JOURNAL

OF INVERTEBRATE

PATHOLOGY

Photoprotection

57, 343-351 (1991)

of Bacillus thuringiensis Ultraviolet irradiation

kurstaki from

EPHRAIM COHEN, *,’ HAREL ROZEN,~ TAMAR JOSEPH,* SERGEI BRAUN,~ AND LEON MARGULIES? *Department of Entomology and fDepartment of Soil and Water, The Faculty of Agriculture, University of Jerusalem, Rehovot 76 100 Israel Received May 14, 1990; accepted August 7, 1990

The Hebrew

Irradiation of Bacillus thuringiensis var. kurstaki HDl at 300- 350 nm for up to 12 hr using a photochemical reactor results in a rapid loss of its toxicity to larvae of Heliothis armigera. Photoprotection of the toxic component was obtained by adsorption of cationic chromophores such as acritIavin (AF), methyl green, and rhodamine B to B. thuringiensis. AF gave the best photoprotection and a level of 0.42 mmol/g dye adsorbed per gram of B. thuringiensis was highly toxic even after 12 hr of ultraviolet (uv) irradiation as compared to the control (77.5 and 5% of insect mortality, respectively). Ultraviolet and Fourier-transform infrared spectroscopic studies indicate molecular interactions between B. thuringiensis and AF. The nature of these interactions and energy or charge transfer as possible mechanisms of photoprotection are discussed. It is speculated that tryptophan residues are essential for the toxic effect of B. thuringiensis. It is suggested that photoprotection is attained as energy is transferred from the excited tryptophan moieties to the chromophore molecules. o IEJI Academic Pm, hc. KEYWORDS: Bacillus thuringiensis; Heliothis armigera; acriflavin; methyl green; rhodamine B; chromophore; Fourier transform infrared spectroscopy; photoprotection; uv irradiation.

INTRODUCTION Strains of spore-forming Bacillus thuringiensis produce proteins toxic to a variety of insect pests, including dipteran (B. thuringiensis israelensis) (de Barjac, 1978), lepidopteran (B. thuringiensis kurstaki) (Whiteley and Schnepf, 1986), and recently coleopteran (B. thuringiensis tenebrionis) species (Krieg et al., 1983). The commercially available microbial pesticides such as B. thuringiensis have been a welcome addition to the broad-spectrum, nonselective, neuroactive pest control agents. However, the B. thuringiensis formulations are inadequately stable under field conditions and rapidly lose their biological activity (Beegle et al., 1981; Ignoffo et al., 1974; Pinnock et al., 1974). Photoinactivation has emerged as the major environmental factor affecting i To whom correspondence should be addressed. 2 Department of Biological Chemistry, Institute of Life Science, The Hebrew University of Jerusalem, Israel.

stability and thus efficacy of entomopathogenie viruses and microorganisms such as B. thuringiensis (Ignoffo et al., 1977). However, other factors such as heat, desiccation, or pH may also play some role (Leong et al., 1980). The effectiveness of several uv-absorbing materials to improve the performance of B. thuringiensis was investigated (Morris, 1983). However, some of the uv photostabilizers may introduce ecological problems related to soil and water pollution. A new approach to extend and maintain the biological activity of photolabile pest control agents has been recently suggested (Margulies et al., 1985). This approach involves a specifically intimate alignment between an organic pesticide and a selected organic chromophore. Such spatial arrangement facilitates transfer of energy or electrons between the excited molecule and the chromophore. In this process a probable photochemical reaction is fully or partially prevented. Photosensitive compounds such as the pyrethroid biores343 0022-201 l/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

344

COHEN

methrin (Margulies et al., 1987) and the nitrogen heterocycle NMHl (Margulies et al., 1988) were successfully protected using a formulation of clay-pesticide-chromophore complex. In the present study we extend the above-mentioned approach to include the photoprotection of a uv-sensitive component in the B. thuringiensis toxin. Fourier transform infrared (FTIR) spectra were used to analyze the interactions between B. thuringiensis and several photostabilizing cationic chromophores. A bioassay with larvae of a major insect pest served to monitor photostabilization following uv irradiation. MATERIALS

AND METHODS

Chemicals

The cationic dyes 3,6-diamino- lo-methyl acridinium (acriflavin) (AF) and methyl green (MG) were purchased from Fluka (Buchs, Switzerland); the supplier of rhodamine B (RB) was Merck (Darmstadt, Germany). The molecular structures of the three dyes are shown in Figure 1. Organisms B. thuringiensis ringiensis kurstaki

fermentation.

