Toxicity of Xanthene Dyes to Entomopathogenic Fungi

Effects of xanthene dyes on mycelial growth and conidial germination in three species of entomopathogenic fungi, Beauveria bassiana, Metarhizium anispoliae and Paecilomyces fumosoroseus, were evaluated in a variety of assay systems. In a disk-diffusion assay, erythrosin B and phloxine B (but not eosin B) produced zones of inhibition in colonies of all three species under continuous exposure to light at disk-loadings of 100mug. None of the dyes produced zones of inhibition in the absence of light at disk loadings of 100mug. Both erythrosin B and phloxine B inhibited mycelial growth of all three species in the light in a dose-dependent manner. Weaker dose-responses for inhibition of growth in the dark were observed for some fungus/dye combinations. Erythrosin B, tested singly, completely inhibited conidial germination in the light in all eight fungal strains tested at 100mug ml-1 medium, but failed to inhibit conidial germination in any of these strains in the dark at the same concentration of dye. For single strains of each of the three fungi, erythrosin B and phloxine B inhibited conidial germination in a dose-dependent manner in the light with IC50s < 6.2mug dye ml-1 medium for all fungus/dye combinations. Phloxine B was a more potent inhibitor of germination than erythrosin B for all three fungal species. At fixed dosages of erythrosin B and phloxine B, inhibition of conidial germination in all three species increased with time of exposure to light. These results constitute the first quantitative demonstration of photodynamic inhibition of conidial germination in fungi by xanthene dyes.     

Compatibility of Photoactive Dyes with Insect Biocontrol Agents

Integrated pest management (IPM) programmes often look for more specific ways to control pests. Biological control agents, such as the bacterium, Bacillus thuringiensis Berliner, and the fungus, Beauveria bassiana (Balsamo) Vuillemin, can control insects with minimal disturbance to the environment because of their host specificity and short half-lives. Often these agents alone cannot prevent yield loss or are too expensive. This study looked at the in vitro combination of these agents and photoactive dyes, especially phloxine B (red dye D&C 28), a Food and Drug Administration approved dye, with the intent to provide better insect control. Photoactive dyes are being tested for the control of many pest insects. Phloxine B and related xanthene dyes, eosin y, fluorescein and rose bengal inhibited the growth of both B. thuringiensis and B. bassiana . Phloxine B was the most inhibitory and fluorescein the least inhibitory dye for both microbes. The magnitude of inhibition increased with increasing concentration of dye and light intensity. Therefore, an adverse effect on the field performance of these biological control agents in combination with xanthene dyes would be expected.     

Insecticidal activity of photoactive dyes to American and migratory grasshoppers (Orthoptera: Acrididae)

Many photoactive dyes are relatively nontoxic to vertebrates despite their insecticidal properties. Several photoactive dyes known to be toxic to some groups of insects were evaluated at various concentrations for toxicity to American and migratory grasshoppers in laboratory and field studies. Rose bengal and phloxine B were effective at inducing mortality of grasshoppers when applied at 2 and 5% to bran bait, though erythrosin B and uranine were ineffective. Partial replacement of phloxine with uranine in dye mixtures resulted in no significant loss of efficacy. Some indication of feeding inhibition was observed at high dye concentrations, so minimum effective dosages, probably 2%, are optimal. Phloxine B and rose bengal appeared to be stable upon exposure to sunlight, and able to withstand at least 24 h of sunlight without significant degradation. Dyes such as phloxine B could be a viable grasshopper control agent for small or medium-sized grasshopper species because mortality can be induced by consumption of a single flake dusted with 5% dye, and yet pose little hazard to vertebrates. Large species such as American grasshopper must consume several flakes before mortality is induced.     

