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.     

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.     

A cost-effective cane molasses medium for enhanced cell-bound phytase production by Pichia anomala

AIM: Formulation of an inexpensive cane molasses medium for improved cell-bound phytase production by Pichia anomala. METHODS AND RESULTS: Cell-bound phytase production by Pichia anomala was compared in synthetic glucose-beef extract and cane molasses media. The yeast was cultivated in 250 ml flasks containing 50 ml of the medium, inoculated with a 12 h-old inoculum (3 x 10(6) CFU ml(-1)) and incubated at 25 degrees C for 24 h at 250 rev min(-1). Different cultural parameters were optimized in cane molasses medium in batch fermentation. The cell-bound phytase content increased significantly in cane molasses medium (176 U g(-1) dry biomass) when compared with the synthetic medium (100 U g(-1) dry biomass). In fed-batch fermentation, a marked increase in biomass (20 g l(-1)) and the phytase yield (3000 U l(-1)) were recorded in cane molasses medium. The cost of production in cane molasses medium was pound 0.006 per 1000 U, which is much lower when compared with that in synthetic medium (pound 0.25 per 1000 U). CONCLUSIONS: An overall 86.6% enhancement in phytase yield was attained in optimized cane molasses medium using fed-batch fermentation when compared with that in synthetic medium. Furthermore, the production in cane molasses medium is cost-effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase yield was improved in cane molasses when compared with the synthetic medium, and the cost of production was also significantly reduced. This enzyme can find application in the animal feed industry for improving the nutritional status of feed and combating environmental pollution.     

A marked enhancement in phytase production by a thermophilic mould Sporotrichum thermophile using statistical designs in a cost-effective cane molasses medium

AIMS: Statistical optimization of phytase production by a thermophilic mould Sporotrichum thermophile in a cost-effective cane molasses medium. METHODS AND RESULTS: Sporotrichum thermophile secreted phytase in cane molasses medium at 45 degrees C and 250 rev min(-1) after 5 days. The important factors identified by Plackett-Burman design (magnesium sulfate, Tween 80, ammonium sulfate and incubation period) were further optimized by response surface methodology (RSM). An overall 107% improvement in phytase production was achieved due to optimization. Supplementation of the medium with inorganic phosphate repressed the enzyme synthesis. When inorganic phosphate was reduced from the cane molasses medium by treatment with calcium chloride, the enzyme production increased. The phytase activity was not affected by the enzyme treatment with trypsin and pepsin. CONCLUSIONS: A twofold increase in phytase production was achieved due to optimization using statistical designs in a cost-effective cane molasses medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase production was doubled due to optimization. The enzyme, being resistant to trypsin and pepsin, thermostable and acid stable, can find application in animal feed industry for improving nutritional status of the feed and combating environmental phosphorus pollution.     

Interaction effects between Beauveria bassiana and imidacloprid against Thrips tabaci (Thysanoptera: Thripidae)

The onion thrips, Thrips tabaci Lindeman is a major pest of many horticultural crops in many parts of the world. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin and imidacloprid are used for the control of the onion thrips. The potential interactions between the fungus and the chemical against the onion thrips adults were evaluated in a laboratory and two greenhouse bioassays. The laboratory bioassay was a leaf dip bioassay for a mixture of B. bassiana and imidacloprid. The first greenhouse bioassay was a topical application of a mixture of the fungus and the chemical while the second bioassay was a drenching application of imidacloprid and a topical application of B. bassiana. In all bioassays, the combined application resulted in higher percentage mortalities than either B. bassiana or imidacloprid alone. In the laboratory bioassay, the highest mortality of 97% was achieved by using B. bassiana at the field rate with imidacloprid at 1/10th the field rate relative to 88%, 94%, 21% for B. bassiana alone, imidacloprid alone and the control, respectively. In the first greenhouse bioassay, the highest mortality of 80% resulted from the combined application of B. bassiana and imidacloprid at field rates compared with 55%, 75%, and 22% for B. bassiana alone, imidacloprid alone and the control, respectively. In the second greenhouse bioassay, the combined application of both control agents at field rates resulted in 85% mortality compared with 52%, 83%, and 18% for B. bassiana alone, imidacloprid alone and the control, respectively. The interaction effect between the two control agents was additive except when B. bassiana at 1/2 field rate was mixed with imidacloprid at 1/10th field rate in the laboratory bioassay, where the interaction was antagonistic. The above results indicate that combining imidacloprid with B. bassiana might reduce the rate of the insecticide application while enhancing the efficacy of the biological control agent.     