B. thu-

HDl was cultured in lliter shake flasks containing 200 ml of medium. The medium at pH 7 had the following ingredients: glucose (0. l%), glycerol (O.l%), corn steep liquor (O.l%), yeast ex-

I 77

MG

AF , \

tW,CH212N

RB

T

+NICHJIZ

0

ET AL.

tract (OS%), NZ-amine B (peptone) (O.l%), MgCl, (0.2%) and CaCO, (0.2%). The flasks were incubated with shaking at 30°C for 40-48 hr, or until good sporulation and lysis of bacterial sells were observed. To scale up B. thuringiensis production, we used a New Brunswick fermentor with a 5-liter capacity. To suppress generation of foam, polystyrene glycol 2025 was added. The bacteria were grown at 30°C with agitation (500 rpm), and an air flow rate of 1 vvm. Spores, toxin crystals, and cells were separated from the culture broth by centrifugation, washed once in distilled water, and dried by lyophilization. The dried material is a mixture of intact spores, lysed bacteria, and free toxin. Insects. The budworm Heliothis armigera (Noctuidae, Lepidoptera) second instar larvae were used as our standard bioassay organism. The insects were reared on a large scale to provide a continuous supply of larvae at a proper physiological age for the bioassay experiments. H. armigera was reared under controlled conditions at 27”C, 70% relative humidity (rh) and a 14-hr light photophase. The semiartificial diet based on Shorey medium (Shorey, 1963), with modifications, consisted of water-soaked crushed beans, alfalfa pellet, dried yeast, sorbic acid, ascorbic acid, formaldehyde, p-hydroxybenzoic acid, gentamicin sulfate (Sigma), and agar. Individual second instar larvae were kept separately until pupation in small Petri plates (5 cm in diameter) to avoid cannibalism. Pupae were segregated according to sex and 10 pairs of emerging moths were placed inside a plastic box (20 x 15 X 20 cm). The adults were provided with a 10% sucrose solution and eggs laid on cotton were removed every 2 days and transferred to the diet.

hpCH31*

Bioassay

/ COOH ‘I

FIG. 1. Molecular structures mophores. AF, acriflavin; MG, rhodamine B.

of cationic chromethyl green; RB,

The B. thuringiensis samples were suspended in distilled water and thoroughly mixed into the diet at a level of 0.2 mg/g. In the bioassay experiments the diet .did not

PHOTOPROTECTION

OF B. thuringiensis

contain antibacterial antibiotics. In preliminary dose-response experiments, this concentration caused 95-100% mortality of H. armigera larvae. Second instar larvae were transferred individually to small plastic vials (55 mm long, 10 mm in diameter) containing 250 mg of diet and kept at 27°C and 70% rh. Mortality of larvae was recorded after 6 days. Adsorption

of chromophores

A suspension of B. thuringiensis (100 mg in 20 ml of distilled water) was dialyzed against 60 ml of various concentrations of a given dye dissolved in water. The dialysis was carried out overnight at room temperature with constant stirring. As controls, 20 ml of distilled water was dialyzed against the same volume of aqueous solutions of the dye at the corresponding concentrations. To remove excess unadsorbed dye, the B. thuringiensis suspension was further dialyzed against 1 liter of distilled water for 24 hr and this procedure was repeated three times. The amount of adsorbed dye was calculated from the concentration changes in solutions before and after dialysis as measured by uv-visible spectroscopy using the extinction coefficients of the relevant dyes: AF, 30,000 M-’ cm-’ at 449 nm; MG, 50,000 M-’ cm-’ at 631 nm; and RB, 100,000M-I cm-’ at 552 nm. Following the extensive dialysis, the B. thuringiensis was removed from the dialysis bag, lyophiiized, and ground. The powder was used in the irradiation and bioassay experiments. Irradiation

Suspensions of either B. thuringiensis or complexes at a level of 1.25 mg/ml of water were prepared and 10 ml was placed inside covered glass Petri plates (5 cm in diameter). The B. thuringiensis preparations were exposed to irradiation at 300-350 nm for various time periods, using a Rayonet RPRlOOphotochemical reactor. The glass plates used prevented an exposure to short-wavelength uv