Evaluation of phloxine B as a photoinsecticide on immature stages of the horn fly, Haematobia irritans (L.) (Diptera: Muscidae)

The use of photoactive substances for controlling adult or immature stages of insect pests is an attractive alternative to chemical insecticides. Phloxine B is an environmentally friendly xanthene derivative that is safe for mammals but toxic for dipterans. In this study we tested the effect of phloxine B as a phototoxic larvicide against immature stages of the blood-sucking horn fly, Haematobia irritans (L.). The mortality rate of phloxine B was very low in the dark during the larval stage (100 h) unless a 0.5-mM dye concentration was used. However, a high mortality rate was attained when larvae III were transferred to containers exposed to 5000 lux during the last 2 h before pupariation. This was concentration-dependent up to 0.1-mM phloxine B. After a 2-h larval exposure to light the phloxine B 50% lethal concentration was 0.043 mM. These results indicate that H . irritans larvae are very sensitive to this dye, which in turn seems a promising component for larvicide formulations to control horn flies.     

Evaluation of phloxine B as a possible control agent against citrus thrips (Thysanoptera: Thripidae)

We evaluated the toxicity of phloxine B photoactive dye combined with a cane molasses bait against citrus thrips, Scirtothrips citri (Moulton). Laboratory bioassays conducted under artificial light showed that thrips mortality followed a log-dose probit-response model with an estimated LC50 of 0.0079% dye. Diluted cane molasses plus 0.01% phloxine B then was used as a standard for comparison of eight additional baits, including three formulations of concentrated citrus peel liquor (CCPL1-3). Citrus thrips mortality ranked highest to lowest with CCPL1 and CCPL3 > CCPL2, Mo-Bait and cane molasses > concentrated beet molasses, concentrated cane molasses, hemicellulose extract, and whey. Several commercial surfactants were tested to see if their addition to the standard increased efficacy. Hyper-Active, Kinetic, and Tween 60 at 1% and Cohort, Hyper-Active, Kinetic, and Silwet at 0.25% when added to the standard, reduced citrus thrips mortality, whereas Tween 60 and Dyne-Amic at 0.25% had no effect. Cane molasses with one or 5% phloxine B dye and CCPL1 with 1% dye were sprayed on citrus trees and allowed to weather in the field. Laboratory bioassays conducted after leaves had weathered for up to 8 d indicated that bait-dye toxicity was persistent. Possible use of the bait-dye mixture in commercial control of citrus thrips is discussed.     

Photoactive dye insecticide formulations: adjuvants increase toxicity to Mexican fruit fly (Diptera: Tephritidae)

The efficacy of photo active dyes as insecticides depends on the ingestion of the dye by the target insect and the activity of the dye at sensitive sites or on essential chemical functions. The site of this activity in insects is not understood, but we have found that certain chemical additives enhance the toxicity of phloxine B in the Mexican fruit fly. A series oftests with commercial adjuvants was performed under laboratory conditions that demonstrated a multifold decrease in the LD50 of phloxine B concentration and a decrease in the time required for photodynamic action to kill the flies. A total of 22 commercial adjuvants was tested. Of these, six were selected for evaluation under field cage conditions in comparison with a non-insecticide control bait (no treatment) and a phloxine B check bait with no adjuvant. Mortality was estimated by counting dead flies, feeding was estimated by fly counts at feeding stations, survival was estimated by trapping flies after the treatment period. In all cases the adjuvants increased the rate of mortality and decreased numbers surviving the treatment. Significant differences between adjuvants and both check and control were observed for mortality rates and the three best adjuvants, SM-9, Kinetic, and Tween60, induced significantly more mortality than the other adjuvants, the control, or the check. Feeding rates and survival rates indicated that the adjuvants increase the effectiveness of phloxine B in a predictable manner. The adjuvants appear to be active inside the insect rather than increasing the solubility of the dye in the bait medium. We propose that the addition of 1% vol:vol of the best adjuvant, Tween60 to the proteinaceous bait with 0.5% phloxine B will enhance toxicity as well as improve mixing and other characteristics of the bait.     

Field evaluation of a phototoxic dye, phloxine B, against three species of fruit flies (Diptera: Tephritidae).

