1989).
5. The extraction and isolation of porphyrins from natu-ral products, and their synthetic preparation (often by modification of natural porphyrins), are relatively simple procedures (Ricchelli et al., 1995). Hence, the overall commercial cost of porphyrins can be fairly low, of the order of 1–2 US$/g, which is a critical factor for large-scale field utilization of porphyric insecticides. It is to be underlined that the uptake of nanomoles of porphyrin is sufficient to cause a rapid mortality of several types of flies even under moder-ate intensities of sunlight (Ben Amor et al., 2000). 6. Porphyrins undergo fast photobleaching in sunlight as
well as when exposed to artificial visible light sources (Rotomskis et al., 1997). The photodegradation pro-ducts do not appear to induce any appreciable toxic or phototoxic effects in a variety of biological sys-tems. Thus, the rapid disappearance of porphyrins from the environment strongly reduces the risk of widespread or persistent contamination.
7. Several porphyrins are presently used as photothera-peutic agents; toxicological studies have shown that these dyes induce important damage to humans only upon uptake of at least 100 mg/kg body weight, that is far greater than the amount which is required for generating an extensive toxicity to insects (Jori and Reddi, 1991).
Two approaches have been devised for defining the scope and potential of porphyric insecticides. One approach involves the administration of a large excess (some hundred milligrams) of 5- amino-levulinic acid (ALA), which is a metabolic precursor of heme, the prosthetic groups of hemoproteins (Rebeiz et al., 1988; Rebeiz et al., 1990b). The excess ALA through a
feed-back mechanism inhibits the final step of the biosyn-thetic pathway, namely the ferrochelatase-catalysed insertion of the Fe2+ion into the tetrapyrrolic
macrocy-cle. This leads to the accumulation of significant amounts of protoporphyrin IX, a well-known photodyn-amic agent. As a consequence, the protoporphyrin-loaded cells become photosensitive, especially at the level of mitochondria which represent the normal site of heme biosynthesis. In general, the photosensitivity develops after a time interval of 3–4 h from the exposure of the insect to ALA. The extent of the protoporphyrin accumulation can be enhanced by the presence of iron chelating agents such as desferroxamine or phenanthro-line (Rebeiz et al., 1990a).
A second strategy is based on the direct administration of a photodynamically active porphyrin in combination with a bait. Typically, 1 h exposure of the insects to haematoporphyrin concentrations of the order of a microgram per ml of bait are sufficient to obtain a 90– 100% decrease in the survival of widely diffused and extremely noxious flies, such asCeratitis capitata(fruit fly),Bactrocera (Dacus) oleae(olive fly) andStomoxys calcitrans (stable fly) (Ben Amor et al. 1998a, 2000). The kinetics of post-irradiation fly death after 1 h irradiation with light intensities corresponding to an early Autumn day at Mediterranean latitudes is shown in Fig. 3. In general, the photosensitivity of porphyrin-loaded insects persists for about 48 h after the adminis-tration of the photosensitizer has been interrupted.
The chemical structure (Fig. 4) also has a profound influence on the photoinsecticidal activity of porphyrins. As shown in Fig. 5, highly water-soluble porphyrins, such as tetracationic meso-substituted-N-methylpyridyl porphine or tetraanionic meso-sulphonatophenyl por-phine, are very inefficient photoinsecticidal agents (Ben
Fig. 3. Percent survival of three diptera,Ceratitis capitata(I), Bac-trocera (Dacus) oleae (×) and Stomoxys calcitrans (j), after 1 h
irradiation with white light at a fluence rate of 1220µE s21m22. The
Fig. 4. Chemical structures of meso-substituted porphyrins: TMP, meso-tetra(4N-methylpyridyl)porphine tetratosylate; DDP, meso-[di-cis(4N-methylpyridyl)] cis-diphenylporphine ditosylate; TRP, tri(4N-methylpyridyl), monophenylporphine tritosylate; TCPP, meso-tetra(4-carboxyphenyl)porphine tetrasodium salt. The counterions are not indicated in the structures.
Fig. 5. Percent survival ofCeratitis capitata after 1 h exposure to white light at a fluence rate of 1220 µE s21 m22in the presence of
3.0µM 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. 7. Percent survival ofCeratitis capitataupon exposure to white light at a fluence rate of 1220µE s21m22in the presence of 8µM
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) andCulex(Carpenter et al., 1984; Respicio et al., 1985) mosquito larvae. Since the carboxylic func-tional groups of xanthenes have pKvalues 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:
gen-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
The photoactivatable insecticides, which act through photodynamic pathways, clearly appear to possess sev-eral favourable features and a broad scope of appli-cations. As the mode of action of such photosensitizers is more deeply elucidated, suitable strategies can be developed to optimize their efficiency and control their biodistribution at both the tissue and the cell level, ther-eby orientating the photosensitizer towards those sites (e.g. cell membranes) which are critical from a func-tional and metabolic point of view, yet their modification is associated with a minimal risk of inducing mutagenic effects and promoting the onset of photoresistance. A detailed control of the in vivo behaviour of ingested pho-tosensitizers would also be of utmost importance for obtaining a synergistic action between two or more sim-ultaneously administered dyes; if the latter have different absorption properties, a more efficient activation of the photoinsecticides by sunlight can be achieved. Thus, the photoinsecticidal action of porphyrins, which possess relatively weak absorption bands in the red spectral region, could be strengthened by their association with tetrapyrrolic analogues, such as phthalocyanines and chlorins, which display an intense absorbance in the 600–800 nm spectral range. In fact, a synergistic action against the house fly has been observed upon the simul-taneous administration of two xanthenes, namely fluor-escein and rose bengal, whose absorption spectra are shifted by about 60 nm (Carpenter et al., 1981).
strategies for minimizing the uptake of such photosensit-izing agents by non-target insects should be developed. The concern about environmental effects of photoin-secticidal agents could be alleviated by the sunlight-induced photobleaching of the sensitizing agents. In fact, while most phenothiazines and acridines are fairly pho-tostable, both porphyrins and xanthenes undergo a fast degradation of the aromatic macrocycle upon illumi-nation with sunlight or equivalent artificial light sources, with a consequent loss of absorption in the near-UV/visible range (van Lier and Spikes, 1989; Spikes, 1992). This feature adds further value to the use of these compounds as photoinsecticidal agents.
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