B. thuringiensis-dye

FROM

IRRADIATION

345

irradiation (less than 300 nm). As controls, Petri plates covered with aluminum foil were also irradiated. The amount of water evaporated during the irradiation period was replenished to ensure the same B. thuringiensis concentration. Spectroscopy

FTIR spectra were measured in KBr pellets using a Nicolet MX-S spectrophotometer interfaced to an Elite Star 16 bits PC and a Goerz SE-284 digital plotter. Ultraviolet-visible absorption spectra of the aqueous solutions and suspensions of the dye and dye-B. thuringiensis complexes were measured in a Uvikon 820 spectrophotometer. To find whether interactions exist between the dye and the B. thuringiensis sample, FTIR spectra of B. thuringiensis were subtracted from that of B. thuringiensisdye complexes. RESULTS

B. thuringiensis exposed to irradiation loses its toxicity toward larvae of H. armigera (Fig. 2). Even 3 hr of irradiation resulted in an appreciable decrease in B. thuringiensis toxicity as 20% of insects survived. Longer exposure times resulted in a lower mortality rate as 70 and 95% of the insects were alive after 6 and 12 hr of irradiation, respectively. The adsorption isotherm of AF at equilibrium shows an increase in dye adsorbed to B. thuringiensis as the dye concentration is increased outside the dialysis bag (Fig. 3). A maximum of about 0.4 mmol AF/g of B. thuringiensis powder was measured above 5 mM concentration of the dye in the equilibrium solution. This level of dye adsorption is most likely dependent on B. thruingiensis surface area and furthermore on surface properties involving the presence of negatively charged functional groups such as glutamic acid and aspartic acid (see Carey et al., 1986 for the amino acid composition of B. thuringiensis kurstaki HDl toxin). Hydrogen bondings and hydrophobic interactions

346

COHEN

ET AL. 0.5

0.4

,

2

‘8. t. -AFH 3 t -AFL

B

0.3

” r ” /i L’

0.2

ii

E $

’

/' i-'

m

b 2 -i, E

a

i-

0.1

;

i

9 2

(

o.oIc

2

0

4

AF IN EQUILIBRIUM FIG. Bacillus

3. Adsorption thuringiensis

6 SOLUTION

a

10

(mM)

isotherm of acritlavin (AF) to kurstaki as a function of the dye

concentration.

2. Protection of Bacillus thuringiensis from photoinactivation by several chromophores. (A) B. thuringiensis-AFL, a complex containing acriflavin at a low concentration (0.06 mmol/g); B. thuringiensisAFH, a complex with a high concentration of acriflavin (0.42 mmol/g). (B) B. fhuringiensis-RB, rhodamine B adsorbed to B. thuringiensis (0.10 mmol/g); B. thuringiensis-MG, methyl green adsorbed to B. thuringiensis (0.53 mmol/g). Samples were irradiated (300350 nm) inside a photochemical reactor for the specitied time and bioassayed with second instar larvae of FIG.

Heliothis

armigera.

may as well play an important role in dye adsorption to B. thuringiensis. The FTIR spectra of B. thuringiensis before and after dialysis against distilled water are shown in Figure 4. Absorption bands at around 1430 and 1080 cm-’ which can be assigned to carboxylate and the amide III vibrations (Koenig and Tabb, 1980; Parker, 1971), correspondingly, are decreased after dialysis. This decrease might be due to the removal of inorganic cations such as Ca*’ and M$+ upon dialysis. These cations present in the growth medium (see Materials and Methods) were detected in large quantities in the original B. thuringiensis powder (unpublished inductively coupled plasma analysis). FTIR

spectra of B. thuringiensis and B. thuringiensis-dye complex between 2000 and 900 cm-’ are present in Figure 5. Molecular interactions between the cationic chromophore and B. thuringiensis proteins are apparent as one compares the subtraction spectrum (B. thuringiensis-dye complex minus B. thuringiensis) with the spectrum of free AF (Fig. 6). These interactions are reflected by disappearance of the 1638 cm-’ AF absorption peak as well as by the appearance of two peaks observed around 1430 and 1050 cm-‘. In addition, an in-

WAVENUMBER

(cm-‘)

Fro. 4. Fourier transform infrared absorption spectra of Bacillus thuringiensis kurstaki before and after dialysis against distilled water.