The xanthene dye phloxine B (D&C Red #28) bait was sprayed against fruit flies in mango orchards in 1996 and 1997. The flies used for testing were Mexican fruit fly, Anastrepha ludens (Loew), West Indian fruit fly, Anastrepha obliqua (Macquart), and Mediterranean fruit fly, Ceratitis capitata (Wiedeman). Results of the experiments indicate that the toxic efficacy of phloxine B against these fruit flies is as good as that of malathion-bait sprays. Results also indicate that type of protein used with phloxine B can dramatically influence its efficacy. Hydrolyzed proteins of corn origin, Mazoferm 802 and Nutriplus, and one from microbial origin, Coltec yeast broth, were best. Phloxine B-bait applications as complete coverage or alternate swaths reduced fly populations as well as 19.5 or 9.8% (AI) malathion-Captor 300. Applications of phloxine B bait at concentrations of 0.12% phloxine B reduced populations as well as those applied at 0.48% (AI). The fruit fly parasitoid Diachasmimorpha longicaudata was adversely affected when exposed to phloxine B-Nutriplus bait but not when exposed to the other proteins.     

Mutagenesis in cultured human diploid cells. IV. Induction of 8-azaguanine resistant mutations by phloxine, a mutagenic red dye.

Disodium 9-(3',4',5',6'-tetrachloro-o-carboxyphenyl)-6-hydroxy-2,4,5,7-tetrabromo-3-isoxanthone (phloxine), a red dye used as a food additive, was tested for its activity to induce 8-azaguanine (8AG) resistant mutations in cultured human embryonic cells. Phloxine had a severe cytotoxic effect on the cells at concentrations of 1 to 10 mug/ml. At concentrations of more than 30 mug/ml of phloxine no further decrease in cell survival was found. This cytotoxic effect of phloxine was not dependent on the duration of treatment. After treatment with phloxine for 2 h division of cells in normal medium was inhibited for 120 h. When cells were treated with phloxine at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected with 30 mug/ml of 8AG, an increase in the induced mutation frequency was found. This increase in mutation frequency was dependent on the concentration of phloxine used as a mutagen and treatment with 100 mug/ml of phloxine increased the frequency to six times that in untreated cultures.     

Phloxine B effect on immature stages of the Mediterranean fruit fly,Ceratitis capitata (Diptera: Tephritidae) (Wiedemann)

A laboratory bioassay was developed to determine both the chemical toxicity and the phototoxicity of the xanthene dye, phloxine B (D&C Red No 28), to the immature stages of the Mediterranean fruit fly, Certitis capitata (Wiedemann). An additional goal was to find out which main tissues are affected first. A low, but significant, level of toxicity was observed when the insects were maintained in the dark: at the point of adult ecdysis, the LC50 was 11.03 mM. As expected, after 8-h exposure of late larva III to light, a high level of mortality was produced (LC50 at ecdysis: 0.45 mM) as a dose-dependent function of dye concentration. At sublethal concentrations of the dye, the surviving insects showed a number of physiological abnormalities. Phloxine B appeared to mainly affect the larval longitudinal muscles as well as the abdominal muscles of ecdysing adults, giving rise to abnormal puparia and failed adult ecdysis, respectively. Moreover, a significant phloxine B-dependent delay in the jumping of surviving larvae for dispersal was documented. This could be attributed to a delay in attaining a threshold weight for jumping and/or to abnormalities in neuromuscular coordination, thus reinforcing the idea of pleiotropic effects of the dye.     

Comparative evaluation of spinosad and phloxine B as toxicants in protein baits for suppression of three fruit fly (Diptera: Tephritidae) species