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.     

Influence of media composition on the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis

Powders of edible leguminous seeds, greengram (Vigna radiata) or soybean (Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/liter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against fifth-instar Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 x 10(6)/mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 x 10(6)/mg, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced delta-endotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard.     

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.     

Optimization of nutritional supplements for enhanced lactic acid production utilizing sugar refinery by-products

The present study investigated the synergistic effect of nutritional supplements (amino acid and Tween 80) on lactic acid production by Lactobacillus delbruckii utilizing a sugar refinery by product (cane molasses) in a submerged fermentation process. Initially, the effect of individual factors on lactic acid yield was studied by supplementing amino acids and their combinations, Tween 80 and cane molasses at varying concentrations in production medium. A combination of l-phenylalanine and l-lysine gave a maximum lactic acid yield of 47.89?±?0.1 g/L on a dry cell weight basis at individual factor level. Similarly, maximum lactic acid yield was obtained by supplementing the production medium with 40.0 g/L and 2.0 g/L Tween 80 and cane molasses, respectively, at individual factor level. In order to further improve the lactic acid yield, nutritional supplements were optimized by central composite rotatable design (CCRD) using Minitab 15 software. Shake flask cultivation under optimized conditions, i.e., cane molasses (32.40 g/L), Tween 80 (2.0 g/L) and l-phenylalanine and l-lysine (34.0 mg/L) gave a lactic acid yield of 64.86?±?0.2 g/L, corresponding to 95.0 % of the predicted yield of 67.78?±?0.3 g/L. Batch cultivation performed in 7.5 L bioreactor (working volume: 3.0 L) under optimized conditions gave maximum lactic acid yield and productivity of 79.12?±?0.2 g/L and 3.40 g/L·h, which is higher than previous studies with reduced fermentation time. Screening of lactic acid producing bacteria and characterization of lactic acid was also done.     

Economical glucoamylase production by alginate-immobilized Thermomucor indicae-seudaticae in cane molasses medium

AIMS: The present investigation is aimed at assessing the suitability of cane molasses as a cheaper carbon and energy source for glucoamylase production using alginate-immobilized Thermomucor indicae-seudaticae. METHODS AND RESULTS: The culture variables for glucoamylase production were optimized by 'one-variable-at-a-time' strategy and response surface methodology (RSM). A high glucoamylase titre was attained when 40 alginate beads (c. 5x10(6) immobilized spores) were used to inoculate 50 ml of cane molasses (8%) medium in 250-ml Erlenmeyer flasks. Response surface optimization of fermentation parameters (cane molasses 7%, inoculum level 44 alginate beads per 50 ml of medium and ammonium nitrate 0.25%) resulted in 1.8-fold higher glucoamylase production (27 U ml(-1)) than that in the unoptimized medium (15 U ml(-1)). Enzyme production was also sustainable in 22 l of laboratory air-lift bioreactor. CONCLUSIONS: Cane molasses served as an excellent carbon and energy source for the economical production of glucoamylase, which was almost comparable with that in sucrose yeast-extract broth. The statistical model developed using RSM allowed determination of optimum levels of the variables for improving glucoamylase production. SIGNIFICANCE AND IMPACT OF THE STUDY: The cost of glucoamylase produced in cane molasses supplemented with ammonium nitrate was considerably lower (euro1.43 per million U) than in synthetic medium containing sucrose and yeast-extract (euro35.66 per million U). The reduction in fermentation time in air-lift bioreactor with sustainable glucoamylase titres suggested the feasibility of scale up of the process.     