PHOTOPROTECTION

OF B. thuringiensis

-e

2000

WAVENUMBER

(cm-‘)

FIG. 5. Fourier transform infrared absorption spectra of Bacillus thuringiensis and B. thuringiensisacriflavin (AF) (0.42 mmohg). Characteristic absorption frequencies of the dye are indicated by arrows.

creased absorption at the area of 1880-1700 cm-’ was detected. It is noteworthy that the 1430and 1050cm-’ peaks appear in the same regions as the carboxylate and amide III vibrations which were shown to change upon dialysis. This seems to suggest that these vibrations are affected by the state of

FROM

347

IRRADIATION

adsorption of inorganic or organic cations to the surface of B. thuringiensis proteins. The peak at 1430 cm- ‘, which is near the carboxylate vibration (1446 cm- ‘), may be the result of interaction between the negatively charged carboxylate group and the positively charged chromophore. On the other hand, the peak at 1050 cm-’ is probably due to an interaction of the dye with the amide moiety. The asymmetrically shaped band at 1750cm-’ is presumably a result of broadening of the amide I vibration band of the protein. This broadening is perhaps due to an interaction with the amine group of the chromophore. Following uv irradiation an increase in absorption of the B. thuringiensis at the uvvisible range is evident (Fig. 7). The distinct peak at 260 nm before irradiation is converted to a shoulder after this treatment. A change in the uv-visible spectrum of the B. thuringiensis-AF complex also occurred after irradiation (Fig. 8). A decrease in the absorption of AF in the B. thuringiensisdye complex was observed and the two ma-

Idsorbed 3n B t

1 w 0

-

Z

a m [r

--

Before ---

After

,rrodmt/on !rrod,ot/on

0

U-J m a

“f reel’

WAVENUMBER

(cm-‘)

FIG. 6. Fourier transform infrared absorption spectra of acriflavin (AF): (a) free and (b) adsorbed on Bacillus thuringiensis. Curve b was obtained by subtracting the spectrum of B. thuringiensis from that of the complex B. thuringiensis-AF (both spectra are shown in Fig. 5). New absorption bands reflecting interactions between the dye and B. thuringiensis are indicated by arrows.

WAVELENGTH

(nm)

7. Electronic absorption spectra of Bacillus before and after irradiation with uv light. The spectra were measured on the same sample in aqueous suspension. FIG.

thuringiensis

COHEN ET AL.

348

~ Before irrod,ot/on --------After ,rrad/otion

f .lJ 0 2 a m IL 0 v, m

2000

-\ ‘\ I

?c

300

WAVELENGTH

1

I 500

400

‘. 600

lnm)

FIG. 8. Electronic absorption spectra of Bacillus Gzuringiensis-acriflavin (AF) (0.42 mmol/g) before and after irradiation with uv light. The spectra were measured on the same sample in aqueous suspension.

IO00

1500

WAVENUMBER

(cm-‘)

FIG. 9. Fourier transform infrared absorption spectra of Bacillus rhuringiensis-acriflavin (AF) (0.42 mmoVg) before and after irradiation with uv light. Characteristic absorption frequencies of the dye are indicated by arrows.

ringiensis, B. thruingiensis-AF (0.42 mmoY g), and B. thuringiensis-AF (0.06 mmol/g)