Spinosad and phloxine B are two more environmentally friendly alternative toxicants to malathion for use in bait sprays for tephritid fruit fly suppression or eradication programs. Laboratory tests were conducted to assess the relative toxicity of these two toxicants for melon fly, Bactrocera cucurbitae Coquillett; oriental fruit fly, Bactrocera dorsalis Hendel; and Mediterranean fruit fly, Ceratitis capitata (Wiedemann) females. Field tests also were conducted with all three species to compare these toxicants outdoors under higher light and temperature conditions. In laboratory tests, spinosad was effective at much lower concentrations with LC50 values at 5 h of 9.16, 9.03, and 4.30 compared with 250.0, 562.1, and 658.9 for phloxine B (27, 62, and 153 times higher) for these three species, respectively. At 16 ppm spinosad, LT50 values were lower for all three species (significantly lower for C. capitata and B. dorsalis) than 630 ppm phloxine B LT50 values. At 6.3 ppm spinosad, the LT50 value for C. capitata (3.94) was still significantly less than the 630 ppm phloxine B LT50 value (6.33). For all species, the 100 ppm spinosad concentrations gave LT50 values of < 2 h. In comparison among species, C. capitata was significantly more sensitive to spinosad than were B. cucurbitae or B. dorsalis, whereas B. cucurbitae was significantly more sensitive to phloxine B than were C. capitata or B. dorsalis. LC50 values were reduced for both toxicants in outdoor tests, with greater reductions for phloxine B than for spinosad for B. dorsalis and B. cucurbitae. Fly behavior, though, is likely to keep flies from being exposed to maximum possible outdoor light intensities. Comparable levels of population suppression for any of the three species tested here will require a much higher concentration of phloxine B than spinosad in the bait.     

Effect of a phloxine B-cucurbitacin bait on diabroticite beetles (Coleoptera: Chrysomelidae)

Cucurbitacin E glycoside, extracted from a bitter mutant of Hawkesbury watermelon [Citrulls lanatus (Thunb.) Matsum. & Nakai (Syn. Citrullus vulgaris Schrad)] is the active ingredient of a feeding stimulant for the corn rootworm complex. It is the primary component of a water-soluble bait that can be combined with toxins for adult diabroticite control. Studies were conducted using phloxine B (D&C Red 28), a xanthene dye, as the toxin. This dye was efficacious against Diabrotica undecimpunctata howardi Barber, spotted cucumber beetle, and Acalymma vittatum (F.), striped cucumber beetle, in cucumber plots and could be recovered from cucumber leaves for 8 d after treatment. The average amount of dye recovered per dead spotted cucumber beetle at 8 d after treatment was 0.173 microg. Concentrated and sugar-free fermented forms of the watermelon extract were developed and compared with the fresh juice in field applications on cucumber plants. There was no significant difference in mortality of beetles from phloxine B-bait prepared with fresh, fermented, or concentrated extract, although in laboratory studies, fermented juice had higher feeding stimulant activity.     

Potential for areawide integrated management of Mediterranean fruit fly (Diptera: Tephritidae) with a braconid parasitoid and a novel bait spray

The braconid wasp, Fopius arisanus (Sonan), a biological control agent for Mediterranean fruit fly, Ceratitis capitata (Wiedemann), was studied in coffee, Coffea arabica L. Fopius arisanus, comprised 79.3% of the total parasitoids (7,014) recovered from fruits collected at three small coffee farms. Data from seasonal host/parasitoid studies at a large coffee plantation also suggested that the most effective natural enemy of C. capitata in coffee may now reside in Hawaii. The original parasitoids introduced into Hawaii for C. capitata control (Diachasmimorpha tryoni (Cameron), Tetrastichus giffardianus Silvestri, and Dirhinus giffardii Silvestri) are now rare. Abundance of F. arisanus with respect to other parasitoids collected was influenced by elevation (274, 457, 610 m). Fopius arisanus was the dominant parasitoid at all three elevations, Diachasmimorpha longicaudata (Ashmead) occurred consistently, and T. giffardianus was abundant only at low elevation. The impacts on C. capitata and F. arisanus populations of bait sprays containing malathion, spinosad, or phloxine B applied to coffee were also evaluated. All three bait sprays suppressed C. capitata populations. Spinosad and phloxine B bait sprays appeared less harmful to the wasp than malathion. Fopius arisanus offers the potential for areawide management of C. capitata that includes biological control and integration with more environmentally safe chemical controls such as spinosad and phloxine B bait sprays.     