Systemic effects of neem on western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

The systemic effects of neem on the western flower thrips, Frankliniella occidentalis (Pergande), were investigated in laboratory trials using green bean, Phaseolus vulgaris L., in arena and microcosm experiments. In arena experiments, systemic effects of neem against western flower thrips larvae on primary bean leaves were observed with maximum corrected mortality of 50.6%. In microcosm experiments using bean seedlings, higher efficacy in the control of western flower thrips were observed with soil applications of neem on a substrate mixture (i.e., Fruhstorfer Erde, Type P, and sand) in a 1:1 ratio (93% corrected mortality) compared with application on the commercial substrate only (76% corrected mortality). However, longer persistence of neem was observed with soil application on the commercial substrate, which showed effects against thrips for up to 6 d after application. In addition to systemic effects observed on all foliage-feeding stages of western flower thrips, mortality on contact and repellent effects were observed on soil-inhabiting stages after soil applications of neem. Finally, bean seedlings grown from seeds pregerminated for 3 d in neem emulsion were also toxic to western flower thrips.     

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.     

Lytic Enzymes for Gram-negative Bacteria Produced by Bacillus subtilis YT-25

The available energy, gross protein value, phosphorus availability and palatability of 16 samples of single cell protein were evaluated in 20 bioassays using total 2,136 depleted chicks.

Four protein samples were products from Aspergillus tamarii grown on waste water of a fish processing factory, three were from Aspergillus oryzae grown on either acetic acid medium or cooked soybean waste, three were from Candida sp. grown on citrus molasses extracted from peel wastes of citrus processing plants, four were from Candida utitis grown on wood molasses produced from various wood wastes, and two were from Pseudomonas sp. and Alteromonas thlasomethanolica grown on methanol.

Five of 16 samples had excellent nutritive value, comparable to single cell proteins available commercially in Europe. All samples were palatable to the chicks, and no sign of acute toxicity was observed.     

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.     

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 Phloxine B, an Insecticidal Photoactive Dye, with Selected Biocontrol Fungi

The incorporation of the photoactive red food dye, phloxine B, into integrated pest management strategies has been suggested for the control of various insect pests. Inclusion of such a chemical pest control agent may interfere with a microbial pest control agent. Thus, representative fungi with potential for biocontrol were tested for their responses to phloxine B (0.01%) at different temperatures. Growth rates at selected temperatures were inhibited by dye in the presence of light for Coniothyrium minitans and Verticillium lecanii. Trichoderma virens growth was inhibited in light and dark. At optimal growth temperatures, Beauveria bassiana growth was inhibited only by the photoactivated dye species, while growth of Stilbella erythrocephala was not affected by the dye even in the presence of light.     

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.     

The Role of Formulation Additives in Increasing the Potency of Cydia pomonella Granulovirus for Codling Moth Larvae,in Laboratory and Field Experiments

Studies were undertaken to improve the biological efficacy of the granulovirus (CpGV) of the codling moth, Cydia pomonella , by evaluating the performance of some formulation additives that might improve virus persistence and/or virus uptake by first instar larvae. Laboratory studies, using a leaf disc bioassay, demonstrated that 15% cane molasses incorporated within a formulation of purified CpGV dramatically reduced the median lethal exposure time (LET 50 ) to CpGV for neonate larvae at a CpGV dosage rate of 10 7 occlusion bodies (OBs) ml -1 . Screening of a range of other compounds showed that sucrose, fructose and sorbitol (at 10% concentrations) and extracts of apple flesh and skin also gave significant reductions in the LET 50 of CpGV formulations containing these ingredients. Pectin, malic acid and &#102 -farnesene did not significantly reduce the LET 50 . In a field trial, molasses included at 15% (v/v) in a CpGV formulation, containing a dosage rate of 10 12 OBs ha -1 , gave as good control of codling moth damage as virus formulations containing the 'sticker' 0.2% skimmed milk at higher dosage rates of 10 13 and 10 14 OBs ha -1 . Studies of CpGV persistence on foliage revealed no significant improvement of virus persistence on apple foliage using 10% or 15% molasses formulations. A second field trial demonstrated that 10% molasses, 10% sorbitol or 0.08% &#102 -farnesene significantly reduced codling moth deep damage to fruit when these ingredients were added to formulations of pure CpGV. Substantial sooty-mould growth ( Cladosporium spp.) was observed on apple foliage treated with formulations containing molasses, indicating that this formulation additive has secondary consequences that would need to be taken into account if molasses was to be used in commercial CpGV formulations. Nonetheless, these studies clearly demonstrate that major biological improvements in CpGV performance can be achieved by the incorporation of formulation additives, including molasses and several other compounds, that probably function as attractants and/or feeding stimulants for codling moth larvae.