covered with aluminum foil for protection jor peaks of the dye at 261 and 449 nm from uv irradiation did not lose their insecshifted to the corresponding 264 and 465 ticidal activity, even after a prolonged exnm, indicating photodecomposition. Fur- posure of 12 hr. Moreover, the B. thurinthermore, AF dissolved in distilled water is giensis toxic protein appears to be insensialso decomposed after uv irradiation. The tive to exposure up to 12 hr to temperatures effect of uv irradiation on the B. thuringien- as high as 80°C (results not shown). Since sis-dye complex is also evident from the temperatures inside the photochemical rechanges observed in the FTIR spectrum actor were not beyond 50°C the effect of (Fig. 9). The disappearance of AF peaks at heat on B. thuringiensis inactivation was 1605, 1495, 1385, and 1330 cm-’ clearly in- excluded. Figure 2B presents results of an dicates partial or total decomposition of the experiment using other chromophores. chromophore molecule following irradiaMG, although adsorbed to B. thuringiensis tion. at a higher ratio (0.53 mmol/g) as compared From the results presented in Fig. 2 (A to AF (0.42 mmol/g), was less efficient, as and B) it is evident that adsorption of AF, larval mortality recorded was lower (35% MG, and RB on the B. thuringiensis surface vs 77.5% after 12 hr of exposure). RB adclearly improves the efficiency of the ento- sorbed at a ratio of 0.10 mmol/g B. thurinmocidal toxin following uv irradiation. A giensis was also less effective than the AF considerable uv photoprotection was ob- dye at 0.06 mmol/g B. thuringiensis (20% served after 6 hr of exposure to uv irradiavs. 48% after 12 hr). The chromophore per tion and this effect is even more emphase had no effect on mortality of H. armigeru sized following 12 hr of irradiation. The larvae. high ratio of AF to B. thuringiensis (0.42 rnmol/g) gave the best protection yet, even DISCUSSION a lower level of the chromophore (0.06 Comnetitiveness of entomotoxin-prommol/g) was still effective (Fig. 2A). B. thua

PHOTOPROTECTION

OF B. thuringiensis

ducing bacteria such as B. thuringiensis with conventional pesticides depends largely on prolonging their persistence under field conditions. Sunlight irradiation appears to be the environmental factor, pivotal in the loss of B. thuringiensis toxicity (Ignoffo et al., 1977; Morris, 1983; Raun et al., 1966). Using a silkworm larvae bioassay, Pozsgay et al. (1987) demonstrated that purified B. thuringiensis kurstaki HDI S-endotoxin was inactivated by uv irradiation. Attempts to achieve photoprotection included encapsulation techniques (Dunkle and Shasha, 1988; Raun and Jackson, 1966), granular formulations (Ahmed et al., 1973) and the addition of a variety of uv screeners to B. thuringiensis formulations (Morris, 1983). In our study a different approach, using selective chromophores which might exchange energy or electrons with an excited component of the B. thuringiensis toxin, was examined (Margulies et al., 1985). Experiments carried out in the laboratory showed that adsorption of the AF cationic dye at a certain level on B. thuringiensis was able to protect the toxin from photoinactivation following an intense irradiation regimen. The same phenomenon, although less prominent, was observed with MG and RB. This raises the possibility of using the above method for B. thuringiensis photoprotection under field conditions, especially in areas with intense sunlight. Different mechanisms for the photoinactivation of the B. thuringiensis toxin have been suggested. Ignoffo and Garcia (1978) invoked the probable involvement of peroxide radicals generated by uv radiation of amino acids in the instability of B. thuringiensis formulations under field conditions. Using Raman spectroscopy, Pozsgayet al. (1987) have shown that exposure of B. thuringiensis kurstaki toxin to 40 hr of uv irradiation resulted in the destruction of 30% of the tryptophan and 20% of the histidine residues. They maintained that a decrease in intensity of bands at 760 and 1555cm-’ is indicative of tryptophan destruction. It is

FROM

IRRADIATION

349

interesting to note that irradiation of the tryptophan solution at 3wOO nm generated toxic compounds, one of which is hydrogen peroxide (McCormick et al., 1976). Pozsgayet al. (1987) suggested a photosensitization mechanism by which an exogenous chromophore absorbs light and transfers energy to a O2 molecule. This in turn creates singlet oxygen which would react with the tryptophan and histidine residues. However, tryptophan absorbs light at longer wavelengths than any other amino acid (maximum absorption at 280 nm) and has considerable absorption above 290 nm (Wetlaufer, 1%2). Since this is the lowest limit of sunlight uv radiation at the earth surface (Kondratyev, 1%9), the possibility of direct photoexcitation cannot be excluded. In any event, it is tempting to speculate that tryptophan is present at the domain that is essential for the interaction of the toxin to insect midgut cell receptors. Alternatively, destruction of tryptophan residues may result in profound changes in the three-dimensional configuration of the toxic protein and consequently to loss of its biological activity. Two main mechanisms involved in B. thuringiensis toxin photoprotection by organic dyes can be considered: (A) Ultraviolet-screening effects; (B) specific interactions between the photolabile site of the toxin and the added chromophore. Morris (1983) pointed out that any ultraviolet screener used in B. thuringiensis formulations should have high absorbance capability in the 330- to 400-nm wavelength range. Apparently, mechanisms other than uv screening are involved since the dyes used in this study have absorption minima in this wavelength range and yet, nevertheless, were capable of photostabilizing the B. thuringiensis toxin. We have suggested energy transfer processes for photostabilization of pesticide-chromophore complexes (Margulies et al., 1985). Such processes might be applicable in the case of the B. thuringiensis toxin photoprotection by certain dyes. It is well established that formation of