Ascorbic Acid Synergistically Potentiates Phloxine B‐induced Photocytotoxicity in Human Acute Promyelocytic Leukemia Cells

Ascorbic acid (AsA) is known as an antioxidant but concomitantly possesses a pro‐oxidant property. Because the impact of AsA on photodynamic therapy response is unclear, we investigated the effect of AsA on photocytotoxicity induced by phloxine B in human acute promyelocytic leukemia HL‐60 cells. AsA synergistically enhanced phloxine B‐induced photocytotoxic effects, including inhibition of cell proliferation, DNA ladder formation, and caspase‐3 activation, whereas AsA itself showed no photocytotoxicity. AsA also enhanced the consumption of the reduced glutathione level compared with the cells treated with phloxine B alone under the light condition. Combination of AsA with phloxine B under the light condition enhanced the phosphorylation of c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase (MAPK). These effects were completely cancelled by catalase. These results suggest that AsA synergistically enhances phloxine B‐induced photocytotoxicity, possibly through the extracellular oxidative stress‐dependent MAPK pathway activation.     

The glycine residues G551 and G1349 within the ATP-binding cassette signature motifs play critical roles in the activation and inhibition of cystic fibrosis transmembrane conductance regulator channels by phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 +/- 1.6 microM: , Ki = 38 +/- 1.4 microM: ) and delF508 CFTR (Ks = 3 +/- 1.8 microM: , Ki = 33 +/- 1 microM: ). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 +/- 1.13 microM: ) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 +/- 1.01 microM: ) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.     

Field tests of environmentally friendly malathion replacements to suppress wild Mediterranean fruit fly (Diptera: Tephritidae) populations

This article reports a large-scale field test of two environmentally friendly malathion replacements on wild populations of the Mediterranean fruit fly, Ceratatis capitata (Wiedemann): spinosad, a bacteria-derived toxin, and phloxine B, a red dye with phototoxic properties. The comparison test was conducted on 11 coffee fields infested with wild populations of Mediterranean fruit fly on the Hawaiian island of Kauai with 8-wk protein bait sprays with and without toxicants. To assess effectiveness, adults were trapped and larval infestation levels were evaluated with fruit collections. Malathion was found to be the most effective treatment. However, the two replacements gave significant levels of control, and because they are environmentally safer, should be considered for eradicating incipient populations of this invasive species of fruit fly. Cage tests were also conducted to ensure that the wild flies consumed the bait and to assess how long the bait-toxicant combination remained effective in the field. Although spinosad and phloxine B were found to be effective up to 1 wk, malathion remained effective at least 2 wk.     

Detection, with the dye phloxine B, of yeast mutants unable to utilize nitrogenous substances as the sole nitrogen source.

Yeast mutants unable to degrade certain nitrogen compounds produce characteristic small red colonies on an agar medium containing the red dye phloxine B, galactose, the test nitrogen compound, and a small amount of ammonium chloride.     

Toxicity and residual effectiveness of insecticides on insecticide-treated spheres for controlling females of Rhagoletis pomonella (Diptera: Tephritidae)

This study evaluated the toxicity of five technical-grade insecticides of four different classes to apple maggot females, Rhagoletis pomonella (Walsh), following a 10-min exposure period in insecticide-coated glass jars, with or without a feeding stimulant (sucrose) present. According to LC90 values for toxicity by ingestion and tarsal contact, imidacloprid was 1.5 times more toxic than dimethoate or abamectin, diazinon was less toxic, and phloxine B (a phototoxic dye) least toxic. Based on LC90 values for tarsal contact alone, dimethoate was 2.3, 4.0, and 18.4 times more toxic than imidacloprid, abamectin, and diazinon, respectively. Contact alone with phloxine B caused no mortality. When exposure was assessed using spheres coated with a latex paint mixture containing sucrose and formulated dimethoate (Digon 400 EC) or imidacloprid (Provado 1.6 F) at concentrations ranging from 5 to 70 g (AI)/cm2, both insecticides showed reduced effectiveness compared with toxicities from glass jar tests, with Digon two times more toxic than Provado. After exposure to artificial rainfall and retreatment with sucrose, Digon- and Provado-treated spheres exhibited greatest residual effectiveness, with diazinon-treated spheres less effective. Spheres treated with formulated abamectin (Agri-Mek 0.15 EC) at 1.0% (AI) performed only slightly better than phloxine B-treated spheres, which completely lost effectiveness after exposure to rainfall. Spheres treated with formulated imidacloprid (Merit 75 WP) at 1.5% (AI) showed equal or better residual efficacy in killing apple maggot flies (> 80% mortality, shorter lethal duration of feeding) over a 12-wk exposure period to outdoor weather than spheres treated with Digon at 1.0% (AI) after both types were retreated with sucrose. Our results indicate that imidacloprid is a promising safe substitute for dimethoate as a fly killing agent on lure-kill spheres. Imidacloprid formulated as Merit 75 WP had greater residual efficacy than imidacloprid formulated as Provado 1.6 F.     