350

COHEN ET AL.

triplet states of aromatic amino acids might be of considerable importance in photochemistry of proteins. Their relatively long lifetimes could enable interactions with various substrates and functional groups (Bent and Hayon, 1976). It was suggested that photolysis of tryptophan involves intersystern crossing from the lowest singlet level at 294 nm to the lowest triplet T, located at 70.5 kcal/mol (404 nm). This triplet is excited by a second photon to a higher triplet state having sufficient energy to reach the ionization potential of the tryptophan molecule (Reinisch et al., 1970). Processes leading to efficient quenching of the T, state can, therefore, prevent the photochemical reaction of tryptophan. Such processes could involve energy or electron transfer from the amino acid to another properly aligned chromophore . We suggest that the observed photostabilization of B. thuringiensis by AF is apparently due to energy transfer from the T, state of tryptophan to the first excited singlet state of the AF chromophore. It is noteworthy that such triplet-singlet energy transfer processes are theoretically allowed (Turro, 1978). This possibility is also supported by a strong overlap in the 400- to 500-nm region between the phosphorescence of tryptophan (Montenay-Garestier, et al., 1976) and the strong absorption band of AF (Fig. 8). Another supporting evidence is based on the observation that acridine dyes can efficiently accept energy from biological chromophores by tripletsinglet energy transfer mechanisms (Monteray-Garestier et al., 1976). Compared to AF, the energy matching requirement is less fulfilled when the dyes MG and RB are considered. Since photostabilization was also observed with these chromophores, other quenching mechanisms such as electron transfer cannot be excluded. All the above-mentioned mechanisms require relatively short distances between the donor and the acceptor. The FTIR results which showed specific interactions between B.

thuringiensis and the AF chromophore firm this requirement.

con-

ACKNOWLEDGMENTS This research was supported by U.S.-Israel Grant DPE-5544-G-SS-7046-00.

CDR

REFERENCES AHMED, S. M., NAGAMMA, M. V., AND MAIUMDER, S. K. 1973. Studies on granular formulations of Bacillus thuringiensis Berliner. Pestic. Sci., 4, 19-23. BEEGLE, C. C., DULMAGE, H. T., WOLFENBARGER, D. A., AND MARTINEZ, E. 1981. Persistence of Bacillus thuringiensis Berliner insecticidal activity on cotton foliage. Environ. Entomol., 10, 4tXl-401. BENT, D. V., AND HAYON, E. 1976. Lifetimes of triplet states of aromatic amino-acids and peptides in water. In “Excited States of Biological Molecules” (J. B. Birks, Ed.) pp. 498-506. Wiley, London. CAREY, P. R., FAST, P., KAPLAN, H., AND POZSGAY, M. 1986. Molecular structure of the protein crystal from Bacillus thuringiensis: A Raman spectroscopic study. Biochim. Biophys. Acta, 872, 169-176. DE BARJAC, H. 1978. Une nouvelle variete de Bacillus thuringiensis tres toxique les mousquites: B. thuringiensis var. israelensis serotype 14. C.R. Acad. Sci., 286, 797-800. DUNKLE, R. L., AND SHASHA, B. S. 1988. Starchencapsulated Bacillus thuringiensis: A potential new method for increasing environmental stability of entomopathogens. Environ. Entomol., 17, 120-126. IGNOFFO, C. M., AND GARCIA, C. 1978. UVphotoinactivation of cells and spores of Bacillus thuringiensis and effects of peroxidase on inactivation. Environ. Entomol., 7, 270-272. IGNOFFO, C. M., HOSTETTER, D. L., AND PINNELL, R. F. 1974. Stability of Bacillus thuringiensis and Baculovirus heliothis on soybean foliage. Environ. Entomol., 3, 117-119. IGNOFFO, C. M., HOSTETTER, D. L., SIKOROWSKI, P. P., SUTTER, G., AND BROOKS, W. M. 1977. Inactivation of representative species of entomopathogenie viruses, a bacterium, fungus and protozoan by a ultraviolet light source. Environ. Entomol., 6,411415. KOENIG, J. L., AND TABB, D. L. 1980. Infrared spectra of globular proteins in aqueous solution. In “Analytical Application of FT-IR to Molecular and Biological Systems” (J. R. Durig, Ed.), pp. 241255. D. Reidel, Dordrecht. KONDRATYEV, K. YA. 1969. “Radiation in the Atmosphere.” Academic Press, New York. KRIEG, A., HUGER, A. M., LANGENBROOK, G. A., AND SCHNETTER, W. 1983. Bacillus thuringiensis var tenebrionis: Ein neuer gegeuber Larvan von Co-