Viability Tests for Thick Walled Fungal Spores (ex: Oospores of Peronospora manshurica)

Oospores of Peronospora manshurica, the causal agent of soybean downy mildew, were stained by a variety of techniques. TTC (tetrazolium chloride) and NBT (nitroblue tetrazolium chloride) primarily stained oospores which were cytologically abnormal and appeared degenerating. Cytological normal oospores were not stained by these compounds presumably because the dyes were excluded from the oospore cytoplasm by the oospore wall or the plasmalemma. Strong autofluorescence of dead/degenerating oospores in the FDA test (fluorescein diacetate) made scoring of the oospore viability by this technique unreliable. Phloxine B was found in a consistent way to stain the degenerating oospores and a small proportion of the oospores which by light microscopic, observations could not be scored cytologically abnormal. Control experiments with live and dead cells of yeast (Saccharomyces cerevisiae) confirm that phloxine B is excluded from live cells and dead cells become stained. The presumed mode of action is that the semipermeability of the plasma membrane of live cells excludes the stain. The phloxine B test described here appears a promising technique for the determination of oospore viability of P. manshurica.     

Phloxine B interacts with the cystic fibrosis transmembrane conductance regulator at multiple sites to modulate channel activity

The fluorescein derivative phloxine B is a potent modulator of the cystic fibrosis transmembrane conductance regulator (CFTR). Low micromolar concentrations of phloxine B stimulate CFTR Cl(-) currents, whereas higher concentrations of the drug inhibit CFTR. In this study, we investigated the mechanism of action of phloxine B. Phloxine B (1 microm) stimulated wild-type CFTR and the most common cystic fibrosis mutation, DeltaF508, by increasing the open probability of phosphorylated CFTR Cl(-) channels. At each concentration of ATP tested, the drug slowed the rate of channel closure without altering the opening rate. Based on the effects of fluorescein derivatives on transport ATPases, these data suggest that phloxine B might stimulate CFTR by binding to the ATP-binding site of the second nucleotide-binding domain (NBD2) to slow the dissociation of ATP from NBD1. Channel block by phloxine B (40 microm) was voltage-dependent, enhanced when external Cl(-) concentration was reduced and unaffected by ATP (5 mm), suggesting that phloxine B inhibits CFTR by occluding the pore. We conclude that phloxine B interacts directly with CFTR at multiple sites to modulate channel activity. It or related agents might be of value in the development of new treatments for diseases caused by the malfunction of CFTR.     

Photodynamic action of proflavine on coliphage T3. II. Protection by L-cysteine

Three kinetically different reactions (Rx1, Rx2, and Rx3) have been distinguished in the photoinactivation of phage T3 in the presence of the dye proflavine. The response of these reactions to the presence of the radical trap l-cysteine has been examined. At dye concentrations equal to or less than 2.2 mug/ml, Rx1 was composed of at least two parallel first-order reactions, one cysteine-insensitive (Rx1A) and one cysteine-inhibited (Rx1B). Rx2 was completely cysteine-insensitive (Rx2A). The cysteine sensitivity of these reactions changed abruptly at dye concentrations above 2.2 mug/ml. Rx1A and Rx1B now operated in tandem, rather than simultaneously, with Rx1B being confined to the first 1 min at most. Rx2, on the other hand, was completely cysteine-inhibited (Rx2B). Rx3 was inhibited roughly 75 to 80% by saturating concentrations of cysteine regardless of the time of addition of cysteine. The dark inactivation associated with Rx3 was inhibited roughly 85% whether the radical trap was added during the light or dark regimes. Changes of initial phage titer did not alter the cysteine sensitivity of a reaction.