PHOTOPROTECTION

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leopteran Wirksamer Pathotyp. Z. Angew. Entomol., 96, 500-508. LEONG, K. L. H., CANO, R. J., AND KUBINSKI, A. M. 1980. Factors affecting BaciZZus thuringiensis total field persistence. Environ. Entomol., 9, 593599. MARGULIES, L., COHEN, E., AND ROZEN, H. 1987. Photostabilization of bioresmethrin by organic cations on a clay surface. Pestic. Sci., 18, 79-87. MARGULIES, L., ROZEN, H., AND COHEN, E. 1985. Energy transfer at the surface of clays and protection of pesticides from photoinactivation. Nature (London), 315, 658-659. MARGULIES, L., ROZEN, H., AND COHEN, E. 1988. Photostabilization of a nitromethylene heterocylce insecticide on the surface of montmorillonite. Clays Clay Miner., 36, 159-164. MCCORMICK, J. P., FISCHER, J. R., PACHLATKO, J. P., AND EISENSTARK, A. 1976. Characterization of a cell-lethal product from photooxidation of tryptophan: Hydrogen peroxide. Science, 191, 468-469. MONTENAY-GARESTIER, T., CHARLIER, M., AND HELENE C. 1976. Aggregate formation, excitedstate interactions, and photochemical reactions in frozen solutions of nucleic acid constituents. In “Photochemistry of Nucleic Acids” (S. Y. Wang, Ed.), Vol. 1, pp. 381-417. Academic Press, New York. MORRIS, 0. N. 1983. Protection of Bacillus thuringiensis from inactivation by sunlight. Canad. Entomol., 115, 1215-1227. PARKER, F. S. 1971. “Applications of Infrared Spectroscopy in Biochemistry, Biology, and Medicine.” Plenum, New York.

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PINNOCK, D. E., BRAND, R. J., JACKSON, K. L., AND MILSTEAD, J. E. 1974. Persistence of Bacillus thuringiensis spores on Cercis occidentalis leaves. J. Znvertebr. Pathol., 23, 341-346. POZSGAY, M., FAST, P., KAPLAN, H., AND CAREY, P. R. 1987. The effect of sunlight on the protein crystals from Bacillus thuringiensis var. kurstaki HDl and NRDl2: A Raman spectroscopy study. J. Znvertebr. Pathol., 50, 246-253. RAUN, E. S., AND JACKSON, R. D. 1966. Encapsulation as a technique for formulating microbial and chemical insecticides. J. Econ. Entomol. 59, 620622. RAUN, E. s., SW-TITER, G. R., AND REVELO, M. A. 1966. Ecological factors affecting the pathogenicity of Bacillus thuringiensis var. thuringiensis to the European corn borer and fall armyworm. J. Znvertebr. Pathol., 8, 365-375. REINISCH, R. F., GLORIA, H. R., AND ANDROES, G. M. 1970. Photoelimination reactions of macromolecules. In “Photochemistry of Macromolecules” (R. F. Reinsch, Ed.), pp. 185-217. Plenum, New York. SHOREY, H. H. 1%3. A simple artilicial medium for the cabbage looper. J. Econ. Entomol., 56,536-537. TURRO, N. J. 1978. “Modern Molecular Photochemistry.” Benjamin, New York. WETLAUFER, D. B. 1%2. Ultraviolet absorption spectra of proteins and aminoacids. Adv. Protein Chem., 17, 303-390. WHITELEY, H. R., AND SCHNEPF, H. E. 1986. The molecular biology of parasporal crystal body in Bacillus thuringiensis. Annu. Rev. Mcrobiol., 40,549 576.